01 fastdigest conventional restriction enzymes

1All Fermentas products are manufactured in class D clean room facilities, qualified and certified as per EU directives and ISPE guidelines. Fermentas quality assurance is carried out according to ISO9001 quality and ISO14001 environmental management systems, guaranteeing batch-to-batch reproducibility. Integration of our clean room and ISO systems ensures stability and the absence of contaminants in all of our products.

FastDigest® and Conventional Restriction Enzymes .................................................. 2PureExtreme® Quality ...................................................................................................... 2Labeled Oligonucleotide (LO) Test .................................................................................. 2Navigation Guide .............................................................................................................. 3Description of Icons ......................................................................................................... 4

FastDigest® Restriction Enzymes .................................................................................... 5Activity and Quality Control Assays ............................................................................... 6Storage and Shipping ...................................................................................................... 6Product List ...................................................................................................................... 7Product Description ....................................................................................................... 11Protocols and Recommendations ................................................................................. 70

1.1. Fast DNA digestion ................................................................................................ 701.2. Reaction set-up for digestion of multiple DNA samples ........................................... 701.3. Double and multiple digestion of DNA ................................................................... 701.4. Scaling up DNA digestion reaction ......................................................................... 70

Reaction Conditions ....................................................................................................... 71Conventional Restriction Enzymes ............................................................................... 75

Activity and Quality Control Assays ............................................................................. 75Storage and Shipping .................................................................................................... 75Product List and Cross-reference to FastDigest® Restriction Enzymes ................... 76Product Description ....................................................................................................... 80

Nicking Enzymes ....................................................................................................... 157Homing Enzyme ......................................................................................................... 159

Protocols and Recommendations ............................................................................... 1601.5. DNA digestion .................................................................................................... 1601.6. Digestion of PCR products .................................................................................. 1601.7. Setting up double digestion ................................................................................. 1601.8. Stability during prolonged incubation ................................................................... 1601.9. Dilution of restriction enzymes ............................................................................. 1601.10. Partial digestion of DNA..................................................................................... 1601.11. Digestion of agarose-embedded DNA ................................................................. 1611.12. Inactivation of restriction enzymes ..................................................................... 161

Reaction Conditions .................................................................................................... 162Reaction Buffers ........................................................................................................ 162Recommended Reaction Conditions ........................................................................... 163Activity of Mesophilic and Thermophilic Enzymes at 37°C ........................................... 167Double Digestion in Universal Tango™ Buffer............................................................... 168Digestion of Agarose-Embedded DNA ........................................................................ 170Cleavage Efficiency Close to the Termini of PCR Fragments ......................................... 171Cleavage of Restriction Targets Located in Close Vicinity within pUC19 Multiple Cloning Site ............................................................................................................... 172

Common Properties ........................................................................................................ 173Classification of Restriction Enzymes ....................................................................... 173Site Preferences by Restriction Endonucleases........................................................ 173Star Activity ...................................................................................................................174Digestion of Methylated DNA ...................................................................................... 175

Effect of Dam Methylation on DNA Cleavage ............................................................... 176Effect of Dcm Methylation on DNA Cleavage ............................................................... 177Effect of CpG Methylation on DNA Cleavage ............................................................... 178Effect of EcoKI and EcoBI Methylation on DNA Cleavage ............................................. 181

Newly Generated Cleavage Sites ................................................................................ 182Recognition Sites Resulting from Ligation of Blunt DNA Ends ....................................... 182Recognition Sites Resulting from Ligation of Protruding Compatible DNA Ends ............. 192Recognition Sites Resulting from Fill-in of 5’-overhang and Self-ligation....................... 202Recognition Sites Resulting from Removal of 3’-overhang and Self-ligation ..................206

Selection Guides .............................................................................................................. 207Alphabetic List of Commercially Available Restriction Enzymes ............................ 207Alphabetic List of Recognition Sequences ................................................................ 219Enzyme Recognizing 2 bp Long Targets ....................................................................222Alphabetic List of Enzymes Recognizing 4 bp Long Targets ....................................222Alphabetic List of Enzymes Recognizing 5 bp Long Targets ....................................222Alphabetic List of Enzymes Recognizing 6 bp Long Targets .................................... 223Alphabetic List of Enzymes Recognizing 7 bp Long Targets .................................... 224Alphabetic List of Enzymes Recognizing 8 bp Long Targets .................................... 224Commercial Restriction Enzymes Generating 5’-protruding Ends .......................... 225Commercial Restriction Enzymes Generating 3’-protruding Ends .......................... 228Commercial Restriction Enzymes Generating Blunt Ends ........................................ 230Commercial Restriction Enzymes Cleaving DNA on the Both Sides of their Recognition Sequence ................................................................................................. 230

Troubleshooting Guide ................................................................................................... 231

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www.fermentas.com www.fermentas.com/doubledigest www.fermentas.com/research www.fermentas.com/reviewer

Restriction enzymes recognize specific nucleo-tide sequences and cleave DNA molecules at a position either within or outside their recognition site. These enzymes are important tools for numerous applications, including studies of DNA primary structure and recombinant DNA tech-nology. The best studied and the most widely

used are type II restriction endonucleases. More than 3800 type II restriction enzymes, exhibiting almost 290 different specificities, have been isolated. Since 1977 Fermentas has discovered approxi-mately 30% of all known restriction enzymes. We are a leading global enzyme manufacturer,

Labeled Oligonucleotide (LO) TestThe Labeled Oligonucleotide Test was designed at Fermentas for detection of trace contaminat-ing activities in enzyme, nucleotide and reagent solutions. This unique test allows us to detect trace activities of endo-, exodeoxyribonucleases and phosphatases in enzyme preparations. Therefore we are able to bring to the market only highest quality and performance products.Single-stranded and double-stranded 5’-[32P]-labeled synthetic oligonucleotides containing no recognition sites for restriction enzymes are used as substrates for the LO test. The labeled oligonucleotides are incubated with excess of restriction enzyme, separated on a polyacrylamide gel under denaturing conditions and analyzed by phospho-imaging. The presence of contaminating endo- and exodeoxyribonucleases results in the degrada-tion of the labeled substrates (see Fig. 1.1), while decreased specific radioactivity indicates the presence of contaminant phosphatases. The restriction enzyme passes this quality control test if there is neither degradation of labeled oligonucleotides nor a decrease in specific radioactivity.

supplying both conventional restriction enzymes and innovative restriction enzyme line for rapid DNA digestion in one universal buffer – FastDigest® restriction enzymes. Fermentas offers 176 FastDigest® and 201 conventional restriction enzymes.

Figure 1.1. Labeled Oligonucleotide (LO) Test.ss – single-stranded radiolabeled oligonucleotideds – double-stranded radiolabeled oligonucleotidePure enzyme – Fermentas NotIContaminated enzyme – competitor’s NotI

Labeled Oligonucleotide Test

Radiolabeled ss and ds oligonucleotides mixed with an excess of enzyme

Incubated at 37°C for 4 hours

Denatured and separated by PAGE

Band pattern imaged and analyzed

Patterns Typical for:

Control Pure enzyme

Contaminated enzyme

ss ds ss ds ss ds

Degradation due to contamination with non-specific nucleases

PureExtreme® Quality

Fermentas Restriction enzymes are produced in clean room class D facilities under the ISO 9001:2008 quality management system and are subjected to extensive quality control.

Fermentas restriction enzymes pass all industry standard quality control assays, as well as the unique Labeled Oligonucleotide

(LO) Test, which is the most sensitive test for the detection of trace activities of endodeoxy-ribonucleases, exodeoxyribonucleases and phosphatases see Fig. 1.1.A warranty is assigned and an expiry date is listed both on the product label and in the Cer-tificate of Analysis supplied with each product. Product lots are monitored to meet the quality specifications up to the expiry date.

FastDigest® and Conventional Restriction Enzymes

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1. & CONVENTIONAL RESTRICTION ENZYMES®

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For patent and license information see p.557 Bulk quantities and custom formulations available upon request

Navigation GuideTable 1.1. FastDigest® and conventional restriction enzymes.To find or select a restriction enzyme Page

By name

Table 1.2. FastDigest® restriction enzymes 7

Table 1.4. Fermentas conventional restriction enzymes 76

Table 1.25. Commercially available restriction enzymes 207

On-line. REsearch™ at www.fermentas.com/research

By recognition sequence

Table 1.26. Recognition sequences of restriction enzymes 219

Table 1.27. Enzyme recognizing 2 bp long targets 222

Table 1.28. Enzymes recognizing 4 bp long targets 222






By number of recognition sites in DNA

Table 14.15. Number of recognition sites in DNA molecules 514

Appendix. Phage and plasmid DNA. 489

On-line. REviewer™ at www.fermentas.com/reviewer

By type of generated DNA ends

Table 1.33. Commercial restriction enzymes generating 5’-protruding ends 225

Table 1.34. Commercial restriction enzymes generating 3’-protruding ends 228

Table 1.35. Commercial restriction enzymes generating blunt ends 230

Table 1.36. Commercial restriction enzymes cleaving DNA on the both sides of their recognition sequence 230


By cleavage efficiency close to termini of DNA

Table 1.3. Reaction conditions for FastDigest® restriction enzymes 71

Table 1.10. Cleavage efficiency close to the termini of PCR fragments 171

Table 1.11. Cleavage of restriction targets located in close vicinity within pUC19 multiple cloning site 172

By sensitivity to DNA methylation

Table 1.12. Fermentas isoschizomers and neoschizomers with differing sensitivities to target methylation 175

Tables 1.13, 1.14. Effect of Dam methylation 176

Tables 1.15, 1.16. Effect of Dcm methylation 177

Tables 1.17, 1.18. Effect of CpG methylation 178

Tables 1.19, 1.20. Effect of EcoBI and EcoKI methylation 181

By newly generated recognition sites

Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends 182

Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends 192

Table 1.23. Newly generated recognition sites resulting from fill-in of 5’-overhang and self-ligation 202

Table 1.24. Newly generated recognition sites resulting from removal of 3’-overhang and self-ligation 206

To perform fast digestion of DNA in universal FastDigest® buffer

Fast DNA digestion reaction set-up Protocols and recommendations for fast DNA digestion 70

Digestion of different DNA substrates

Table 1.3. Reaction conditions for FastDigest® restriction enzymes 71Digestion close to DNA terminiInactivation of enzyme after digestionIncubation time without star activityCompatibility of FastDigest® enzyme buffer with downstream applications 2.1. Activity of DNA/RNA modifying enzymes in Fermentas buffers 274

Troubleshooting Table 1.37. Troubleshooting guide for DNA digestion 232

To perform DNA digestion with conventional restriction enzymesDNA digestion reaction set-up Protocols and recommendations for DNA digestion with conventional restriction enzymes 160

Double/multiple digestion of DNAOn-line. DoubleDigest™ at www.fermentas.com/doubledigest

Table 1.8. Double digestion in universal Tango™ buffer 168

Table 1.6. Reaction conditions for restriction enzymes 163

Reaction conditions for individual enzymesTable 1.6. Reaction conditions for restriction enzymes 163

Table 1.5. Reaction buffers for restriction enzymes 162

Digestion close to DNA terminiTable 1.10. Cleavage efficiency close to the termini of PCR fragments 171

Table 1.11. Cleavage of restriction targets located in close vicinity within pUC19 multiple cloning site 172

Digestion of agarose-embeded DNA 1.11 Table 1.9. Digestion of agarose-embedded DNA 161

Inactivation of enzyme after digestion1.12. Inactivation of restriction enzymes 161

Table 1.6. Reaction conditions for restriction enzymes 163

How to identify and avoid star activity Description of star activity of restriction enzymes 174

Compatibility of conventional restriction enzyme buffers with downstream applications 2.1. Activity of DNA/RNA modifying enzymes in Fermentas buffers 274

Partial digestion of DNA 1.10. Partial digestion of DNA 160

How to dilute restriction enzymes 1.9. Dilution of restriction enzymes 160

Stability during prolonged incubation Table 1.6. Reaction conditions for restriction enzymes 163

Enzyme activity at non-optimal temperature Table 1.7. Activity of mesophilic and thermophilic enzymes at 37°C 167

Troubleshooting guide Table 1.37. Troubleshooting guide for DNA digestion 232

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Description of Icons

Additives. Indicates additives required to obtain the stated enzyme activity. Solutions of S-adenosyl-methionine and oligonucleotides are supplied with enzymes. DTT (#R0861) is available separately (p.476).

Incubation Temperature. Indicates the optimal incubation temperature in degree Celsius (°C).

Ligation Efficiency. Indicates the ligation efficiency of DNA fragments generated by digestion with the restriction enzyme (see p.6 and p.75).

Star Activity. Indicates restriction enzymes prone to star activity (see p.174).

Sensitivity to Methylation. DNA cleavage by the restriction enzyme is blocked or impaired by Dam, Dcm or CpG methylation within the target sequences (see p.175).

High Concentration. Indicates enzymes available at a high concentration (50 u/µl).

Genome Qualified. Indicates that the restriction enzyme cleaves agarose-embedded DNA (see p.161).

Recombinant Enzyme. Indicates enzymes purified from recombinant E.coli.

Blue/White Certified. Enzyme was tested by the Blue/White cloning assay (see p.6 and p.75).

FastDigest® Enzyme. Enzyme is available in FastDigest® format (see p.5).

Buffers. Letters in the buffer icon indicate the recommended buffer for a specific restriction enzyme. FastDigest® and FastDigest® Green Buffers are indicated by “F” icon. B (blue), G (green), O (orange), R (red) and Tango™ (yellow) correspond to the color codes of the Five Buffer System for conventional restriction enzymes. The “Unique” icon indicates conventional restriction enzymes that require a special buffer, which is supplied with the enzyme (see p.162).

Thermal Inactivation. Indicates conditions for thermal inactivation of the enzyme (see p.161).Indicates that only small amounts of restriction enzyme (up to 10 units) can be thermally inactivated.

LO Certified. Enzyme was tested by the labeled oligonucleotide (LO) assay (see p.2 and p.75).

Sensitivity to Overlapping Dam, Dcm or CpG Methylation. The target site may be methylated in certain sequence contexts (overlapping methylation). This will result in blocked or impaired DNA cleavage (see p.175).

Single letter codeR = G or A; H = A, C or T; Y = C or T; V = A, C or G;W = A or T; B = C, G or T;M = A or C; D = A, G or T;K = G or T; N = G, A, T or C.S = C or G;

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DescriptionFastDigest® enzymes are an advanced line of restriction enzymes for rapid DNA digestion. All FastDigest® enzymes are 100% active in the universal FastDigest® and FastDigest® Green buffers and are able to digest DNA in 5-15 min-utes. This enables any combination of restriction enzymes to work simultaneously in one reaction tube and eliminates the need for sequential digestions. As an added convenience, the Fast-Digest® Green Buffer allows for direct loading of the reaction mixture on a gel.FastDigest® enzymes can be used to digest plasmid, genomic and viral DNA as well as PCR products and do not show star activity even in prolonged incubations.The FastDigest® line is ideal for use in applications that require high purity reaction components, performance reliability and simple reaction set-up.

Universal FastDigest® BufferTwo versions of the universal FastDigest® buffer are supplied with each enzyme: 10X FastDi-gest® Buffer and 10X FastDigest® Green Buffer. All FastDigest® enzymes are 100% active in both FastDigest® and FastDigest® Green buffers. Enzymes used in common downstream applications such as ligation, blunting and dephosphorylation also have 100% activity in both buffers. As an added convenience, the FastDigest® Green Buffer contains a density reagent and two tracking dyes that allow for direct loading of the reaction mixture on gels. The blue dye migrates with 3-5 kb DNA fragments in 1% agarose gel and has an excitation peak at 424 nm while the yellow dye migrates faster than 10 bp DNA fragments in 1% agarose gel and has an excitation peak at 615 nm. The presence of the dyes in the FastDigest® Green buffer does not interfere with DNA digestion or with downstream applications but may hinder digestion product analysis by fluorescence excitation. For such applications we recommend use of the colorless FastDigest® Buffer.

Features• 100% activity of all FastDigest® enzymes in

the new green universal digestion buffer.• 100% buffer compatibility with all down-

stream applications.• Complete digestion in 5-15 minutes.• Direct loading of reaction mixture on gels.

Applications• Fast clone analysis.• Fast preparation of DNA for cloning.• Digestion of PCR products.• Fast RFLP genotyping.• Digestion of difficult-to-cleave DNA.

FastDigest® Restriction Enzymes

M C 1 2 C 3 4 M

FastDigest® Buffer

FastDigest® GreenBuffer

Figure 1.2. Five minute triple digestion followed by ligation of plasmid DNA in the universal

FastDigest® and FastDigest® Green buffers.

M – GeneRuler™ Express DNA Ladder (#SM1551).C – undigested plasmid DNA control.1 – plasmid DNA triple digested with FastDigest® EcoRI, FastDigest® KpnI and FastDigest® SmaI in FastDigest® Buffer.2 – the reaction mixture after ligation in FastDigest® Buffer.3 – plasmid triple digested with FastDigest® EcoRI, FastDigest® KpnI and FastDigest® SmaI in FastDigest® Green Buffer.4 – the reaction mixture after ligation in FastDigest® Green Buffer.

1 2

Figure 1.3. FastDigest® Green Buffer.1 – Reaction mixture containing FastDigest® Green Buffer before electrophoresis.2 – Reaction mixture containing FastDigest® Green Buffer after electrophoresis.

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Activity and Quality Control Assays

Activity Assay1 µl of FastDigest® enzyme is formulated to cleave 1 µg of substrate DNA in 5 or 15 min at recommended temperature in 1X FastDigest® buffer. 1 µl of FastDigest® restriction endonu-clease corresponds to 1 FastDigest® unit (FDU) of enzyme. In general, enzymes are assayed with λ phage DNA at 37°C. However, some exceptions apply:

• Restriction enzymes that show optimum activity at temperatures other than 37°C are assayed under their optimal temperature.

• FastDigest® enzymes that do not have recog-nition sites on λ DNA are assayed with other specific DNA substrates, as indicated in the Certificates of Analysis.

• FastDigest® enzymes with only a few recog-nition sites on the λ DNA are assayed using λ DNA cut with another restriction enzyme.

• FastDigest® enzymes sensitive to Dam or Dcm methylation are assayed using DNA purified from dam–, dcm– strain of E.coli.

Quality Control

Labeled Oligonucleotide (LO) Test

1µl of FastDigest® enzyme is incubated with 5’-[32P]-labeled single-stranded and double-stranded synthetic oligonucleotides in 1X FastDigest® buffer for 1 hour at the recommended temperature. The restriction enzyme passes this quality control test if there is no degradation of labeled oligonucleotides and no decrease in their specific radioactivity.

Prolonged Incubation / Star Activity AssayThe star activity assay is designed to test alterations in the DNA digestion pattern after prolonged incubation with FastDigest® enzymes.1 µl of FastDigest® enzyme is incubated with 1 µg of substrate DNA at the recommended temperature for up to 16 hours. After electro-phoretic separation of the DNA fragments, the characteristic banding patterns are examined for alterations. The maximal incubation time that does not cause any star activity is indicated for each FastDigest® enzyme in the product description and the Certificate of Analysis sup-plied with each enzyme.

Ligation and Recleavage AssayThe ligation and recleavage assay confirms the integrity of DNA ends. DNA fragments obtained after overdigestion with 1 µl of FastDigest® enzyme for 1 hour are ligated with T4 DNA ligase and then recut with the same enzyme. A FastDigest® restriction enzyme conforms to the quality criterion if the observed ligation efficiency is identical to that of conventional enzyme.The percentage of DNA that can be successfully ligated and then re-cleaved for each FastDigest® restriction enzyme is indicated in the in the pro-duct description and the Certificate of Analysis supplied with each enzyme.

Blue/White (B/W) Cloning AssayThe Blue/White cloning assay is designed to test the integrity of DNA ends. pUC57 DNA is digested at unique sites within the lacZ reporter gene with 1 µl of a FastDigest® restriction enzyme in 1X FastDigest® buffer. After a 1 hour incubation, the plasmid DNA is recircularized by ligation and transformed into E.coli XL1-Blue competent cells. The cells are then plated onto X-Gal/IPTG/Amp agar. An intact lacZ gene will give rise to a blue colony. If the termini of the linearized pUC57 are altered by contaminating exodeoxynucleases, the lacZ reading frame is interrupted, which results in the appearance of white colonies. An enzyme conforms to this quality criterion if the number of white colonies does not exceed 3%. For FastDigest® restric-tion enzymes lacking recognition sites in pUC57 DNA, the assay is performed with the mixture of pUC57/HindIII, pUC57/PstI and pUC57/Eco32I DNA fragments representing three different types of termini (3’-overhang, 5’-overhang and blunt ends).

Storage and ShippingAll FastDigest® restriction enzymes should be stored at -20°C. During shipment on dry ice, enzymes may freeze. This does not affect their quality – all Fermentas enzymes are 100% active after at least three freeze-thaw cycles.For 24-48 hour delivery, enzymes may be shipped on blue ice since their quality is not affected by short exposure to 4°C.

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Product List

Table 1.2. FastDigest® restriction enzymes.

FastDigest® restriction enzyme

Prototype Specificity 5’→3’ Cat. # Page

FastDigest® AatII AatII GACGT↓C FD0994 11FastDigest® AccI (XmiI) AccI GT↓MKAC FD1484 11FastDigest® Acc65I KpnI (GGTAC↓C) G↓GTACC FD0904 11FastDigest® AciI (SsiI) AciI CCGC(-3/-1)↓ FD1794 12FastDigest® AclI (Psp1406I) AclI AA↓CGTT FD0944 12FastDigest® AcuI (Eco57I) Eco57I CTGAAG(16/14)↓ FD0344 12FastDigest® AfeI (Eco47III) Eco47III AGC↓GCT FD0324 13FastDigest® AflII (BspTI) AflII C↓TTAAG FD0834 13FastDigest® AgeI (BshTI) AgeI A↓CCGGT FD1464 13FastDigest® AjuI AjuI ↓(7/12)GAA(N)7TTGG(11/6)↓ FD1954 14FastDigest® AleI (OliI) OliI CACNN↓NNGTG FD1634 14FastDigest® AluI AluI AG↓CT FD0014 14FastDigest® Alw21I HgiAI GWGCW↓C FD0024 15FastDigest® Alw26I BsmAI GTCTC(1/5)↓ FD0034 15FastDigest® AlwNI (CaiI) AlwNI CAGNNN↓CTG FD1394 15FastDigest® ApaI ApaI GGGCC↓C FD1414 16FastDigest® ApaLI (Alw44I) ApaLI G↓TGCAC FD0044 16FastDigest® AscI (SgsI) AscI GG↓CGCGCC FD1894 16FastDigest® AseI (VspI) VspI AT↓TAAT FD0914 17FastDigest® AsiSI (SfaAI) SgfI GCGAT↓CGC FD2094 17FastDigest® AvaI (Eco88I) AvaI C↓YCGRG FD0384 17FastDigest® AvaII (Eco47I) AvaII G↓GWCC FD0314 18FastDigest® AvrII (XmaJI) AvrII C↓CTAGG FD1564 18FastDigest® BamHI BamHI G↓GATCC FD0054/5 18FastDigest® BanI (BshNI) HgiCI G↓GYRCC FD1004 19FastDigest® BbsI (BpiI) BbvII GAAGAC(2/6)↓ FD1014 19FastDigest® BbvI (Lsp1109I) BbvI GCAGC(8/12)↓ FD2074 19FastDigest® BclI BclI T↓GATCA FD0724 20FastDigest® BfaI (FspBI) MaeI C↓TAG FD1764 20FastDigest® BglI BglI GCCNNNN↓NGGC FD0074 20FastDigest® BglII BglII A↓GATCT FD0083/4 21FastDigest® BlpI (Bpu1102I) EspI GC↓TNAGC FD0094 21FastDigest® Bme1580I (BseSI) BseSI GKGCM↓C FD1444 21FastDigest® BmtI (BspOI) NheI (G↓CTAGC) GCTAG↓C FD2044 22FastDigest® BplI BplI ↓(8/13)GAG(N)5CTC(13/8)↓ FD1314 22FastDigest® BpmI (GsuI) BpmI CTGGAG(16/14)↓ FD0464 22FastDigest® Bpu10I Bpu10I CCTNAGC(-5/-2)↓ FD1184 23FastDigest® BsaAI (Ppu21I) BsaAI YAC↓GTR FD1974 23FastDigest® BsaBI (BseJI) BsaBI GATNN↓NNATC FD1414 23FastDigest® BsaHI (Hin1I) AcyI GR↓CGYC FD0474 24FastDigest® BsaJI (BseDI) SecI C↓CNNGG FD1084 24FastDigest® BseGI FokI (GGATG(9/13)↓) GGATG(2/0)↓ FD0874 24FastDigest® BseNI BsrI ACTGG(1/-1)↓ FD0884 25FastDigest® BseXI BbvI GCAGC(8/12)↓ FD1454 25FastDigest® Bsh1236I FnuDII CG↓CG FD0924 25FastDigest® BsiEI (Bsh1285I) McrI CGRY↓CG FD0894 26FastDigest® BsiWI (Pfl23II) SplI C↓GTACG FD0854 26FastDigest® BslI (BseLI) BsiYI CCNNNNN↓NNGG FD1204 26

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FastDigest® BsmBI (Esp3I) Esp3I CGTCTC(1/5)↓ FD0454 27FastDigest® BsmFI (FaqI) FinI GGGAC(10/14)↓ FD1814 27FastDigest® Bsp119I AsuII TT↓CGAA FD0124 27FastDigest® Bsp120I ApaI (GGGCC↓C) G↓GGCCC FD0134 28FastDigest® Bsp1286I (SduI) SduI GDGCH↓C FD0654 28FastDigest® Bsp1407I Bsp1407I T↓GTACA FD0933/4 28FastDigest® BspCNI (BseMII) BseMII CTCAG(10/8)↓ FD1404 29FastDigest® BspHI (PagI) BspHI T↓CATGA FD1284 29FastDigest® BspMI (BveI) BspMI ACCTGC(4/8)↓ FD1744 29FastDigest® BsrBI (MbiI) BsrBI CCGCTC(-3/-3)↓ FD1274 30FastDigest® BsrDI (BseMI) BsrDI GCAATG(2/0)↓ FD1264 30FastDigest® BsrFI (Cfr10I) Cfr10I R↓CCGGY FD0184 30FastDigest® BssHII (PteI) BsePI G↓CGCGC FD2134 31FastDigest® BstXI BstXI CCANNNNN↓NTGG FD1024 31FastDigest® BstZ17I (Bst1107I) SnaI GTA↓TAC FD0704 31FastDigest® Bsu36I (Eco81I) SauI CC↓TNAGG FD0374 32FastDigest® ClaI (Bsu15I) ClaI AT↓CGAT FD0143/4 32FastDigest® Csp6I RsaI (GT↓AC) G↓TAC FD0214 32FastDigest® DdeI (HpyF3I) DdeI C↓TNAG FD1884 32FastDigest® DpnI DpnI Gm6A↓TC FD1703/4 33FastDigest® DraI AhaIII TTT↓AAA FD0224 33FastDigest® DraIII (AdeI) DraIII CACNNN↓GTG FD1234 34FastDigest® DrdI (AasI) DrdI GACNNNN↓NNGTC FD1724 34FastDigest® EagI (Eco52I) XmaIII C↓GGCCG FD0334 34FastDigest® Eam1105I Eam1105I GACNNN↓NNGTC FD0244 35FastDigest® EarI (Eam1104I) Ksp632I CTCTTC(1/4)↓ FD0234 35FastDigest® Ecl136II SacI (GAGCT↓C) GAG↓CTC FD0254 35FastDigest® Eco31I Eco31I GGTCTC(1/5)↓ FD0293/4 36FastDigest® Eco91I BstEII G↓GTNACC FD0394 36FastDigest® EcoNI (XagI) EcoNI CCTNN↓NNNAGG FD1304 36FastDigest® EcoO109I DraII RG↓GNCCY FD0264 37FastDigest® EcoRI EcoRI G↓AATTC FD0274/5 37FastDigest® EcoRV (Eco32I) EcoRV GAT↓ATC FD0303/4 37FastDigest® EheI NarI (GG↓CGCC) GGC↓GCC FD0443/4 38FastDigest® Fnu4HI (SatI) Fnu4HI GC↓NGC FD1644 38FastDigest® FokI FokI GGATG(9/13)↓ FD2144 38FastDigest® FspI (NsbI) MstI TGC↓GCA FD1224 39FastDigest® FspAI FspAI RTGC↓GCAY FD1664 39FastDigest® HaeII (BfoI) HaeII RGCGT↓Y FD2184 39FastDigest® HaeIII (BsuRI) HaeIII GG↓CC FD0154 40FastDigest® HgaI (CseI) HgaI GACGC(5/10)↓ FD1904 40FastDigest® HhaI HhaI GCG↓C FD1854 40FastDigest® HincII HindII GTY↓RAC FD0494 41FastDigest® HindIII HindIII A↓AGCTT FD0504/5 41FastDigest® HinfI HinfI G↓ANTC FD0804 41FastDigest® HinP1I (Hin6I) HhaI (GCG↓C) G↓CGC FD0484 42FastDigest® HpaI (KspAI) HpaI GTT↓AAC FD1034 42FastDigest® HpaII HpaII C↓CGG FD0514 42FastDigest® Hpy8I MjaIV GTN↓NAC FD1574 43FastDigest® HpyF10VI MwoI GCNNNNN↓NNGC FD1734 43

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FastDigest® KpnI KpnI GGTAC↓C FD0524 43FastDigest® Kpn2I BspMII T↓CCGGA FD0534 44FastDigest® MauBI MauBI CG↓CGCGCG FD2084 44FastDigest® MboI MboI ↓GATC FD0814 44FastDigest® MboII MboII GAAGA(8/7)↓ FD0824 45FastDigest® MfeI (MunI) MfeI C↓AATTG FD0753/4 45FastDigest® MluI MluI A↓CGCGT FD0564 45FastDigest® MlyI (SchI) PleI (GAGTC(4/5)↓) GAGTC(5/5)↓ FD1374 46FastDigest® MnlI MnlI CCTC(7/6)↓ FD1074 46FastDigest® MreI Sse232I CG↓CCGGCG FD2024 46FastDigest® MscI (MlsI) BalI TGG↓CCA FD1214 47FastDigest® MseI (SaqAI) MseI T↓TAA FD2174 47FastDigest® MslI (RseI) MslI CAYNN↓NNRTG FD2004 47FastDigest® MspI HpaII C↓CGG FD0544 48FastDigest® MssI PmeI GTTT↓AAAC FD1344 48FastDigest® MvaI EcoRII (↓CCWGG) CC↓WGG FD0554 48FastDigest® Mva1269I BsmI GAATGC(1/-1)↓ FD0964 49FastDigest® NaeI (PdiI) NaeI GCC↓GGC FD1524 49FastDigest® NciI (BcnI) CauII CC↓SGG FD0064 49FastDigest® NcoI NcoI C↓CATGG FD0573/4/5 50FastDigest® NdeI NdeI CA↓TATG FD0583/4/5 50FastDigest® NheI NheI G↓CTAGC FD0973/4 50FastDigest® NlaIII (Hin1II) NlaIII CATG↓ FD1834 51FastDigest® NlaIV (BspLI) NlaIV GGN↓NCC FD1154 51FastDigest® NmuCI Tsp45I ↓GTSAC FD1514 51FastDigest® NotI NotI GC↓GGCCGC FD0593/4/6 52FastDigest® NruI (RruI) NruI TCG↓CGA FD2154 52FastDigest® NsiI (Mph1103I) AvaIII ATGCA↓T FD0734 52FastDigest® NspI (XceI) NspI RCATG↓Y FD1474 53FastDigest® PacI PacI TTAAT↓TAA FD2204 53FastDigest® PdmI XmnI GAANN↓NNTTC FD1534 53FastDigest® PflMI (Van91I) PflMI CCANNNN↓NTGG FD0714 54FastDigest® PfoI PfoI T↓CCNGGA FD1754 54FastDigest® PmlI (Eco72I) PmaCI CAC↓GTG FD0364 54FastDigest® PpuMI (Psp5II) PpuMI RG↓GWCCY FD0764 55FastDigest® PshAI (BoxI) PshAI GACNN↓NNGTC FD1434 55FastDigest® PsiI (AanI) PsiI TTA↓TAA FD2064 55FastDigest® PspFI BseYI CCCAGC(-5/-1)↓ FD2224 56FastDigest® PstI PstI CTGCA↓G FD0614/5 56FastDigest® PsuI XhoII R↓GATCY FD1554 56FastDigest® PsyI Tth111I GACN↓NNGTC FD1334 57FastDigest® PvuI PvuI CGAT↓CG FD0624 57FastDigest® PvuII PvuII CAG↓CTG FD0634 57FastDigest® RsaI RsaI GT↓AC FD1124 58FastDigest® RsrII (CpoI) RsrII CG↓GWCCG FD0744 58FastDigest® SacI SacI GAGCT↓C FD1133/4 58FastDigest® SalI SalI G↓TCGAC FD0644 59FastDigest® SanDI (KflI) SanDI GG↓GWCCC FD2164 59FastDigest® SapI (LguI) SapI GCTCTTC(1/4)↓ FD1934 59FastDigest® Sau3AI (Bsp143I) MboI ↓GATC FD0784 60

10

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Alphabetic list of commercially available restriction enzymes see p.207.





FastDigest® Sau96I (Cfr13I) AsuI G↓GNCC FD0194 60FastDigest® SbfI (SdaI) Sse8387I CCTGCA↓GG FD1194 60FastDigest® ScaI ScaI AGT↓ACT FD0434 61FastDigest® ScrFI (Bme1390I) ScrFI CC↓NGG FD1424 61FastDigest® SexAI (CsiI) SexAI A↓CCWGGT FD2114 61FastDigest® SfaNI (BmsI) SfaNI GCATC(5/9)↓ FD2124 62FastDigest® SfcI (BfmI) SfeI C↓TRYAG FD1164 62FastDigest® SfiI SfiI GGCCNNNN↓NGGCC FD1824 62FastDigest® SmaI SmaI CCC↓GGG FD0663/4 63FastDigest® SnaBI (Eco105I) SnaBI TAC↓GTA FD0404 63FastDigest® SpeI (BcuI) SpeI A↓CTAGT FD1253/4 63FastDigest® SphI (PaeI) SphI GCATG↓C FD0604 64FastDigest® SspI SspI AAT↓ATT FD0774 64FastDigest® StuI (Eco147I) StuI AGG↓CCT FD0424 64FastDigest® StyI (Eco130I) StyI C↓CWWGG FD0414 65FastDigest® SwaI (SmiI) SwaI ATTT↓AAAT FD1244 65FastDigest® TaaI Tsp4CI ACN↓GT FD1364 65FastDigest® TaiI MaeII (A↓CGT) ACGT↓ FD1144 66FastDigest® TaqI TaqI T↓CGA FD0674 66FastDigest® TatI TatI W↓GTACW FD1294 66FastDigest® TauI TauI GCSG↓C FD1654 67FastDigest® TfiI (PfeI) TfiI G↓AWTC FD1784 67FastDigest® Tru1I MseI T↓TAA FD0984 67FastDigest® Tsp509I (TasI) TspEI ↓AATT FD1354 68FastDigest® TspRI (TscAI) TspRI CASTG(2/-7)↓ FD2104 68FastDigest® XapI ApoI R↓AATTY FD1383/4 68FastDigest® XbaI XbaI T↓CTAGA FD0684/5 69FastDigest® XhoI XhoI C↓TCGAG FD0694/5 69

11

Protocols and Recommendations p.70

1



FastDigest® AccI (XmiI)

Digestion time with 1 µl of FastDigest® AccI (XmiI) bp from end of DNA required for complete digestion

Thermal inactivation

Incubation time without star activityLambda DNA, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl

5 min 5 min 60 min 5 min 2 bp 65°C, 5 min 16 hours

5’...G T↓M K A C...3’

3’...C A K M↑T G...5’

#FD1484 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml

Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA in 5 min at 37°C in 1X FastDigest® Buffer.

Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.

Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – blocked (p.178).

λ DN

A, 1

.0%

aga

rose

FastDigest® Acc65I

Digestion time with 1 µl of FastDigest® Acc65I bp from end of DNA required for complete digestion




5’...G↓ G T A C C...3’

3’...C C A T G↑ G...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/BamHI fragments in 5 min at 37°C in 1X FastDigest® Buffer.


Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).

λ DN

A, 0

.7%

aga

rose

NoteFastDigest® Acc65I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

FastDigest® AatII

Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).

Digestion time with 1 µl of FastDigest® AatII bp from end of DNA required for complete digestion




5’...G A C G T↓C...3’

3’...C↑T G C A G...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pUC19 DNA/SmaI fragments in 15 min at 37°C in 1X FastDigest® Buffer.


pUC1

9 DN

A/Sm

aI, 1

.0%

aga

rose

Product Description

1 cl

eava

ge s

ite9

clea

vage

site

s2

clea

vage

site

s

12

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1


FastDigest® AclI (Psp1406I)

Digestion time with 1 µl of FastDigest® AclI (Psp1406I) bp from end of DNA required for complete digestion




5’...A A↓C G T T...3’

3’...T T G C↑A A...5’




Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not deter-mined.

λ DN

A, 0

.7%

aga

rose

FastDigest® AciI (SsiI)

Digestion time with 1 µl of FastDigest® AciI (SsiI ) bp from end of DNA required for complete digestion




5’...C↓C G C...3’

3’...G G C↑G...5’





λ DN

A, 2

.0%

aga

rose

FastDigest® AcuI (Eco57I)

Digestion time with 1 µl of FastDigest® AcuI (Eco57I) bp from end of DNA required for complete digestion




5’...C T G A A G (N)16↓...3’

3’...G A C T T C (N)14↑...5’

#FD0344 20 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml20X SAM (0.2 mM) 20 µl


Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.SAM 0.01 mM.

Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.

λ DN

A, 1

.0%

aga

rose

Note• FastDigest® AcuI (Eco57I) requires only

Mg2+ for its activity, but is stimulated by S-adenosylmethionine. Still, complete cleavage of some substrates with FastDi-gest® AcuI (Eco57I) is difficult to achieve.

• For cleavage with FastDigest® AcuI (Eco57I) at least two copies of its recognition sequence are required.

• FastDigest® AcuI (Eco57I) may remain associated with the cleaved DNA. This may cause DNA band shifting during electropho-resis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.

516

clea

vage

site

s7

clea

vage

site

s40

cle

avag

e si

tes

13


1



FastDigest® AfeI (Eco47III)

Digestion time with 1 µl of FastDigest® AfeI (Eco47III ) bp from end of DNA required for complete digestion




5’...A G C↓G C T...3’

3’...T C G↑C G A...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/Eco81I fragments in 5 min at 37°C in 1X FastDigest® Buffer.


Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.

λ DN

A, 0

.7%

aga

rose

FastDigest® AgeI (BshTI)

Digestion time with 1 µl of FastDigest® AgeI (BshTI) bp from end of DNA required for complete digestion




5’...A↓C C G G T...3’

3’...T G G C C↑A...5’




Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not deter-mined.

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® AflII (BspTI) bp from end of DNA required for complete digestion



5 min 5 min 5 min 5 min 4 bp No 16 hours

5’...C↓T T A A G...3’

3’...G A A T T↑C...5’




Methylation EffectsDam, Dcm, CpG, EcoBI, EcoKI – no effect.

λ DN

A, 0

.7%

aga

rose

FastDigest® AflII (BspTI)

2 cl

eava

ge s

ites

3 cl

eava

ge s

ites

13 c

leav

age

site

s

14

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Digestion time with 1 µl of FastDigest® AluI bp from end of DNA required for complete digestion




FastDigest® AluI

5’...A G↓C T...3’

3’...T C↑G A...5’




Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® AleI (OliI ) bp from end of DNA required for complete digestion




5’...C A C N N↓N N G T G...3’

3’...G T G N N↑N N C A C...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of FX174 DNA in 5 min at 37°C in 1X FastDigest® Buffer.


Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap – blocked (p.181).

FastDigest® AleI (OliI)

FX1

74 D

NA, 1

.0%

aga

rose

Note• FastDigest® AjuI requires S-adenosylme-

thionine for activity. Still, complete cleavage of some substrates with FastDigest® AjuI is difficult to achieve.

• FastDigest® AjuI produces double-strand cuts on both sides of the interrupted rec-ognition site. In certain sequence contexts, the cleavage position may be shifted by one base pair. However, the cleavage position indicated above will predominate.

Digestion time with 1 µl of FastDigest® AjuI bp from end of DNA required for complete digestion



5 min 5 min 10 min 5 min ND 65°C, 5 min 16 hours

5’...↓ 7(N) G A A(N)7 T T G G (N)11↓...3’

3’...↑ 12(N) C T T(N)7 A A C C (N)6↑ ...5’




Methylation EffectsDam, Dcm, CpG, EcoKI, EcoBI – no effect.

FX1

74 D

NA, 1

.0%

aga

rose

FastDigest® AjuI

1 cl

eava

ge s

ite1

clea

vage

site

143

clea

vage

site

s

15


1



FastDigest® AlwNI (CaiI)

Digestion time with 1 µl of FastDigest® AlwNI (CaiI) bp from end of DNA required for complete digestion




5’...C A G N N N↓C T G...3’

3’...G T C↑N N N G A C...5’




Methylation EffectsDam, CpG, EcoKI – no effect.Dcm: may overlap – blocked (p.177).EcoBI: may overlap – effect not determined.

λ DN

A, 0

.7%

aga

rose Note

FastDigest® AlwNI (CaiI) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Digestion time with 1 µl of FastDigest® Alw21I bp from end of DNA required for complete digestion




5’...G W G C W↓C...3’

3’...C↑W C G W G...5’





λ DN

A, 1

.0%

aga

rose

FastDigest® Alw21I

28 c

leav

age

site

s

FastDigest® Alw26I

Digestion time with 1 µl of FastDigest® Alw26I bp from end of DNA required for complete digestion




5’...G T C T C(N)1↓...3’

3’...C A G A G(N)5↑...5’




Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).

λ DN

A, 1

.0%

aga

rose

37 c

leav

age

site

s41

cle

avag

e si

tes

16

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FastDigest® ApaLI (Alw44I)

Digestion time with 1 µl of FastDigest® ApaLI (Alw44I) bp from end of DNA required for complete digestion




5’...G↓T G C A C...3’

3’...C A C G T↑G...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/SmaI fragments in 5 min at 37°C in 1X FastDigest® Buffer.



λ DN

A, 0

.7%

aga

rose

FastDigest® AscI (SgsI)

Digestion time with 1 µl of FastDigest® AscI (SgsI) bp from end of DNA required for complete digestion




5’...G G↓C G C G C C...3’

3’...C C G C G C↑G G...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/CpoI fragments in 5 min at 37°C in 1X FastDigest® Buffer.



λ DN

A, 0

.7%

aga

rose

FastDigest® ApaI

Digestion time with 1 µl of FastDigest® ApaI bp from end of DNA required for complete digestion




5’...G G G C C↓C...3’

3’...C↑C C G G G...5’





λ DN

A, 0

.7%

aga

rose

NoteFastDigest® ApaI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).1

clea

vage

site

4 cl

eava

ge s

ites

2 cl

eava

ge s

ites

17


1



FastDigest® AseI (VspI)

Digestion time with 1 µl of FastDigest® AseI (VspI) bp from end of DNA required for complete digestion




5’...A T↓T A A T...3’

3’...T A A T↑T A...5’





λ DN

A , 1

.0%

aga

rose

FastDigest® AvaI (Eco88I)

Digestion time with 1 µl of FastDigest® AvaI (Eco88I) bp from end of DNA required for complete digestion




5’...C↓Y C G R G...3’

3’...G R G C Y↑C...5’




Methylation EffectsDam, Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – cleavage impaired (p.178).

λ DN

A, 0

.7%

aga

rose

FastDigest® AsiSI (SfaAI)

Digestion time with 1 µl of FastDigest® AsiSI (SfaAI) bp from end of DNA required for complete digestion



no cleavage sites 5 min 60 min 5 min 5 bp 80°C, 5 min 6 hours

5’...G C G A T↓ C G C...3’

3’...C G C↑ T A G C G...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of linearized pJET1 DNA with inserted SfaAI recognition site in 5 min at 37°C in 1X FastDigest® Buffer.



pJET

1-Sf

aAI D

NA/S

daI,

1.0%

aga

rose

17 c

leav

age

site

s1

clea

vage

site

8 cl

eava

ge s

ites

18

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FastDigest® AvrII (XmaJI)

Digestion time with 1 µl of FastDigest® AvrII (XmaJI) bp from end of DNA required for complete digestion




5’...C↓C T A G G...3’

3’...G G A T C↑C...5’





λ DN

A, 1

.0%

aga

rose

FastDigest® BamHI

Digestion time with 1 µl of FastDigest® BamHI bp from end of DNA required for complete digestion



5 min 5 min 5 min 5 min 2 bp 80°C, 5 min 1 hour

5’...G↓G A T C C...3’

3’...C C T A G↑G...5’

#FD0054 800 µlSupplied with:10X FastDigest® buffer 2x1 ml10X FastDigest® Green Buffer 2x1 ml


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/Bsp120I fragments in 5 min at 37°C in 1X FastDigest® Buffer.



λ DN

A, 0

.7%

aga

rose

FastDigest® AvaII (Eco47I)

Digestion time with 1 µl of FastDigest® AvaII (Eco47I) bp from end of DNA required for complete digestion




5’...G↓G W C C ...3’

3’...C C W G↑G...5’




Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – blocked (p.177, 179).

λ DN

A, 1

.0%

aga

rose

NoteFastDigest® AvaII (Eco47I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

35 c

leav

age

site

s2

clea

vage

site

s5

clea

vage

site

s

19


1



FastDigest® BanI (BshNI)

Digestion time with 1 µl of FastDigest® BanI (BshNI) bp from end of DNA required for complete digestion




5’...G↓G Y R C C...3’

3’...C C R Y G↑G...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dcm– in 5 min at 37°C in 1X FastDigest® Buffer.


Methylation EffectsDam, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).EcoKI: may overlap – effect not determined.

λ DN

A (d

cm– ),

1.0

% a

garo

se

NoteFastDigest® BanI (BshNI) cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Digestion time with 1 µl of FastDigest® BbvI (Lsp1109I) bp from end of DNA required for complete digestion




5’...G C A G C (N)8 ↓...3’

3’...C G T C G (N)12↑...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pBR322 DNA in 5 min at 37°C in 1X FastDigest® Buffer.



pBR3

22 D

NA, 0

.7%

aga

rose

FastDigest® BbvI (Lsp1109I)

FastDigest® BbsI (BpiI)

Digestion time with 1 µl of FastDigest® BbsI (BpiI) bp from end of DNA required for complete digestion




5’...G A A G A C(N)2↓...3’

3’...C T T C T G(N)6↑...5’





λ DN

A. 0

.7%

aga

rose

25 c

leav

age

site

s24

cle

avag

e si

tes

21 c

leav

age

site

s

20

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FastDigest® BglI

Digestion time with 1 µl of FastDigest® BglI bp from end of DNA required for complete digestion




5’...G C C N N N N↓N G G C...3’

3’...C G G N↑N N N N C C G...5’





λ DN

A, 0

.7%

aga

rose

FastDigest® BclI

Digestion time with 1 µl of FastDigest® BclI bp from end of DNA required for complete digestion




5’...T↓G A T C A...3’

3’...A C T A G↑T...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dam– in 5 min at 37°C in 1X FastDigest® Buffer.


Methylation EffectsDam: completely overlaps – blocked (p.176).Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – blocked (p.181).

λ DN

A (d

am–),

0.7%

aga

rose

NoteFastDigest® BclI is blocked by dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

FastDigest® BfaI (FspBI)

Digestion time with 1 µl of FastDigest® BfaI (FspBI) bp from end of DNA required for complete digestion




5’...C↓T A G...3’

3’...G A T↑C...5’





λ DN

A, 0

.7%

aga

rose

8 cl

eava

ge s

ites

13 c

leav

age

site

s29

cle

avag

e si

tes

21


1



FastDigest® BglII

Digestion time with 1 µl of FastDigest® BglII bp from end of DNA required for complete digestion




5’...A↓G A T C T...3’

3’...T C T A G↑A...5’

#FD0083 100 µl #FD0084 200 µl

Both supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml



Methylation EffectsDam, Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – cleavage impaired (p.181).

λ DN

A, 0

.7%

aga

rose

FastDigest® BlpI (Bpu1102I)

Digestion time with 1 µl of FastDigest® BlpI (Bpu1102I) bp from end of DNA required for complete digestion




5’...G C↓T N A G C...3’

3’...C G A N T↑C G...5’





λ DN

A, 0

.7%

aga

rose

FastDigest® Bme1580I (BseSI)

Digestion time with 1 µl of FastDigest® Bme1580I (BseSI) bp from end of DNA required for complete digestion




5’...G K G C M↓C...3’

3’...C↑M C G K G...5’




Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – cleavage impaired (p.181).

λ DN

A, 0

.7%

aga

rose

6 cl

eava

ge s

ites

6 cl

eava

ge s

ites

10 c

leav

age

site

s

22

1. F

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ICTI

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1


FastDigest® BpmI (GsuI)

Digestion time with 1 µl of FastDigest® BpmI (GsuI) bp from end of DNA required for complete digestion




5’...C T G G A G (N)16↓...3’

3’...G A C C T C (N)14↑...5’




Methylation EffectsDam, CpG, EcoKI, EcoBI – no effect.Dcm: may overlap – blocked (p.177).

λ DN

A, 1

.0%

aga

rose

Note• FastDigest® BpmI (GsuI) requires only

Mg2+ for its activity, but is stimulated by S-adenosylmethionine.

• FastDigest® BpmI (GsuI) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

FastDigest® BplI

Digestion time with 1 µl of FastDigest® BplI bp from end of DNA required for complete digestion



15 min 20 min 30 min 30 min ND 65°C, 5 min 16 hours

5’...↓ 8(N) G A G(N)5 C T C(N)13↓...3’

3’...↑ 13(N) C T C(N)5 G A G(N)8↑ ...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/XhoI fragments in 15 min at 37°C in 1X FastDigest® Buffer.


λ DN

A/Xh

oI, 0

.7%

aga

rose Note

FastDigest® BplI requires only Mg2+ for its acti-vity, but is stimulated by S-adenosylmethionine. Still, complete cleavage of some substrates with FastDigest® BplI is difficult to achieve.


Digestion time with 1 µl of FastDigest® BmtI (BspOI) bp from end of DNA required for complete digestion




5’...G C T A G↓C...3’

3’...C↑G A T C G...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/HindIII fragments in 5 min at 37°C in 1X FastDigest® Buffer.



λ DN

A, 0

.7%

aga

rose

FastDigest® BmtI (BspOI)

1 cl

eava

ge s

ite1

clea

vage

site

25 c

leav

age

site

s

23


1



FastDigest® BsaBI (BseJI)

Digestion time with 1 µl of FastDigest® BsaBI (BseJI) bp from end of DNA required for complete digestion



5 min 5 min 5 min 5 min 3 bp No 1 hour

5’...G A T N N↓N N A T C...3’

3’...C T A N N↑N N T A G...5’




Methylation EffectsDam: may overlap – blocked (p.176).Dcm, EcoKI, CpG – no effect.EcoBI: may overlap – effect not determined.

λ DN

A (d

am–),

1.0%

aga

rose

NoteFastDigest® BsaBI (BseJI) is blocked by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

FastDigest® BsaAI (Ppu21I)

Digestion time with 1 µl of FastDigest® BsaAI (Ppu21I) bp from end of DNA required for complete digestion




5’...Y A C↓G T R...3’

3’...R T G↑C A Y...5’




Methylation EffectsDam, Dcm – no effect.CpG: completely overlaps – blocked (p.178).EcoKI, EcoBI: may overlap – effect not determined.

λ DN

A, 0

.7%

aga

rose

FastDigest® Bpu10I

Digestion time with 1 µl of FastDigest® Bpu10I bp from end of DNA required for complete digestion




5’...C C↓T N A G C...3’

3’...G G A N T↑C G...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of M13mp18 DNA in 15 min at 37°C in 1X Fast-Digest® Buffer.



M13

mp1

8 DN

A, 0

.7%

aga

rose

NoteFor cleavage with FastDigest® Bpu10I at least two copies of its recognition sequence are required.

4 cl

eava

ge s

ites

14 c

leav

age

site

s21

cle

avag

e si

tes

24

1. F

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t® R

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ICTI

ON E

NZYM

ES

1


FastDigest® BsaJI (BseDI)

Digestion time with 1 µl of FastDigest® BsaJI (BseDI) bp from end of DNA required for complete digestion




5’...C↓C N N G G...3’

3’...G G N N C↑C...5’





λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® BseGI bp from end of DNA required for complete digestion




5’...G G A T G N N↓...3’

3’...C C T A C↑N N ...5’





FastDigest® BseGI

λ DN

A, 1

.0%

aga

rose

FastDigest® BsaHI (Hin1I)

Digestion time with 1 µl of FastDigest® BsaHI (Hin1I) bp from end of DNA required for complete digestion




5’...G R↓C G Y C...3’

3’...C Y G C↑R G...5’

#FD0474 100 µl Supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml



Methylation EffectsDam, EcoKI – no effect.Dcm: may overlap – cleavage impaired (p.177).CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.

λ DN

A (d

cm– ),

1.0

% a

garo

se

NoteFastDigest® BsaHI (Hin1I) cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

40 c

leav

age

site

s10

5 cl

eava

ge s

ites

150

clea

vage

site

s

25


1



FastDigest® Bsh1236I

Digestion time with 1 µl of FastDigest® Bsh1236I bp from end of DNA required for complete digestion




5’...C G↓C G...3’

3’...G C↑G C...5’





λ DN

A, 1

.4%

aga

rose

FastDigest® BseNI

Digestion time with 1 µl of FastDigest® BseNI bp from end of DNA required for complete digestion




5’...A C T G G N↓...3’

3’...T G A C↑C N ...5’




Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not deter-mined.

λ DN

A, 1

.0%

aga

rose

FastDigest® BseXI

Digestion time with 1 µl of FastDigest® BseXI bp from end of DNA required for complete digestion




5’...G C A G C(N)8↓ ...3’

3’...C G T C G(N)12↑...5’





pBR3

22 D

NA, 1

.4%

aga

rose

110

clea

vage

site

s21

cle

avag

e si

tes

157

clea

vage

site

s

26

1. F

astD

iges

t® R

ESTR

ICTI

ON E

NZYM

ES

1


FastDigest® BsiWI (Pfl23II)

Digestion time with 1 µl of FastDigest® BsiWI (Pfl23II) bp from end of DNA required for complete digestion




5’...C↓G T A C G...3’

3’...G C A T G↑C...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/Psp1406I fragments in 15 min at 37°C in 1X FastDigest® Buffer.


Methylation EffectsCpG: completely overlaps – blocked (p.178).Dam, Dcm, EcoKI, EcoBI – no effect.

λ DN

A, 0

.7%

aga

rose

FastDigest® BslI (BseLI)

Digestion time with 1 µl of FastDigest® BslI (BseLI) bp from end of DNA required for complete digestion




5’...C C N N N N N↓N N G G...3’

3’...G G N N↑N N N N N C C...5’





λ DN

A (d

cm– ),

1.4

% a

garo

se

NoteFastDigest® BslI (BseLI) cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

FastDigest® BsiEI (Bsh1285I)

Digestion time with 1 µl of FastDigest® BsiEI (Bsh1285I) bp from end of DNA required for complete digestion




5’...C G R Y↓C G...3’

3’...G C↑Y R G C...5’




Methylation EffectsDam: may overlap – cleavage impaired (p.176).Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).

λ DN

A, 1

.0%

aga

rose

NoteFastDigest® BsiEI (Bsh1285I) cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).22

cle

avag

e si

tes

1 cl

eava

ge s

ite17

6 cl

eava

ge s

ites

27


1



Digestion time with 1 µl of FastDigest® Bsp119I bp from end of DNA required for complete digestion




5’...T T↓C G A A...3’

3’...A A G C↑T T...5’




Methylation EffectsDam, Dcm, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).EcoKI: may overlap – effect not determined.

FastDigest® Bsp119I

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® BsmFI (FaqI) bp from end of DNA required for complete digestion




5’...G G G A C(N)10↓...3’

3’...C C C T G(N)14↑...5’




FastDigest® BsmFI (FaqI)

λ DN

A, 1

.0%

aga

rose

Note• FastDigest® BsmFI (FaqI) requires only Mg2+

for its activity, but is stimulated by S-ade-nosylmethionine. Still, complete cleavage of some substrates is difficult to achieve.

• For cleavage with FastDigest® BsmFI (FaqI) at least two copies of its recognition sequence are required.

Methylation EffectsDam, Dcm, EcoBI, EcoKI – no effect.CpG: may overlap – blocked (p.179).

FastDigest® BsmBI (Esp3I)

Digestion time with 1 µl of FastDigest® BsmBI (Esp3I) bp from end of DNA required for complete digestion




5’...C G T C T C(N)1↓...3’

3’...G C A G A G(N)5↑...5’



Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.DTT (#R0861/2) 1 mM.


λ DN

A, 1

.0%

aga

rose Note

The enzyme requires DTT (#R0861/2). Freshly made DTT should be added to the reaction buffer.

14 c

lava

ge s

ites

38 c

leav

age

site

s7

clea

vage

site

s

28

1. F

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ICTI

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NZYM

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1






5’...T↓G T A C A...3’

3’...A C A T G↑T...5’

#FD0933 50 µl#FD0934 100 µl






λ DN

A, 0

.7%

aga

rose





5’...G↓G G C C C...3’

3’...C C C G G↑G...5’






λ DN

A, 0

.7%

aga

rose

NoteFastDigest® Bsp120I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Digestion time with 1 µl of FastDigest® Bsp1286I (SduI) bp from end of DNA required for complete digestion




5’...G D G C H↓C...3’

3’...C↑H C G D G...5’




Methylation EffectsDam, Dcm, CpG – no effect.EcoKI, EcoBI: may overlap – effect not determined.

λ DN

A, 1

.0%

aga

rose

FastDigest® Bsp1286I (SduI)

1 cl

eava

ge s

ite38

cle

avag

e si

tes

5 cl

eava

ge s

ites

29


1



Digestion time with 1 µl of FastDigest® BspCNI (BseMII) bp from end of DNA required for complete digestion




5’...C T C A G(N)10↓...3’

3’...G A G T C(N)8↑ ...5’




Methylation EffectsDam, Dcm, CpG, EcoBI, EcoKI – no effect.

FastDigest® BspCNI (BseMII)

λ DN

A, 1

.0%

aga

rose

NoteFastDigest® BspCNI (BseMII) requires S-adenosylmethionine for activity. Sinefungin can replace SAM in the restriction reaction. In this case DNA is not methylated and more than 95% of the ligated BspCNI (BseMII) fragments can be recut by this enzyme.

Digestion time with 1 µl of FastDigest® BspHI (PagI) bp from end of DNA required for complete digestion



5 min 10 min 5 min 10 min 3 bp 80°C, 5 min 0.5 hour

5’...T↓C A T G A...3’

3’...A G T A C↑T...5’




Methylation EffectsDcm, CpG, EcoKI – no effect.Dam, EcoBI: may overlap – cleavage impaired (p.176, 181).

FastDigest® BspHI (PagI)

FX1

74 D

NA, 1

.0%

aga

rose

NoteFastDigest® BspHI (PagI) cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Digestion time with 1 µl of FastDigest® BspMI (BveI) bp from end of DNA required for complete digestion




5’...A C C T G C(N)4↓...3’

3’...T G G A C G(N)8↑...5’

#FD1744 50 µlSupplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml20X Oligonucleotide (0.01 mM) 20 µl


Reaction Conditions1X FastDigest® Buffer or 1X FastDigest® Green Buffer at 37°C.Oligonucleotide 0.5 µM.

Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI, EcoKI: may overlap – effect not determined.

FastDigest® BspMI (BveI)

λ DN

A, 0

.7%

aga

rose

sequence in the reaction mixture signifi-cantly improves cleavage of plasmid DNAs, especially of those with a single FastDi-gest® BspMI (BveI) site. Still, complete cleavage of some substrates is difficult to achieve.

Note• At least two copies of FastDigest® BspMI

(BveI) recognition site are required for efficient cleavage.

• Inclusion of 0.5 µM oligonucleotide with the FastDigest® BspMI (BveI) recognition

• FastDigest® BspMI (BveI) may remain associated with the cleaved DNA. This may cause DNA band shifting during electropho-resis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.

79 c

leav

age

site

s3

clea

vage

site

s41

cle

avag

e si

tes

30

1. F

astD

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t® R

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ICTI

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NZYM

ES

1


Digestion time with 1 µl of FastDigest® BsrDI (BseMI) bp from end of DNA required for complete digestion




5’...G C A A T G N N↓...3’

3’...C G T T A C↑N N ...5’





FastDigest® BsrDI (BseMI)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® BsrBI (MbiI) bp from end of DNA required for complete digestion




5’...C C G↓C T C...3’

3’...G G C↑G A G...5’




Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: completely overlaps – cleavage impaired (p.178).EcoBI: may overlap – effect not determined.

FastDigest® BsrBI (MbiI)

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® BsrFI (Cfr10I) bp from end of DNA required for complete digestion




5’...R↓C C G G Y...3’

3’...Y G G C C↑R...5’





FastDigest® BsrFI (Cfr10I)

λ DN

A, 1

.0%

aga

rose

NoteFor cleavage with FastDigest® BsrFI (Cfr10I) at least two copies of its recognition sequence are required.

17 c

leav

age

site

s44

cle

avag

e si

tes

61 c

leav

age

site

s

31


1



Digestion time with 1 µl of FastDigest® BstZ17I (Bst1107I) bp from end of DNA required for complete digestion




5’...G T A↓T A C...3’

3’...C A T↑A T G...5’





FastDigest® BstZ17I (Bst1107I)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® BstXI bp from end of DNA required for complete digestion



5 min 5 min 5 min 5 min 4 bp 80°C, 5 min 0.5 hours

5’...C C A N N N N N↓N T G G...3’

3’...G G T N↑N N N N N A C C...5’




Methylation EffectsDam, CpG, EcoKI, EcoBI – no effect.Dcm: may overlap – cleavage impaired (p.177).

FastDigest® BstXI

λ DN

A, 0

.7%

aga

rose

NoteFastDigest® BstXI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Digestion time with 1 µl of FastDigest® BssHII (PteI) bp from end of DNA required for complete digestion




5’...G↓C G C G C...3’

3’...C G C G C↑G...5’





FastDigest® BssHII (PteI)

λ DN

A, 0

.7%

aga

rose

6 cl

eava

ge s

ites

13 c

leav

age

site

s3

clea

vage

site

s

32

1. F

astD

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t® R

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ICTI

ON E

NZYM

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1


Digestion time with 1 µl of FastDigest® Csp6I bp from end of DNA required for complete digestion




5’...G↓T A C...3’

3’...C A T↑G...5’





FastDigest® Csp6I

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® Bsu36I (Eco81I) bp from end of DNA required for complete digestion




5’...C C↓T N A G G...3’

3’...G G A N T↑C C...5’





FastDigest® Bsu36I (Eco81I)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® ClaI (Bsu15I) bp from end of DNA required for complete digestion




5’...A T↓C G A T...3’

3’...T A G C↑T A...5’

#FD0143 50 µl#FD0144 100 µl




Methylation EffectsDam: may overlap – blocked (p.176).CpG: completely overlaps – blocked (p.178).Dcm, EcoKI – no effect.EcoBI: may overlap – effect not determined.

FastDigest® ClaI (Bsu15I)

λ DN

A, 0

.7%

aga

rose

NoteFastDigest® ClaI (Bsu15I) is blocked by overlapping dam methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

2 cl

eava

ge s

ites

15 c

leav

age

site

s11

3 cl

eava

ge s

ites

33


1



Digestion time with 1 µl of FastDigest® DpnI bp from end of DNA required for complete digestion



not determined 5 min not applicable 10 min 1 bp 80°C, 5 min 16 hours

Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pBR322 DNA (dam methylated) in 5 min at 37°C in 1X FastDigest® Buffer.


Methylation EffectsDam: does not cut dam– DNA (p.176).Dcm, EcoKI, CpG – no effect.EcoBI: may overlap – effect not determined.

FastDigest® DpnI

pBR3

22 D

NA, 1

.4%

aga

rose

Note• FastDigest® DpnI requires the presence

of N6-methyladenine within the recognition sequence to cleave DNA.

• DNA purified from a dam+ strain will be a substrate for FastDigest® DpnI.

• FastDigest® DpnI will only cleave fully-adenomethylated dam sites.

• FastDigest® DpnI, FastDigest® Sau3AI (Bsp143I) and FastDigest® MboI all recog-nize the same sequence but have different methylation sensitivities (pp.176-181) and cleavage positions.

5’...G m6A↓ T C...3’

3’...C T↑m6A G...5’

#FD1703 50 µl #FD1704 100 µl


Digestion time with 1 µl of FastDigest® DdeI (HpyF3I) bp from end of DNA required for complete digestion




5’...C↓T N A G...3’

3’...G A N T↑C...5’





FastDigest® DdeI (HpyF3I)

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® DraI bp from end of DNA required for complete digestion




5’...T T T↓A A A...3’

3’...A A A↑T T T...5’




Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – blocked (p.181).

FastDigest® DraI

λ DN

A, 0

.7%

aga

rose

104

clea

vage

site

s22

cle

avag

e si

tes

13 c

leav

age

site

s

34

1. F

astD

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t® R

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ICTI

ON E

NZYM

ES

1


Digestion time with 1 µl of FastDigest® EagI (Eco52I) bp from end of DNA required for complete digestion




5’...C↓G G C C G...3’

3’...G C C G G↑C...5’





FastDigest® EagI (Eco52I)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® DraIII (AdeI) bp from end of DNA required for complete digestion




5’...C A C N N N↓G T G...3’

3’...G T G↑N N N C A C...5’




Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap – effect not determined.

FastDigest® DraIII (AdeI)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® DrdI (AasI) bp from end of DNA required for complete digestion




5’...G A C N N N N↓N N G T C...3’

3’...C T G N N↑N N N N C A G...5’




Methylation EffectsDam, Dcm, EcoBI, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).

λ DN

A, 0

.7%

aga

rose

FastDigest® DrdI (AasI)

10 c

leav

age

site

s3

clea

vage

site

s2

clea

vage

site

s

35


1



Digestion time with 1 µl of FastDigest® Ecl136II bp from end of DNA required for complete digestion




5’...G A G↓C T C...3’

3’...C T C↑G A G...5’




Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – effect not determined.

FastDigest® Ecl136II

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® EarI (Eam1104I) bp from end of DNA required for complete digestion




5’...C T C T T C(N)1↓...3’

3’...G A G A A G(N)4↑...5’





FastDigest® EarI (Eam1104I)

FX1

74 D

NA, 1

.0%

aga

rose

NoteCertain sites in λ DNA are difficult to cleave with FastDigest® EarI (Eam1104I), the same as with its prototype Ksp632I.

Digestion time with 1 µl of FastDigest® Eam1105I bp from end of DNA required for complete digestion




5’...G A C N N N↓N N G T C...3’

3’...C T G N N↑N N N C A G...5’





FastDigest® Eam1105I

λ DN

A, 0

.7%

aga

rose

9 cl

eava

ge s

ites

2 cl

eava

ge s

ites

2 cl

eava

ge s

ites

36

1. F

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1


Digestion time with 1 µl of FastDigest® Eco91I bp from end of DNA required for complete digestion




5’...G↓G T N A C C...3’

3’...C C A N T G↑G...5’





FastDigest® Eco91I

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® EcoNI (XagI) bp from end of DNA required for complete digestion




5’...C C T N N↓N N N A G G...3’

3’...G G A N N N↑N N T C C...5’





FastDigest® EcoNI (XagI)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® Eco31I bp from end of DNA required for complete digestion




5’...G G T C T C(N)1↓...3’

3’...C C A G A G(N)5↑...5’

#FD0293 50 µl #FD0294 100 µl


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dcm–/HindIII fragments in 5 min at 37°C in 1X FastDigest® Buffer.


Methylation EffectsDam, EcoKI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).EcoBI: may overlap – effect not determined.

FastDigest® Eco31I

λ DN

A (d

cm–),

0.7%

aga

rose

NoteFastDigest® Eco31I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

2 cl

eava

ge s

ites

13 c

leav

age

site

s9

clea

vage

site

s

37


1



Digestion time with 1 µl of FastDigest® EcoRV (Eco32I) bp from end of DNA required for complete digestion




5’...G A T↓A T C...3’

3’...C T A↑T A G...5’

#FD0303 200 µl #FD0304 400 µl





FastDigest® EcoRV (Eco32I)

λ DN

A, 1

.0%

aga

rose

Digestion time with 1 µl of FastDigest® EcoO109I bp from end of DNA required for complete digestion




5’...R G↓G N C C Y...3’

3’...Y C C N G↑G R...5’





FastDigest® EcoO109I

λ DN

A, 0

.7%

aga

rose Note

FastDigest® EcoO109I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Digestion time with 1 µl of FastDigest® EcoRI bp from end of DNA required for complete digestion




5’...G↓A A T T C...3’

3’...C T T A A↑G...5’






FastDigest® EcoRI

λ DN

A, 0

.7%

aga

rose

3 cl

evag

e si

tes

5 cl

eava

ge s

ites

21 c

leav

age

site

s

38

1. F

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NZYM

ES

1


Digestion time with 1 µl of FastDigest® EheI bp from end of DNA required for complete digestion




5’...G G C↓G C C...3’

3’...C C G↑C G G...5’

#FD0443 20 µl #FD0444 50 µl


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/PstI fragments in 5 min at 37°C in 1X FastDigest® Buffer.



FastDigest® EheI

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® FokI bp from end of DNA required for complete digestion




5’...G G A T G(N)9↓...3’

3’...C C T A C(N)13↑...5’

#FD2144 100 µlBoth supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml




FastDigest® FokI

λ DN

A, 1

.0%

aga

rose

Note• At least two copies of FastDigest® FokI

recognition site are required for an efficient cleavage.

• FastDigest® FokI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.

Digestion time with 1 µl of FastDigest® Fnu4HI (SatI) bp from end of DNA required for complete digestion




5’...G C↓N G C...3’

3’...C G N↑C G...5’





FastDigest® Fnu4HI (SatI)

λ DN

A, 1

.4%

aga

rose

NoteAt least two copies of FastDigest® Fnu4HI (SatI) recognition site are required for an efficient cleavage.

1 cl

eava

ge s

ite38

0 cl

eava

ge s

ites

150

clea

vage

site

s

39


1



Digestion time with 1 µl of FastDigest® FspI (NsbI) bp from end of DNA required for complete digestion




5’...T G C↓G C A...3’

3’...A C G↑C G T...5’





FastDigest® FspI (NsbI)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® FspAI bp from end of DNA required for complete digestion




5’...R T G C↓G C A Y...3’

3’...Y A C G↑C G T R...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA/Psp1406I fragments in 5 min at 37°C in 1X FastDigest® Buffer.



FastDigest® FspAI

λ DN

A, 0

.5%

aga

rose

5’...R G C G C↓Y...3’

3’...Y↑C G C G R...5’


λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® HaeII (BfoI) bp from end of DNA required for complete digestion






Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).EcoBI: may overtlap – not determined.

FastDigest® HaeII (BfoI)

15 c

leav

age

site

s2

clea

vage

site

s48

cle

avag

e si

tes

40

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1


Digestion time with 1 µl of FastDigest® HhaI bp from end of DNA required for complete digestion




5’...G C G↓C...3’

3’...C↑G C G...5’





FastDigest® HhaI

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® HaeIII (BsuRI) bp from end of DNA required for complete digestion




5’...G G↓C C...3’

3’...C C↑G G...5’





FastDigest® HaeIII (BsuRI)

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® HgaI (CseI) bp from end of DNA required for complete digestion




5’...G A C G C(N)5↓...3’

3’...C T G C G(N)10↑...5’




Methylation EffectsDam, Dcm, EcoKI: never overlaps – no effect.CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.

FastDigest® HgaI (CseI)

pBR3

22 D

NA, 1

.4%

aga

rose

Note• For cleavage with FastDigest® HgaI (CseI) at

least two copies of its recognition sequence are required.

• FastDigest® HgaI (CseI) may remain associ-ated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.

149

clea

vage

site

s11

cle

avag

e si

tes

215

clea

vage

site

s

41


1



Digestion time with 1 µl of FastDigest® HincII bp from end of DNA required for complete digestion




5’...G T Y↓R A C...3’

3’...C A R↑Y T G...5’





FastDigest® HincII

λ DN

A, 1

.0%

aga

rose

Digestion time with 1 µl of FastDigest® HindIII bp from end of DNA required for complete digestion




5’...A↓A G C T T...3’

3’...T T C G A↑A...5’






FastDigest® HindIII

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® HinfI bp from end of DNA required for complete digestion




5’...G↓A N T C...3’

3’...C T N A↑G...5’




Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – blocked (p.181).

FastDigest® HinfI

λ DN

A, 1

.4%

aga

rose

35 c

leav

age

site

s7

clea

vage

site

s14

8 cl

eava

ge s

ites

42

1. F

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ICTI

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NZYM

ES

1


Digestion time with 1 µl of FastDigest® HinP1I (Hin6I) bp from end of DNA required for complete digestion




5’...G↓C G C...3’

3’...C G C↑G...5’





FastDigest® HinP1I (Hin6I)

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® HpaII bp from end of DNA required for complete digestion




5’...C↓C G G...3’

3’...G G C↑C...5’





FastDigest® HpaII

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® HpaI (KspAI) bp from end of DNA required for complete digestion




5’...G T T↓A A C...3’

3’...C A A↑T T G...5’




Methylation EffectsDam, Dcm, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI: may overlap – blocked (p.181).

FastDigest® HpaI (KspAI)

λ DN

A, 0

.7%

aga

rose

215

clea

vage

site

s14

cle

avag

e si

tes

328

clea

vage

site

s

43


1



Digestion time with 1 µl of FastDigest® Hpy8I bp from end of DNA required for complete digestion




5’...G T N↓N A C...3’

3’...C A N↑N T G...5’




Methylation EffectsDam, Dcm, – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI – effect not determined.

FastDigest® Hpy8I

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® HpyF10VI bp from end of DNA required for complete digestion




5’...G C N N N N N↓N N G C...3’

3’...C G N N↑N N N N N C G...5’





FastDigest® HpyF10VI

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® KpnI bp from end of DNA required for complete digestion




5’...G G T A C↓C...3’

3’...C↑C A T G G...5’




Methylation EffectsDam, Dcm, CpG, EcoKI, EcoKI, EcoBI – no effect.

FastDigest® KpnI

λ DN

A, 0

.7%

aga

rose

125

clea

vage

site

s34

6 cl

eava

ge s

ites

2 cl

eava

ge s

ites

44

1. F

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NZYM

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1


Digestion time with 1 µl of FastDigest® Kpn2I bp from end of DNA required for complete digestion




5’...T↓C C G G A...3’

3’...A G G C C↑T...5’





FastDigest® Kpn2I

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® MauBI bp from end of DNA required for complete digestion




5’...C G↓C G C G C G...3’

3’...G C G C G C↑G C...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of linear-ized pJET1 DNA with inserted MauBI recognition sites in 5 min at 37°C in 1X FastDigest® Buffer.



FastDigest® MauBI

pJET

1-M

auBI

DNA

/Bam

HI, 0

.7%

aga

rose

Note• FastDigest® MboI is blocked by overlapping

dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

• FastDigest® MboI, FastDigest® Sau3AI (Bsp143I) and FastDigest® DpnI all recog-nize the same sequence but have different methylation sensitivities (pp.176-181) and cleavage positions.

Digestion time with 1 µl of FastDigest® MboI bp from end of DNA required for complete digestion




5’...↓G A T C ...3’

3’... C T A G↑...5’




Methylation EffectsDam: completely overlaps – blocked (p.176).EcoBI: may overlap – blocked (p.181).Dcm, CpG, EcoKI – no effect.

FastDigest® MboI

λ DN

A (d

am–),

1.4%

aga

rose

24 c

leav

age

site

s2

cleav

age

sites

116

clea

vage

site

s

45


1



Digestion time with 1 µl of FastDigest® MfeI (MunI) bp from end of DNA required for complete digestion




5’...C↓A A T T G...3’

3’...G T T A A↑C...5’

#FD0753 20 µl #FD0754 50 µl





FastDigest® MfeI (MunI)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® MluI bp from end of DNA required for complete digestion




5’...A↓C G C G T...3’

3’...T G C G C↑A...5’





FastDigest® MluI

λ DN

A, 0

.7%

aga

rose

Note• FastDigest® MboII is blocked by overlapping

dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

• FastDigest® MboII produces DNA fragments that have a single-base 3’-extension which are more difficult to ligate than blunt-ended fragments.

• FastDigest® MboII may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.

Digestion time with 1 µl of FastDigest® MboII bp from end of DNA required for complete digestion




5’...G A A G A(N)8↓...3’

3’...C T T C T(N)7↑...5’




Methylation EffectsDam: may overlap – blocked (p.176).Dcm, CpG, EcoKI, EcoBI – no effect.

FastDigest® MboII

λ DN

A (d

am–),

1.4%

aga

rose

130

clea

vage

site

s8

clea

vage

site

s7

clea

vage

site

s

46

1. F

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NZYM

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1


Digestion time with 1 µl of FastDigest® MreI bp from end of DNA required for complete digestion




5’...C G↓ C C G G C G...3’

3’...G C G G C C↑G C...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of linearized pJET1 DNA with inserted MreI recognition site in 5 min at 37°C in 1X FastDigest® Buffer.



FastDigest® MreI

pJET

1-M

reI D

NA/E

am11

05I, 1

.0%

aga

rose

Digestion time with 1 µl of FastDigest® MlyI (SchI) bp from end of DNA required for complete digestion




5’...G A G T C(N)5↓...3’

3’...C T C A G(N)5↑...5’





FastDigest® MlyI (SchI)

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® MnlI bp from end of DNA required for complete digestion




5’...C C T C(N)7↓...3’

3’...G G A G(N)6↑...5’




FastDigest® MnlI

λ DN

A, 1

.4%

aga

rose

NoteFastDigest® MnlI produces DNA fragments that have a single-base 3’-extension which are more difficult to ligate than blunt-ended fragments.


61 c

leav

age

site

s26

2 cl

eava

ge s

ites

1 cle

avag

e sit

e

47


1



Digestion time with 1 µl of FastDigest® MslI (RseI) bp from end of DNA required for complete digestion




5’...C A Y N N↓ N N R T G...3’

3’...G T R N N↑N N Y A C...5’




Methylation EffectsDam, Dcm, CpG – no effect.EcoKI: may overlap – blocked (p.181).EcoBI: may overlap – effect not determined.

FastDigest® MslI (RseI)

λ DN

A, 1

.2%

aga

rose

Digestion time with 1 µl of FastDigest® MscI (MlsI) bp from end of DNA required for complete digestion




5’...T G G↓C C A...3’

3’...A C C↑G G T...5’





FastDigest® MscI (MlsI)

λ DN

A (d

cm–),

0.7%

aga

rose

NoteFastDigest® MscI (MlsI) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

pBR3

22 D

NA, 1

.0%

aga

rose

Digestion time with 1 µl of FastDigest® MseI (SaqAI) bp from end of DNA required for complete digestion






Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – effect not determined.

FastDigest® MseI (SaqAI)

5’...T↓T A A...3’

3’...A A T↑T...5’


18 c

leav

age

site

s15

cle

avag

e si

tes

62 c

leav

age

site

s

48

1. F

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1


Digestion time with 1 µl of FastDigest® MssI bp from end of DNA required for complete digestion




5’...G T T T↓A A A C...3’

3’...C A A A↑T T T G...5’





FastDigest® MssI

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® MspI bp from end of DNA required for complete digestion




5’...C↓C G G...3’

3’...G G C↑C...5’





FastDigest® MspI

λ DN

A, 1

.4%

aga

rose

NoteFastDigest® MspI is an isoschizomer of HpaII. When the external C in the sequence CCGG is methylated, these enzymes cannot cleave. However, unlike HpaII, FastDigest® MspI can cleave the sequence when the internal C residue is methylated.

Digestion time with 1 µl of FastDigest® MvaI bp from end of DNA required for complete digestion




5’...C C↓W G G...3’

3’...G G W↑C C...5’





FastDigest® MvaI

λ DN

A, 1

.4%

aga

rose

328

clea

vage

site

s2

clea

vage

site

s70

cle

avag

e si

tes

49


1



Digestion time with 1 µl of FastDigest® Mva1269I bp from end of DNA required for complete digestion




5’...G A A T G C N↓...3’

3’...C T T A C↑G N ...5’





FastDigest® Mva1269I

λ DN

A, 1

.0%

aga

rose

Digestion time with 1 µl of FastDigest® NciI (BcnI) bp from end of DNA required for complete digestion




5’...C C↓S G G...3’

3’...G G S↑C C...5’





FastDigest® NciI (BcnI)

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® NaeI (PdiI ) bp from end of DNA required for complete digestion



not determined 5 min 5 min 5 min 3 bp 65°C, 15 min 16 hours

5’...G C C↓G G C...3’

3’...C G G↑C C G...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pBR322 DNA/NdeI fragments in 5 min at 37°C in 1X FastDigest® Buffer.


FastDigest® NaeI (PdiI)

pBR3

22 D

NA/N

deI,

0.7%

aga

rose

Note• For cleavage with FastDigest® NaeI (PdiI) at

least two copies of its recognition sequence are required.

• Certain sites in pBR322 are difficult to cleave with FastDigest® NaeI (PdiI), the same as with its prototype NaeI.


46 c

leav

age

site

s4

clea

vage

site

s11

4 cl

eava

ge s

ites

50

1. F

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NZYM

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1


Digestion time with 1 µl of FastDigest® NdeI bp from end of DNA required for complete digestion




5’...C A↓T A T G...3’

3’...G T A T↑A C...5’

#FD0583 100 µl #FD0584 300 µl


#FD0585 1000 µlSupplied with:10X FastDigest® Buffer 2X1 ml10X FastDigest® Green Buffer 2X1 ml




FastDigest® NdeI

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® NcoI bp from end of DNA required for complete digestion




5’...C↓C A T G G...3’

3’...G G T A C↑C...5’

#FD0573 20 µl #FD0574 100 µl#FD0575 300 µl

All supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml




FastDigest® NcoI

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest ® NheI bp from end of DNA required for complete digestion




5’...G↓C T A G C...3’

3’...C G A T C↑G...5’

#FD0973 50 µl#FD0974 100 µl





FastDigest® NheI

λ DN

A 0.

7% a

garo

se

4 cl

eava

ge s

ites

7 cl

eava

ge s

ites

1 cl

eava

ge s

ite

51


1



Digestion time with 1 µl of FastDigest® NmuCI bp from end of DNA required for complete digestion




5’...↓G T S A C ...3’

3’... C A S T G↑...5’





FastDigest® NmuCI

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® NlaIV (BspLI) bp from end of DNA required for complete digestion




5’...G G N↓N C C...3’

3’...C C N↑N G G...5’





FastDigest® NlaIV (BspLI)

λ DN

A, 1

.4%

aga

rose Note

FastDigest® NlaIV (BspLI) cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Digestion time with 1 µl of FastDigest® NlaIII (Hin1II) bp from end of DNA required for complete digestion




5’... C A T G↓ ...3’

3’...↑G T A C ...5’





FastDigest® NlaIII (Hin1II)

λ DN

A, 1

.4%

aga

rose

NoteMore stable when stored at -70°C. At -20°C the half-life of FastDigest® NlaIII (Hin1II) is 6 months.

181

clea

vage

site

s82

cle

avag

e si

tes

81 c

leav

age

site

s

52

1. F

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ICTI

ON E

NZYM

ES

1


Digestion time with 1 µl of FastDigest® NsiI (Mph1103I) bp from end of DNA required for complete digestion




5’...A T G C A↓T...3’

3’...T↑A C G T A...5’





FastDigest® NsiI (Mph1103I)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® NruI (RruI) bp from end of DNA required for complete digestion




5’...T C G↓C G A...3’

3’...A G C↑G C T...5’




Methylation EffectsDam, Dcm, EcoBI, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).

FastDigest® NruI (RruI)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® NotI bp from end of DNA required for complete digestion




5’...G C↓G G C C G C...3’

3’...C G C C G G↑C G...5’

#FD0593 20 µl #FD0594 50 µl#FD0596 250 µl

All supplied with:10X FastDigest® Buffer 1 ml10X FastDigest® Green Buffer 1 ml

Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pTZ19RJL2 DNA/BseLI fragments in 5 min at 37°C in 1X FastDigest® Buffer.



FastDigest® NotI

pTZ1

9RJL

2/Bs

eLI D

NA, 1

.0%

aga

rose

1 cl

eava

ge s

ite5

clea

vage

site

s14

cle

avag

e si

tes

53


1



Digestion time with 1 µl of FastDigest® PdmI bp from end of DNA required for complete digestion




5’...G A A N N↓N N T T C...3’

3’...C T T N N↑N N A A G...5’





FastDigest® PdmI

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® NspI (XceI) bp from end of DNA required for complete digestion




5’...R C A T G↓Y...3’

3’...Y↑G T A C R...5’




Methylation EffectsDam, Dcm, EcoKI, EcoBI, CpG – no effect.

FastDigest® NspI (XceI)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® PacI bp from end of DNA required for complete digestion




5’...T T A A T↓T A A...3’

3’...A A T↑T A A T T...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of linear-ized pJET1 DNA with inserted PacI recognition site in 5 min at 37°C in 1X FastDigest® Buffer.


Methylation EffectsDam, Dcm, EcoBI, CpG – no effect.EcoKI: may overlap – effect not determined.

FastDigest® PacI

pJET

1-Pa

cI D

NA/S

daI,

1.0%

aga

rose

32 c

leav

age

site

s1

clea

vage

site

24 c

leav

age

site

s

54

1. F

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ICTI

ON E

NZYM

ES

1


Digestion time with 1 µl of FastDigest® PmlI (Eco72I) bp from end of DNA required for complete digestion




5’...C A C↓G T G...3’

3’...G T G↑C A C...5’





FastDigest® PmlI (Eco72I)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® PfoI bp from end of DNA required for complete digestion




5’...T↓C C N G G A...3’

3’...A G G N C C↑T...5’




Methylation EffectsDam: may overlap – cleavage impaired (p.176).Dcm, CpG: may overlap – blocked (p.177, 179).EcoKI, EcoBI – no effect.

FastDigest® PfoI

λ DN

A, 0

.7%

aga

rose

NoteFastDigest® PfoI cleavage is impaired by overlapping dam methylation and blocked by overlapping dcm methylation. To avoid dam or dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Digestion time with 1 µl of FastDigest® PflMI (Van91I) bp from end of DNA required for complete digestion




5’...C C A N N N N↓N T G G...3’

3’...G G T N↑N N N N A C C...5’





FastDigest® PflMI (Van91I)

λ DN

A, 0

.7%

aga

rose

NoteFastDigest® PflMI (Van91I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

14 c

leav

age

site

s15

cle

avag

e si

tes

3 cl

eava

ge s

ites

55


1



Digestion time with 1 µl of FastDigest® PsiI (AanI) bp from end of DNA required for complete digestion




5’...T T A↓T A A ...3’

3’...A A T↑A T T ...5’





FastDigest® PsiI (AanI)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® PshAI (BoxI) bp from end of DNA required for complete digestion




5’...G A C N N↓N N G T C...3’

3’...C T G N N↑N N C A G...5’





FastDigest® PshAI (BoxI)

λ DN

A, 1

.0%

aga

rose

Digestion time with 1 µl of FastDigest® PpuMI (Psp5II) bp from end of DNA required for complete digestion




5’...R G↓G W C C Y...3’

3’...Y C C W G↑G R...5’





FastDigest® PpuMI (Psp5II)

λ DN

A, 0

.7%

aga

rose Note

FastDigest® PpuMI (Psp5II) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).3

clea

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site

s7

clea

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site

s12

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56

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Digestion time with 1 µl of FastDigest® PsuI bp from end of DNA required for complete digestion




5’...R↓G A T C Y...3’

3’...Y C T A G↑R...5’





FastDigest® PsuI

λ DN

A, 0

.7%

aga

rose

Note• Surrounding sequences: the presence of

adjacent runs of G-C base pairs confers sig-nificant resistance to cleavage (Armstrong, K. and Bauer, W.R., NAR, 10, 993-1007, 1982).

• FastDigest® PstI will not cut AGCTGCAG when methylated by AluI methyltransferase.

Digestion time with 1 µl of FastDigest® PstI bp from end of DNA required for complete digestion




5’...C T G C A↓G...3’

3’...G↑A C G T C...5’






FastDigest® PstI

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® PspFI bp from end of DNA required for complete digestion



5 min 5 min 5 min 5 min 4 80°C, 5 min 16 hours

5’...C C C A G↓C...3’

3’...G↑G G T C G...5’





λ DN

A, 0

.7%

aga

rose

FastDigest® PspFI

32 c

leav

age

site

s28

cle

avag

e si

tes

21 c

leav

age

site

s

57


1



Digestion time with 1 µl of FastDigest® PvuII bp from end of DNA required for complete digestion



5 min 5 min 5 min 5 min 1 bp No 0.5 hour

5’...C A G↓C T G...3’

3’...G T C↑G A C...5’





FastDigest® PvuII

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® PsyI bp from end of DNA required for complete digestion




5’...G A C N↓N N G T C...3’

3’...C T G N N↑N C A G...5’





FastDigest® PsyI

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® PvuI bp from end of DNA required for complete digestion




5’...C G A T↓C G...3’

3’...G C↑T A G C...5’





FastDigest® PvuI

λ DN

A, 0

.7%

aga

rose

2 cl

eava

ge s

ites

3 cl

eava

ge s

ites

15 c

leav

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site

s

58

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Digestion time with 1 µl of FastDigest® SacI bp from end of DNA required for complete digestion




5’...G A G C T↓C...3’

3’...C↑T C G A G...5’

#FD1133 100 µl#FD1134 200 µl





FastDigest® SacI

λ DN

A, 0

.7%

aga

rose

Note• FastDigest® SacI is sensitive to cytosine

methylation at GAGmCTC but not GAGCTmC and insensitive to adenine methylation at GmAGCTC. AluI methyltransferase (AGmCT) can be used to block FastDigest® SacI.

• FastDigest® SacI is inhibited by common clini-cal anticoagulants found in some preparations of anticoagulated peripheral blood and bone marrow. (Coad, J.E., et al., Inhibition of restric-tion endonucleases by common clinical antico-agulants, Anal. Biochem., 205, 368-369,1992).

Digestion time with 1 µl of FastDigest® RsrII (CpoI) bp from end of DNA required for complete digestion




5’...C G↓G W C C G...3’

3’...G C C W G↑G C...5’





FastDigest® RsrII (CpoI)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® RsaI bp from end of DNA required for complete digestion




5’...G T↓A C...3’

3’...C A↑T G...5’





FastDigest® RsaI

λ DN

A, 1

.4%

aga

rose

113

clea

vage

site

s5

clea

vage

site

s2

clea

vage

site

s

59


1



Digestion time with 1 µl of FastDigest® SapI (LguI) bp from end of DNA required for complete digestion




5’...G C T C T T C(N)1↓...3’

3’...C G A G A A G(N)4↑...5’





FastDigest® SapI (LguI)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® SalI bp from end of DNA required for complete digestion




5’...G↓T C G A C...3’

3’...C A G C T↑G...5’




FastDigest® SalI

λ DN

A, 0

.7%

aga

rose


Digestion time with 1 µl of FastDigest® SanDI (KflI) bp from end of DNA required for complete digestion




5’...G G↓G W C C C...3’

3’...C C C W G↑G G...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of linear-ized pJET1 DNA with inserted SanDI recognition sites in 5 min at 37°C in 1X FastDigest® Buffer.


Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm: may overlap – effect not determined.CpG: may overlap – cleavage impaired (p.179).

FastDigest® SanDI (KflI)

pJET

1-Sa

nDI D

NA/B

amHI

, 0.7

% a

garo

se

2 cl

eava

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ites

3 cle

avag

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es10

cle

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60

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Digestion time with 1 µl of FastDigest® SbfI (SdaI) bp from end of DNA required for complete digestion




5’...C C T G C A↓G G...3’

3’...G G↑A C G T C C...5’





FastDigest® SbfI (SdaI)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® Sau96I (Cfr13I) bp from end of DNA required for complete digestion




5’...G↓G N C C...3’

3’...C C N G↑G...5’





FastDigest® Sau96I (Cfr13I)

λ DN

A, 1

.0%

aga

rose

NoteFastDigest® Sau96I (Cfr13I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Digestion time with 1 µl of FastDigest® Sau3AI (Bsp143I) bp from end of DNA required for complete digestion




5’...↓G A T C ...3’

3’... C T A G↑...5’





FastDigest® Sau3AI (Bsp143I)

λ DN

A, 1

.4%

aga

rose

NoteFastDigest® DpnI, FastDigest® Sau3AI (Bsp143I) and FastDigest® MboI all recognize the same sequence but have different methylation sensi-tivities (pp.176-181) and cleavage positions.

116

clea

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site

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5 cl

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61


1



Digestion time with 1 µl of FastDigest® SexAI (CsiI ) bp from end of DNA required for complete digestion




5’...A↓C C W G G T...3’

3’...T G G W C C↑A...5’




Methylation EffectsDam, Dcm, CpG – no effect.EcoBI, EcoKI: may overlap – effect not determined.

FastDigest® SexAI (CsiI)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® ScrFI (Bme1390I) bp from end of DNA required for complete digestion




5’...C C↓N G G...3’

3’...G G N↑C C...5’





FastDigest® ScrFI (Bme1390I)

λ DN

A (d

cm–),

1.4%

aga

rose

NoteFastDigest® ScrFI (Bme1390I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Digestion time with 1 µl of FastDigest® ScaI bp from end of DNA required for complete digestion




5’...A G T↓A C T...3’

3’...T C A↑T G A...5’





FastDigest® ScaI

λ DN

A, 0

.7%

aga

rose

5 cl

eava

ge s

ites

184

clea

vage

site

s5

clea

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62

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Digestion time with 1 µl of FastDigest® SfcI (BfmI) bp from end of DNA required for complete digestion




5’...C↓T R Y A G...3’

3’...G A Y R T↑C...5’





FastDigest® SfcI (BfmI)

λ DN

A,1.

0% a

garo

se

Note• FastDigest® SfiI cleavage is impaired by

overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

• For cleavage with FastDigest® SfiI at least two copies of its recognition sequence are required. The two sites can be on either the same or different DNA molecules. (Wertzell, L.M. et al., J. Mol. Biol., 248, 581-595, 1995.)

Digestion time with 1 µl of FastDigest® SfiI bp from end of DNA required for complete digestion



no cleavage sites 15 min 15 min 15 min 5 bp No 16 hours

5’...G G C C N N N N↓N G G C C...3’

3’...C C G G N↑N N N N C C G G...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pUC19 DNA with inserted SfiI recognition sites in 15 min at 50°C in 1X FastDigest® Buffer.


Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179). pU

C19-

SfiI D

NA, 0

.7%

aga

rose

FastDigest® SfiI

Digestion time with 1 µl of FastDigest® SfaNI (BmsI) bp from end of DNA required for complete digestion




5’...G C A T C(N)5↓...3’

3’...C G T A G(N)9↑...5’





FastDigest® SfaNI (BmsI)

λ DN

A, 1

.4%

aga

rose Note

At least two copies of FastDigest® SfaNI (BmsI) recognition site are required for efficient cleavage.

169

clea

vage

site

s38

cle

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2 cl

eava

ge s

ites

63


1



Digestion time with 1 µl of FastDigest® SpeI (BcuI) bp from end of DNA required for complete digestion


Incubation time without star activityLambda, 1µg/20µl Plasmid DNA, 1µg/20µl PCR product, ~0.2µg/30µl Genomic DNA, 1µg/10µl

no cleavage sites 5 min 5 min 5 min 1 bp No 16 hours

5’...A↓C T A G T...3’

3’...T G A T C↑A...5’

#FD1253 20 µl #FD1254 50 µl


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of pUC19 DNA with inserted BcuI recognition site/Psp1406I fragments in 5 min at 37°C in 1X FastDigest® Buffer. The control DNA is.



FastDigest® SpeI (BcuI)

pUC1

9-Bc

uI DN

A/Ps

p140

6I, 1

.0%

aga

rose

Digestion time with 1 µl of FastDigest® SnaBI (Eco105I) bp from end of DNA required for complete digestion




5’...T A C↓G T A...3’

3’...A T G↑C A T...5’





FastDigest® SnaBI (Eco105I)

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® SmaI bp from end of DNA required for complete digestion




5’...C C C↓G G G...3’

3’...G G G↑C C C...5’

#FD0663 100 µl#FD0664 200 µl





FastDigest® SmaI

λ DN

A, 0

.7%

aga

rose

3 cl

eava

ge s

ites

1 cl

eava

ge s

ite1

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64

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Digestion time with 1 µl of FastDigest® StuI (Eco147I) bp from end of DNA required for complete digestion




5’...A G G↓C C T...3’

3’...T C C↑G G A...5’





FastDigest® StuI (Eco147I)

λ DN

A, 0

.7%

aga

rose Note

FastDigest® StuI (Eco147I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Digestion time with 1 µl of FastDigest® SspI bp from end of DNA required for complete digestion




5’...A A T↓A T T...3’

3’...T T A↑T A A...5’





FastDigest® SspI

λ DN

A, 1

.0%

aga

rose

Digestion time with 1 µl of FastDigest® SphI (PaeI) bp from end of DNA required for complete digestion




5’...G C A T G↓C...3’

3’...C↑G T A C G...5’





FastDigest® SphI (PaeI)

λ DN

A, 0

.7%

aga

rose

6 cl

eava

ge s

ites

20 c

leav

age

site

s6

clea

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site

s

65


1



Digestion time with 1 µl of FastDigest® TaaI bp from end of DNA required for complete digestion




5’...A C N↓G T...3’

3’...T G↑N C A...5’





FastDigest® TaaI

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® StyI (Eco130I) bp from end of DNA required for complete digestion




5’...C↓C W W G G...3’

3’...G G W W C↑C...5’





FastDigest® StyI (Eco130I)

λ DN

A, 1

.0%

aga

rose

Digestion time with 1 µl of FastDigest® SwaI (SmiI) bp from end of DNA required for complete digestion



no cleavage sites 20 min 30 min 30 min 2 bp 65°C, 15 min 1 hour

5’...A T T T↓A A A T...3’

3’...T A A A↑T T T A...5’


Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of M13mp18 DNA/MvaI fragments in 5 min at 37°C in 1X FastDigest® Buffer.



FastDigest® SwaI (SmiI)

M13

mp1

8 DN

A/M

vaI,

1.0%

aga

rose

10 c

leav

age

site

s1

clea

vage

site

187

clea

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site

s

66

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Digestion time with 1 µl of FastDigest® TatI bp from end of DNA required for complete digestion




5’...W↓G T A C W...3’

3’...W C A T G↑W...5’





FastDigest® TatI

λ DN

A, 1

.0%

aga

rose

Digestion time with 1 µl of FastDigest® TaqI bp from end of DNA required for complete digestion




5’...T↓C G A...3’

3’...A G C↑T...5’




FastDigest® TaqI

λ DN

A (d

am–),

1.4%

aga

rose

NoteFastDigest® TaqI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).


Digestion time with 1 µl of FastDigest® TaiI bp from end of DNA required for complete digestion




5’... A C G T↓...3’

3’...↑T G C A ...5’





FastDigest® TaiI

λ DN

A, 1

.4%

aga

rose

143

clea

vage

site

s12

1 cl

eava

ge s

ites

24 c

leav

age

site

s

67


1



Digestion time with 1 µl of FastDigest® Tru1I bp from end of DNA required for complete digestion




5’...T↓T A A ...3’

3’...A A T↑T...5’





FastDigest® Tru1I

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® TfiI (PfeI) bp from end of DNA required for complete digestion




5’...G↓A W T C...3’

3’...C T W A↑G...5’




Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – blocked (p.179).EcoBI: may overlap – effect not determined.

FastDigest® TfiI (PfeI)

λ DN

A, 1

.4%

aga

rose

Digestion time with 1 µl of FastDigest® TauI bp from end of DNA required for complete digestion




5’...G C S G↓C...3’

3’...C↑G S C G...5’




Methylation EffectsDam, Dcm, EcoBI, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).

FastDigest® TauI

λ DN

A, 1

.4%

aga

rose

NoteFastDigest® TauI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.

181

clea

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site

s87

cle

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tes

195

clea

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site

s

68

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Digestion time with 1 µl of FastDigest® XapI bp from end of DNA required for complete digestion




5’...R↓A A T T Y...3’

3’...Y T T A A↑R...5’

#FD1383 50 µl#FD1384 100 µl





FastDigest® XapI

λ DN

A, 0

.7%

aga

rose

Digestion time with 1 µl of FastDigest® TspRI (TscAI) bp from end of DNA required for complete digestion




5’... N N C A S T G N N↓...3’

3’...↑N N G T S A C N N ...5’





FastDigest® TspRI (TscAI)

λ DN

A, 1

.4%

aga

rose

Note• FastDigest® TspRI (TscAI) produces DNA

fragments with a 9-base 3’-extension. This may result in atypical DNA band patterns.

• FastDigest® TspRI (TscAI) may remain associated with the cleaved DNA. This may cause DNA band shifting during electropho-resis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.

Digestion time with 1 µl of FastDigest® Tsp509I (TasI) bp from end of DNA required for complete digestion




5’...↓A A T T ...3’

3’... T T A A↑...5’





FastDigest® Tsp509I (TasI)

pBR3

22 D

NA, 1

.0%

aga

rose

8 cl

eava

ge s

ites

119

clea

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site

s58

cle

avag

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69


1



Digestion time with 1 µl of FastDigest® XbaI bp from end of DNA required for complete digestion




5’...T↓C T A G A...3’

3’...A G A T C↑T...5’



Formulation1 µl of enzyme (1 FDU) cleaves 1 µg of lambda DNA dam–/SmaI fragments in 5 min at 37°C in 1X FastDigest® Buffer.



FastDigest® XbaI

λ DN

A (d

am–),

0.7%

aga

rose

NoteFastDigest® XbaI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Digestion time with 1 µl of FastDigest® XhoI bp from end of DNA required for complete digestion




5’...C↓T C G A G...3’

3’...G A G C T↑C...5’






FastDigest® XhoI

λ DN

A, 0

.7%

aga

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1 cl

eava

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70

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Protocols and Recommendations

1.1. Fast DNA digestion1. Prepare the reaction mixture at room tem-

perature in the order indicated:

ComponentVolume

Plasmid DNA

PCR product

Genomic DNA

Water, nuclease-free* (#R0581)

15 µl 17 µl 30 µl

10X FastDigest® Buffer or 10X FastDigest® Green Buffer

2 µl 2 µl ** 5 µl

DNA* 2 µl (up to 1 µg)

10 µl (~0.2 µg)

10 µl (5 µg)

FastDigest® enzyme 1 µl 1 µl 5 µl

Total volume 20 µl 30 µl 50 µl

2. Mix gently and spin down.3. Incubate at 37°C in a heat block or water

thermostat for 5-15 min.***4. Inactivate the enzyme (optional).***Note* The volume of water should be corrected

to keep the indicated total reaction volume. The volume of DNA can be scaled up to 10 µl or down to 0.5 µl depending on the DNA concentration.

** Only 2 µl of 10X FastDigest® buffer or 10X FastDigest® Green Buffer is required for unpurified PCR product in a 30 µl reac-tion volume.

*** See the Certificate of Analysis or Table 1.3 (p.71) for enzyme and substrate specific incubation time, recommended tem-perature and enzyme inactivation conditions.

• For fast simultaneous plasmid vector linearization and dephosphorylation protocol see p.338.

1.2. Reaction set-up for diges-tion of multiple DNA samples1. Pipette 2 µl of each DNA* sample into

labeled tubes.2. Prepare a master mix for n+1 samples.

Example of master mix (for 10 samples of plasmid DNA):

Water, nuclease-free* (#R0581) (10 + 1) x 15 µl = 165 µl


(10 + 1) x 2 µl = 22 µl

FastDigest® enzyme (10 + 1) x 1 µl = 11 µl

3. Add 18 µl of master mix* into tubes contain-ing DNA.

Note* The volume of DNA can be scaled up to

10 µl or down to 0.5 µl depending on the DNA concentration. The volume of water and master mix should be corrected to keep the indicated total reaction volume.

1.3. Double and multiple diges-tion of DNA FastDigest® enzymes allow simultaneous digestion of DNA with two or more enzymes in one digestion reaction.• Use 1 µl of each enzyme and scale up the

reaction conditions appropriately.• The combined volume of all added enzymes

should not exceed 1/10 of the total reaction volume.

• If enzymes require different incubation tem-perature perform sequential DNA cleavage: complete the first digestion reaction at the lower temperature, add the second enzyme and increase the digestion temperature for the second enzyme cleavage.

1.4. Scaling up DNA digestion reaction

DNA 1 µg 2 µg 3 µg 4 µg 5 µg

FastDigest® enzyme 1 µl 2 µl 3 µl 4 µl 5 µl


2 µl 2 µl 3 µl 4 µl 5 µl

Total volume 20 µl 20 µl 30 µl 40 µl 50 µl

71

1



Reaction Conditions

Table 1.3. Reaction conditions for FastDigest® restriction enzymes.



Specificity 5’→3’

Reaction temp.

Digestion time with 1 µl of FastDigest® enzyme, min bp from end of

DNA required for complete

digestion

Thermalinactivation

Incubationtime

withoutstar

activity

Lambda DNA

1µg/20µl

Plasmid DNA

1µg/20µl

PCR product

~0.2µg/30µl

Genomic DNA

1µg/10µl

FastDigest® AatII GACGT↓C 37°C 15 20 15 15 5 80°C, 5 min 16 h

FastDigest® AccI (XmiI) GT↓MKAC 37°C 5 5 60 5 2 65°C, 5 min 16 h

FastDigest® Acc65I G↓GTACC 37°C 5 5 5 5 3 65°C, 5 min 16 h

FastDigest® AciI (SsiI) CCGC(-3/-1)↓ 37°C 5 5 5 5 2 65°C, 5 min 4 h

FastDigest® AclI (Psp1406I) AA↓CGTT 37°C 5 5 5 5 3 65°C, 5 min 16 h

FastDigest® AcuI (Eco57I)* CTGAAG(16/14)↓ 37°C 15 30 15 15 2 65°C, 5 min 16 h

FastDigest® AfeI (Eco47III) AGC↓GCT 37°C 5 5 5 5 4 65°C, 5 min 16 h

FastDigest® AflII (BspTI) C↓TTAAG 37°C 5 5 5 5 4 NO 16 h

FastDigest® AgeI (BshTI) A↓CCGGT 37°C 5 5 5 5 3 80°C, 5 min 16 h

FastDigest® AjuI* ↓(7/12)GAA(N)7TTGG(11/6)↓ 37°C 5 5 10 5 ND 65°C, 5 min 16 h

FastDigest® AleI (OliI) CACNN↓NNGTG 37°C 5 5 10 5 3 65°C, 5 min 6 h

FastDigest® AluI AG↓CT 37°C 15 15 15 15 4 65°C, 5 min 16 h

FastDigest® Alw21I GWGCW↓C 37°C 5 5 5 5 1 80°C, 20 min 16 h

FastDigest® Alw26I GTCTC(1/5)↓ 37°C 5 5 5 5 1 65°C, 5 min 16 h

FastDigest® AlwNI (CaiI) CAGNNN↓CTG 37°C 5 5 5 5 4 65°C, 10 min 16 h

FastDigest® ApaI GGGCC↓C 37°C 5 5 20 10 2 65°C, 5 min 16 h

FastDigest® ApaLI (Alw44I) G↓TGCAC 37°C 5 60 10 5 4 80°C, 5 min 16h

FastDigest® AscI (SgsI) GG↓CGCGCC 37°C 5 5 5 5 2 65°C, 20 min 16 h

FastDigest® AseI (VspI) AT↓TAAT 37°C 5 5 5 30 2 65°C, 5 min 16 h

FastDigest® AsiSI (SfaAI) GCGAT↓CGC 37°C no sites 5 60 5 5 80°C, 5 min 6 h

FastDigest® AvaI (Eco88I) C↓YCGRG 37°C 5 5 5 5 2 65°C, 5 min 16 h

FastDigest® AvaII (Eco47I) G↓GWCC 37°C 5 5 5 5 1 80°C, 20 min 16 h

FastDigest® AvrII (XmaJI) C↓CTAGG 37°C 5 10 5 10 2 NO 16 h

FastDigest® BamHI G↓GATCC 37°C 5 5 5 5 2 80°C, 5 min 1 h

FastDigest® BanI (BshNI) G↓GYRCC 37°C 5 60 5 15 2 65°C, 10 min 16 h

FastDigest® BbsI (BpiI) GAAGAC(2/6)↓ 37°C 5 5 5 5 1 65°C, 10 min 16 h

FastDigest® BbvI (Lsp1109I) GCAGC(8/12)↓ 37°C 5 5 5 10 2 65°C, 5 min 1 h

FastDigest® BclI T↓GATCA 37°C 5 5 15 5 3 80°C, 20 min 16 h

FastDigest® BfaI (FspBI) C↓TAG 37°C 5 15 5 20 3 80°C, 5 min 16 h

FastDigest® BglI GCCNNNN↓NGGC 37°C 5 5 5 5 3 65°C, 5 min 2 h

FastDigest® BglII A↓GATCT 37°C 5 20 30 20 3 NO 16 h

FastDigest® BlpI (Bpu1102I) GC↓TNAGC 37°C 5 5 5 5 2 80°C, 5 min 16 h

FastDigest® Bme1580I (BseSI) GKGCM↓C 37°C 5 5 5 30 3 80°C, 15 min 16 h

FastDigest® BmtI (BspOI) GCTAG↓C 37°C 5 5 15 5 3 80°C, 5min 1 h

FastDigest® BplI* ↓(8/13)GAG(N)5CTC(13/8)↓ 37°C 15 20 30 30 ND 65°C, 5 min 16 h

FastDigest® BpmI (GsuI) CTGGAG(16/14)↓ 30°C 15 30 15 15 2 65°C, 5 min 16 h

FastDigest® Bpu10I CCTNAGC(-5/-2)↓ 37°C 15 15 30 15 3 80°C, 5 min 1 h

FastDigest® BsaAI (Ppu21I) YAC↓GTR 37°C 5 5 5 5 1 65°C, 5 min 6 h

FastDigest® BsaBI (BseJI) GATNN↓NNATC 65°C 5 5 5 5 3 NO 1 h

FastDigest® BsaHI (Hin1I) GR↓CGYC 37°C 5 5 5 5 1 65°C, 5 min 16 h

FastDigest® BsaJI (BseDI) C↓CNNGG 37°C 5 5 5 5 3 80°C, 5 min 16 h

FastDigest® BseGI GGATG(2/0)↓ 37°C 5 5 5 5 2 80°C, 5 min 16 h

FastDigest® BseNI ACTGG(1/-1)↓ 65°C 5 5 5 5 2 80°C, 5 min 16 h

FastDigest® BseXI GCAGC(8/12)↓ 65°C 15 15 15 15 3 80°C, 15 min 16 h

FastDigest® Bsh1236I CG↓CG 37°C 5 5 5 5 1 80°C, 10 min 16 h

FastDigest® BsiEI (Bsh1285I) CGRY↓CG 37°C 15 15 15 15 3 80°C, 15 min 1 h

FastDigest® BsiWI (Pfl23II) C↓GTACG 37°C 15 15 15 15 3 65°C, 5 min 16 h

FastDigest® BslI (BseLI) CCNNNNN↓NNGG 37°C 5 5 5 5 2 NO 16 h

FastDigest® BsmBI (Esp3I)* CGTCTC(1/5)↓ 37°C 5 5 5 5 1 65°C, 10 min 6 h

FastDigest® BsmFI (FaqI)* GGGAC(10/14)↓ 37°C 15 15 15 15 3 65°C, 5 min 16 h

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Reaction temp.



digestion

Thermalinactivation

Incubationtime

withoutstar

activity

Lambda DNA

1µg/20µl

Plasmid DNA

1µg/20µl

PCR product

~0.2µg/30µl

Genomic DNA

1µg/10µlFastDigest® Bsp119I TT↓CGAA 37°C 5 5 5 5 1 80°C, 5 min 16 h

FastDigest® Bsp120I G↓GGCCC 37°C 5 5 15 5 3 80°C, 10 min 16 h

FastDigest® Bsp1286I (SduI) GDGCH↓C 37°C 5 5 5 5 2 80°C, 15 min 1 h

FastDigest® Bsp1407I T↓GTACA 37°C 5 5 5 5 3 80°C, 10 min 16 h

FastDigest® BspCNI (BseMII)* CTCAG(10/8)↓ 55°C 15 30 15 15 2 80°C, 5 min 16 h

FastDigest® BspHI (PagI) T↓CATGA 37°C 5 10 5 10 3 80°C, 5 min 0.5 h

FastDigest® BspMI (BveI)* ACCTGC(4/8)↓ 37°C 15 15 60 20 5 65°C, 5 min 16 h

FastDigest® BsrBI (MbiI) CCGCTC(-3/-3)↓ 37°C 5 5 5 5 3 65°C, 5 min 16 h

FastDigest® BsrDI (BseMI) GCAATG(2/0)↓ 55°C 15 15 15 15 3 80°C, 5 min 16 h

FastDigest® BsrFI (Cfr10I) R↓CCGGY 37°C 5 5 10 5 2 NO 2 h

FastDigest® BssHII (PteI) G↓CGCGC 37°C 5 5 5 5 3 80°C, 5 min 16 h

FastDigest® BstXI CCANNNNN↓NTGG 37°C 5 5 5 5 4 80°C, 5 min 0.5 h

FastDigest® BstZ17I (Bst1107I) GTA↓TAC 37°C 5 5 5 10 3 NO 6 h

FastDigest® Bsu36I (Eco81I) CC↓TNAGG 37°C 5 5 5 5 2 80°C, 10 min 16 h

FastDigest® ClaI (Bsu15I) AT↓CGAT 37°C 5 5 5 5 2 65°C, 15 min 16 h

FastDigest® Csp6I G↓TAC 37°C 5 5 5 5 2 80°C, 10 min 16 h

FastDigest® DdeI (HpyF3I) C↓TNAG 37°C 5 5 5 10 3 65°C, 5 min 16 h

FastDigest® DpnI Gm6A↓TC 37°C ND 5not

applicable10 1 80°C, 5 min 16 h

FastDigest® DraI TTT↓AAA 37°C 5 5 5 5 4 65°C, 5 min 16 h

FastDigest® DraIII (AdeI) CACNNN↓GTG 37°C 5 5 5 5 2 80°C, 5 min 6 h

FastDigest® DrdI (AasI) GACNNNN↓NNGTC 37°C 5 5 5 5 1 80°C, 10 min 1 h

FastDigest® EagI (Eco52I) C↓GGCCG 37°C 5 20 20 5 3 65°C, 5 min 16 h

FastDigest® Eam1105I GACNNN↓NNGTC 37°C 5 5 5 5 3 65°C, 5 min 16 h

FastDigest® EarI (Eam1104I) CTCTTC(1/4)↓ 37°C 5 5 5 5 3 80°C, 5 min 6 h

FastDigest® Ecl136II GAG↓CTC 37°C 5 5 5 5 1 65°C, 5 min 6 h

FastDigest® Eco31I GGTCTC(1/5)↓ 37°C 5 5 5 5 3 65°C, 5 min 16 h

FastDigest® Eco91I G↓GTNACC 37°C 5 5 5 5 2 65°C, 10 min 2 h

FastDigest® EcoNI (XagI) CCTNN↓NNNAGG 37°C 5 5 5 20 2 65°C, 5 min 16 h

FastDigest® EcoO109I RG↓GNCCY 37°C 5 10 15 5 2 65°C, 5 min 16 h

FastDigest® EcoRI G↓AATTC 37°C 5 5 20 5 2 80°C, 5 min 0.5 h

FastDigest® EcoRV (Eco32I) GAT↓ATC 37°C 5 5 5 5 2 NO 16 h

FastDigest® EheI GGC↓GCC 37°C 5 5 5 5 2 65°C, 5 min 6 h

FastDigest® Fnu4HI (SatI) GC↓NGC 37°C 5 5 10 5 3 65°C, 5 min 16 h

FastDigest® FokI GGATG(9/13)↓ 37°C 5 5 5 5 1 65°C, 5 min 1 h

FastDigest® FspI (NsbI) TGC↓GCA 37°C 5 5 5 5 3 65°C, 15 min 16 h

FastDigest® FspAI RTGC↓GCAY 37°C 5 5 5 5 4 65°C, 5 min 16 h

FastDigest® HaeII (BfoI) RGCGC↓Y 37°C 5 5 5 5 2 65°C, 10 min 1h

FastDigest® HaeIII (BsuRI) GG↓CC 37°C 5 5 5 5 3 NO 16 h

FastDigest® HgaI (CseI) GACGC(5/10)↓ 37°C 15 15 30 15 2 80°C, 5 min 16 h

FastDigest® HhaI GCG↓C 37°C 5 5 5 5 1 NO 16 h

FastDigest® HincII GTY↓RAC 37°C 5 5 5 10 1 65°C, 5 min 16 h

FastDigest® HindIII A↓AGCTT 37°C 5 5 20 10 3 80°C, 10 min 16 h

FastDigest® HinfI G↓ANTC 37°C 5 5 5 5 2 65°C, 20 min 16 h

FastDigest® HinP1I (Hin6I) G↓CGC 37°C 5 10 5 5 2 80°C, 10 min 16 h

FastDigest® HpaI (KspAI) GTT↓AAC 37°C 5 5 5 5 3 65°C, 20 min 0.5 h

FastDigest® HpaII C↓CGG 37°C 5 5 5 5 3 65°C, 5 min 16 h

FastDigest® Hpy8I GTN↓NAC 37°C 5 5 5 10 2 80°C, 5 min 16 h

FastDigest® HpyF10VI GCNNNNN↓NNGC 37°C 5 5 5 5 2 80°C, 5 min 16 h

FastDigest® KpnI GGTAC↓C 37°C 5 5 5 5 3 80°C, 5 min 16 h

FastDigest® Kpn2I T↓CCGGA 37°C 5 5 5 5 2 80°C, 5 min 16 h

FastDigest® MauBI CG↓CGCGCG 37°C no sites 5 10 5 5 65°C, 5 min 16 h

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Reaction temp.



digestion

Thermalinactivation

Incubationtime

withoutstar

activity

Lambda DNA

1µg/20µl

Plasmid DNA

1µg/20µl

PCR product

~0.2µg/30µl

Genomic DNA

1µg/10µlFastDigest® MboI ↓GATC 37°C 5 5 5 10 1 65°C, 15 min 16 h

FastDigest® MboII GAAGA(8/7)↓ 37°C 5 5 5 5 2 65°C, 5 min 16 h

FastDigest® MfeI (MunI) C↓AATTG 37°C 5 5 5 5 3 NO 16 h

FastDigest® MluI A↓CGCGT 37°C 5 5 5 15 3 80°C, 5 min 16 h

FastDigest® MlyI (SchI) GAGTC(5/5)↓ 37°C 5 5 30 5 2 80°C, 5 min 1 h

FastDigest® MnlI CCTC(7/6)↓ 37°C 5 5 5 5 2 65°C, 5 min 16 h

FastDigest® MreI CG↓CCGGCG 37°C no sites 5 5 5 3 80°C, 5 min 16 h

FastDigest® MscI (MlsI) TGG↓CCA 37°C 5 5 30 20 3 80°C, 20 min 16 h

FastDigest® MseI (SaqAI) T↓TAA 37°C 5 5 5 5 4 65°C, 5 min 16 h

FastDigest® MslI (RseI) CAYNN↓NNRTG 37°C 5 5 5 5 4 65°C, 20 min 16 h

FastDigest® MspI C↓CGG 37°C 5 5 5 5 3 NO 16 h

FastDigest® MssI GTTT↓AAAC 37°C 5 5 5 5 2 65°C, 10 min 16 h

FastDigest® MvaI CC↓WGG 37°C 5 5 5 10 4 NO 1 h

FastDigest® Mva1269I GAATGC(1/-1)↓ 37°C 5 5 5 5 3 65°C, 5 min 16 h

FastDigest® NaeI (PdiI) GCC↓GGC 37°C ND 5 5 5 3 65°C, 15 min 16 h

FastDigest® NciI (BcnI) CC↓SGG 37°C 5 5 5 5 3 80°C, 20 min 16 h

FastDigest® NcoI C↓CATGG 37°C 5 10 10 5 3 65°C, 15 min 16 h

FastDigest® NdeI CA↓TATG 37°C 5 5 60 30 3 65°C, 5 min 6 h

FastDigest® NheI G↓CTAGC 37°C 5 15 5 5 5 65°C, 5 min 6 h

FastDigest® NlaIII (Hin1II) CATG↓ 37°C 5 10 5 10 4 80°C, 5 min 16 h

FastDigest® NlaIV (BspLI) GGN↓NCC 37°C 5 5 5 5 2 65°C, 20 min 16 h

FastDigest® NmuCI ↓GTSAC 37°C 5 5 5 10 2 65°C, 5 min 16 h

FastDigest® NotI GC↓GGCCGC 37°C no sites 30 5 10 2 80°C, 5 min 16 h

FastDigest® NruI (RruI) TCG↓CGA 37°C 5 5 5 5 5 NO 4 h

FastDigest® NsiI (Mph1103I) ATGCA↓T 37°C 5 5 5 10 3 65°C, 15 min 6 h

FastDigest® NspI (XceI) RCATG↓Y 37°C 5 5 5 5 2 65°C, 5 min 16 h

FastDigest® PacI TTAAT↓TAA 37°C no sites 5 5 5 2 65°C, 10 min 16 h

FastDigest® PdmI GAANN↓NNTTC 37°C 5 5 5 5 2 65°C, 5 min 16 h

FastDigest® PflMI (Van91I) CCANNNN↓NTGG 37°C 5 5 5 5 3 65°C, 10 min 6 h

FastDigest® PfoI T↓CCNGGA 37°C 5 5 5 5 3 65°C, 5 min 16 h

FastDigest® PmlI (Eco72I) CAC↓GTG 37°C 5 5 5 5 2 80°C, 10 min 1 h

FastDigest® PpuMI (Psp5II) RG↓GWCCY 37°C 5 5 5 20 2 80°C, 5 min 1 h

FastDigest® PshAI (BoxI) GACNN↓NNGTC 37°C 5 5 5 5 3 80°C, 20 min 16 h

FastDigest® PsiI (AanI) TTA↓TAA 37°C 5 5 5 5 5 65°C, 5 min 16 h

FastDigest® PspFI CCCAGC(-1/-5)↓ 37°C 5 5 5 5 4 80°C, 5 min 16 h

FastDigest® PstI CTGCA↓G 37°C 5 5 30 5 3 NO 16 h

FastDigest® PsuI R↓GATCY 37°C 5 5 5 5 2 80°C, 5 min 16 h

FastDigest® PsyI GACN↓NNGTC 37°C 5 5 5 5 2 80°C, 5 min 4 h

FastDigest® PvuI CGAT↓CG 37°C 5 15 5 5 4 80°C, 5 min 16 h

FastDigest® PvuII CAG↓CTG 37°C 5 5 5 5 1 NO 0.5 h

FastDigest® RsaI GT↓AC 37°C 5 5 5 5 3 NO 16 h

FastDigest® RsrII (CpoI) CG↓GWCCG 37°C 5 5 5 5 1 65°C, 5 min 16 h

FastDigest® SacI GAGCT↓C 37°C 5 15 30 10 1 65°C, 5 min 16 h

FastDigest® SalI G↓TCGAC 37°C 5 5 60 5 3 65°C, 10 min 16 h

FastDigest® SanDI (KflI) GG↓GWCCC 37°C 5 5 5 10 5 NO 16 h

FastDigest® SapI (LguI) GCTCTTC(1/4)↓ 37°C 5 5 5 5 2 65°C, 5 min 16 h

FastDigest® Sau3AI (Bsp143I) ↓GATC 37°C 5 5 10 5 4 65°C, 20 min 16 h

FastDigest® Sau96I (Cfr13I) G↓GNCC 37°C 5 5 5 5 2 NO 16 h

FastDigest® SbfI (SdaI) CCTGCA↓GG 37°C 5 5 5 5 3 NO 1 h

FastDigest® ScaI AGT↓ACT 37°C 5 5 5 5 4 65°C, 10 min 16 h

FastDigest® ScrFI (Bme1390I) CC↓NGG 37°C 5 5 5 5 4 NO 16 h

FastDigest® SexAI (CsiI) A↓CCWGGT 37°C 5 10 5 15 4 65°C, 5 min 16 h

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Note* – for digestion with FastDigest® BsmBI (Esp3I) add 1mM DTT. for digestion with FastDigest® AcuI (Eco57I), FastDigest® AjuI and FastDigest® BspCNI (BseMII) add 0.01 mM SAM. for digestion with FastDigest® BplI and FastDigest® BsmFI (FaqI) add 0.05 mM SAM. for digestion with FastDigest® BspMI (BveI) add 0.5 µM oligonucleotide.NO – no thermal inactivation. Spin column purification or phenol/chloroform extraction and ethanol precipitation of DNA is recommended.

ND – not determined.




Reaction temp.



digestion

Thermalinactivation

Incubationtime

withoutstar

activity

Lambda DNA

1µg/20µl

Plasmid DNA

1µg/20µl

PCR product

~0.2µg/30µl

Genomic DNA

1µg/10µlFastDigest® SfaNI (BmsI) GCATC(5/9)↓ 37°C 5 5 5 5 1 65°C, 5 min 16 h

FastDigest® SfcI (BfmI) C↓TRYAG 37°C 5 5 5 5 2 65°C, 20 min 16 h

FastDigest® SfiI GGCCNNNN↓NGGCC 50°C no sites 15 15 15 5 NO 16 h

FastDigest® SmaI CCC↓GGG 37°C 5 5 5 5 1 65°C, 5 min 16 h

FastDigest® SnaBI (Eco105I) TAC↓GTA 37°C 5 5 5 5 3 65°C, 5 min 16 h

FastDigest® SpeI (BcuI) A↓CTAGT 37°C no sites 5 5 5 1 NO 16 h

FastDigest® SphI (PaeI) GCATG↓C 37°C 5 5 5 5 5 65°C, 5 min 16 h

FastDigest® SspI AAT↓ATT 37°C 5 5 5 30 2 65°C, 5 min 1 h

FastDigest® StuI (Eco147I) AGG↓CCT 37°C 5 5 5 5 3 80°C, 10 min 16 h

FastDigest® StyI (Eco130I) C↓CWWGG 37°C 5 5 20 20 3 65°C, 5 min 16 h

FastDigest® SwaI (SmiI) ATTT↓AAAT 37°C no sites 20 30 30 2 65°C, 15 min 1 h

FastDigest® TaaI ACN↓GT 65°C 5 5 5 5 3 NO 16 h

FastDigest® TaiI ACGT↓ 65°C 5 5 5 5 2 NO 6 h

FastDigest® TaqI T↓CGA 65°C 5 5 5 5 4 NO 16 h

FastDigest® TatI W↓GTACW 65°C 15 15 15 20 5 NO 1 h

FastDigest® TauI GCSG↓C 55°C 15 60 15 30 5 NO 16 h

FastDigest® TfiI (PfeI) G↓AWTC 37°C 5 5 10 5 2 65°C, 5 min 16 h

FastDigest® Tru1I T↓TAA 65°C 5 5 5 10 3 NO 2 h

FastDigest® Tsp509I (TasI) ↓AATT 65°C 5 5 5 5 3 NO 6 h

FastDigest® TspRI (TscAI) CASTG(2/-7)↓ 65°C 5 5 5 5 2 NO 6 h

FastDigest® XapI R↓AATTY 37°C 5 5 5 10 4 80°C, 5 min 6 h

FastDigest® XbaI T↓CTAGA 37°C 5 5 5 10 2 65°C, 20 min 16 h

FastDigest® XhoI C↓TCGAG 37°C 5 5 5 10 2 80°C, 5 min 16 h

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1. CONVENTIONAL RESTRICTION ENZYMES


Activity and Quality Control Assays

Activity AssayOne unit is the amount of enzyme required to hydrolyze 1 µg of substrate DNA in 60 min in 50 µl of reaction mixture under the recom-mended conditions. To determine restriction enzyme activity, concentrated enzymes are first diluted to approximately 0,5-1 units/µl with an enzyme dilution buffer (20 mM potassium phosphate (pH 7.4), 200 mM KCl, 1 mM EDTA, 7 mM 2-mercaptoethanol, 10% glycerol and 0.2 mg/ml BSA) and then used to cleave DNA.In general, enzymes are assayed with λ phage DNA at 37°C. However, some exceptions apply:• Restriction enzymes that show optimum

activity at temperatures other than 37°C are assayed under their optimal temperature.

• Restriction enzymes without recognition sites on λ DNA are assayed with another specific DNA substrate.

• Restriction enzymes with only a few recognition sites on the λ DNA are assayed using λ DNA hydrolyzed with another restriction enzyme.

• Restriction enzymes sensitive to Dam or Dcm methylation are assayed with λ DNA (dam–, dcm–).

Quality Control

Labeled Oligonucleotide (LO) TestThe labelled oligonucleotides are incubated with an excess of restriction enzyme, separated on a polyacrylamide gel under denaturing conditions and analyzed by phospho-imaging. The restric-tion enzyme passes this quality control test if there is no degradation of labelled oligonucle-otides and no decrease in specific radioactivity (see Fig. 1.1 on p.2).

Non-specific Nuclease and Cross-contamination AssayVarying amounts of restriction enzyme (2-20 units) are incubated for 16 hours with 1 µg of substrate DNA under the recommended assay conditions. After electrophoretic separation of the DNA fragments, the characteristic banding patterns are examined for alterations. To pass the test, the restriction enzyme must yield an unaltered banding pattern under conditions of up to 160-fold overdigestion (10 units x 16 hours). For information regarding restriction enzyme star activity, see the product description or Certificate of Analysis supplied with each enzyme.

Ligation and Recleavage AssayThe ligation and re-cleavage assay tests the integrity of DNA ends. DNA fragments obtained after 2-, 10- and 50-fold overdigestion (units/µg of DNA x hours) are ligated with T4 DNA ligase and then recut with the same restriction enzyme. Only DNA fragments with intact 5’- and 3’-termini are ligated by T4 DNA ligase, and only molecules with reconstructed recognition sites can then be cleaved by the same restric-tion enzyme. A restriction enzyme conforms to the quality criterion if the ligation efficiency of DNA fragments generated by digestion with the restriction enzyme doesn’t depend on the fold of DNA overdigestion with the assayed enzyme.The percentage of DNA that can be success-fully ligated and then re-cleaved is presented for each restriction enzyme both in its catalog description and the Certificate of Analysis sup-plied with each enzyme.

Fermentas conventional restriction endonucleases are classic restriction enzymes, which normally digest DNA in one hour and each require a specific reaction buffer for 100% activity.

Storage and ShippingAll conventional restriction enzymes should be stored at -20°C. During shipment on dry ice, enzymes may freeze. This does not affect their quality – all Fermentas enzymes are 100% active after at least three freeze-thaw cycles. For 24-48 hour delivery, enzymes may be shipped on blue ice since their quality is not affected by short exposure to 4°C.

Conventional Restriction Enzymes

Blue/White (B/W) Cloning AssayThe Blue/White cloning assay is designed to test the integrity of DNA ends. pUC57 DNA is digested at unique sites within the lacZ reporter gene with 10 units of a restriction enzyme in its optimal buffer. After a 16 hour incubation, the plasmid DNA is recircularized by ligation and transformed into E.coli XL1-Blue competent cells. The cells are then plated onto X-Gal/IPTG/Amp agar. An intact lacZ gene will produce a blue colony. If the termini of the linearized pUC57 are altered by contaminating exodeoxy-nucleases, the lacZ reading frame is inter-rupted, which results in the appearance of white colonies. The higher the ratio of blue:white colonies, the higher the quality of the restriction enzyme. An enzyme conforms to this quality criterion if the number of white colonies does not exceed 3%. Details of the assay are given in the Certificate of Analysis of each product. The test is applicable for enzymes recognizing unique sites within the lacZ reporter gene, and for those lacking recognition sites in pUC57. In the latter case, the assay is performed with the mixture of pUC57/HindIII, pUC57/PstI and pUC57/Eco32I DNA fragments representing three different types of termini (3’-overhang, 5’-overhang and blunt ends).

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Product List and Cross-reference to FastDigest® Restriction Enzymes

Table 1.4. Fermentas conventional restriction enzymes.

Conventional restriction enzyme



AanI (PsiI) FastDigest® PsiI (AanI) PsiI TTA↓TAA ER2061 80

AarI AarI CACCTGC(4/8)↓ ER1581/2 80AasI (DrdI) FastDigest® DrdI (AasI) DrdI GACNNNN↓NNGTC ER1721 80

AatII FastDigest® AatII AatII GACGT↓C ER0991/2 81

Acc65I (Asp718I) FastDigest® Acc65I KpnI (GGTAC↓C) G↓GTACC ER0901/2 81

AdeI (DraIII ) FastDigest® DraIII (AdeI) DraIII CACNNN↓GTG ER1231 82

AjiI (BmgBI) BtrI CAC↓GTC ER1941 82AjuI FastDigest® AjuI AjuI ↓(7/12)GAA(N)7TTGG(11/6)↓ ER1951 83

AlfI AlfI ↓(10/12)GCA(N)6TGC(12/10)↓ ER1801 83AloI AloI ↓(7/12-13)GAAC(N)6TCC(12-13/7)↓ ER1491 84AluI FastDigest® AluI AluI AG↓CT ER0011/2 84

Alw21I (BsiHKAI) FastDigest® Alw21I HgiAI GWGCW↓C ER0021 84

Alw26I (BsmAI) FastDigest® Alw26I BsmAI GTCTC(1/5)↓ ER0031 85

Alw44I (ApaLI) FastDigest® ApaLI (Alw44I) ApaLI G↓TGCAC ER0041 85

ApaI FastDigest® ApaI ApaI GGGCC↓C ER1411/5 85

BamHI FastDigest® BamHI BamHI G↓GATCC ER0051/2/3/5 86

BauI (BssSI) BsiI CACGAG(-5/-1)↓ ER1841 87BclI FastDigest® BclI BclI T↓GATCA ER0721/2/5 87

BcnI (NciI) FastDigest® NciI (BcnI) CauII CC↓SGG ER0061 88

BcuI (SpeI) FastDigest® SpeI (BcuI) SpeI A↓CTAGT ER1251/2 88

BdaI BdaI ↓(10/12)TGA(N)6TCA(12/10)↓ ER1961 88BfiI (BmrI) BfiI ACTGGG(5/4)↓ ER1591 89

BfmI (SfcI) FastDigest® SfcI (BfmI) SfeI C↓TRYAG ER1161/2 89

BfuI (BciVI) BciVI GTATCC(6/5)↓ ER1501 90BglI FastDigest® BglI BglI GCCNNNN↓NGGC ER0071/2 90

BglII FastDigest® BglII BglII A↓GATCT ER0081/2 90

Bme1390I (ScrFI) FastDigest® ScrFI (Bme1390I) ScrFI CC↓NGG ER1421/2 91

BoxI (PshAI) FastDigest® PshAI (BoxI) PshAI GACNN↓NNGTC ER1431 91

BpiI (BbsI) FastDigest® BbsI (BpiI) BbvII GAAGAC(2/6)↓ ER1011/2 92

BplI FastDigest® BpII BplI ↓(8/13)GAG(N)5CTC(13/8)↓ ER1311/2 92

Bpu10I FastDigest® Bpu10l Bpu10I CCTNAGC(-5/-2)↓ ER1181 93

Bpu1102I (BlpI) FastDigest® BlpI (Bpu1102I) EspI GC↓TNAGC ER0091/2 93

BseDI (BsaJI) FastDigest® BsaJI (BseDI) SecI C↓CNNGG ER1081/2 94

BseGI (BtsCI) FastDigest® BseGI FokI (GGATG(9/13)↓) GGATG(2/0)↓ ER0871/2 94

BseJI (BsaBI) FastDigest® BsaBI (BseJI) BsaBI GATNN↓NNATC ER1711 94

BseLI (BslI) FastDigest® BslI (BseLI) BsiYI CCNNNNN↓NNGG ER1201 95

BseMI (BsrDI) FastDigest® BsrDI (BseMI) BsrDI GCAATG(2/0)↓ ER1261/2 95

BseMII (BspCNI) FastDigest® BspCNI (BseMII) BseMII CTCAG(10/8)↓ ER1401 95

BseNI (BsrI) FastDigest® BseNI BsrI ACTGG(1/-1)↓ ER0881/2 96

BseSI (Bme1580I) FastDigest® Bme1580I (BseSI) BseSI GKGCM↓C ER1441 96

BseXI (BbvI) FastDigest® BseXI BbvI GCAGC(8/12)↓ ER1451/2 96

Bsh1236I (BstUI) FastDigest® Bsh1236I FnuDII CG↓CG ER0921/2 97

Bsh1285I (BsiEI) FastDigest® BsiEI (Bsh1285I) McrI CGRY↓CG ER0891 97

BshNI (BanI) FastDigest® BanI (BshNI) HgiCI G↓GYRCC ER1001 97

BshTI (AgeI) FastDigest® AgeI (BshTI) AgeI A↓CCGGT ER1461/2 98

Bsp68I (NruI) FastDigest® NruI (RruI) NruI TCG↓CGA ER0111 98

Bsp119I (BstBI) FastDigest® Bsp119I AsuII TT↓CGAA ER0121 99

Bsp120I (PspOMI) FastDigest® Bsp120I ApaI (GGGCC↓C) G↓GGCCC ER0131 99

Bsp143I (Sau3AI) FastDigest® Sau3AI (Bsp143I) MboI ↓GATC ER0781/2 99

Bsp1407I (BsrGI) FastDigest® Bsp1407I Bsp1407I T↓GTACA ER0931/2 100


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BspLI (NlaIV) FastDigest® NlaIV (BspLI) NlaIV GGN↓NCC ER1151/2 100

BspOI (BmtI) FastDigest® BmtI (BspOI) NheI (G↓CTAGC) GCTAG↓C ER2041 100

BspPI (AlwI) BinI GGATC(4/5)↓ ER1321/2 101BspTI (AflII) FastDigest® AflII (BspTI) AflII C↓TTAAG ER0831 101

Bst1107I (BstZ17I) FastDigest® BstZ17I (Bst1107I) SnaI GTA↓TAC ER0701 102

BstXI FastDigest® BstXI BstXI CCANNNNN↓NTGG ER1021/2 102

Bsu15I (ClaI) FastDigest® ClaI (Bsu15I) ClaI AT↓CGAT ER0141/2/5 103

BsuRI (HaeIII) FastDigest® HaeIII (BsuRI) HaeIII GG↓CC ER0151 103

BveI (BspMI) FastDigest® BspMI (BveI) BspMI ACCTGC(4/8)↓ ER1741 104

CaiI (AlwNI) FastDigest® AlwNI (CaiI) AlwNI CAGNNN↓CTG ER1391 104

CfrI (EaeI) CfrI Y↓GGCCR ER0161 105Cfr9I (XmaI) SmaI (CCC↓GGG) C↓CCGGG ER0171/2 105Cfr10I (BsrFI) FastDigest® BsrFI (Cfr10I) Cfr10I R↓CCGGY ER0181 105

Cfr13I (Sau96I) FastDigest® Sau96I (Cfr13I) AsuI G↓GNCC ER0191 106

Cfr42I (SacII) SacII CCGC↓GG ER0201/2/5 106CpoI (RsrII) FastDigest® RsrII (CpoI) RsrII CG↓GWCCG ER0741/2 106

CseI (HgaI) FastDigest® HgaI (CseI) HgaI GACGC(5/10)↓ ER1901 107

Csp6I (CviQI) FastDigest® Csp6I RsaI (GT↓AC) G↓TAC ER0211 107

DpnI FastDigest® DpnI DpnI Gm6A↓TC ER1701/2/5 108

DraI FastDigest® DraI AhaIII TTT↓AAA ER0221/3 108

Eam1104I (EarI) FastDigest® EarI (Eam1104I) Ksp632I CTCTTC(1/4)↓ ER0231/2 109

Eam1105I (AhdI) FastDigest® Eam1105I Eam1105I GACNNN↓NNGTC ER0241 109

Ecl136II (EcoICRI) FastDigest® Ecl136II SacI (GAGCT↓C) GAG↓CTC ER0251 109

Eco24I (BanII) HgiJII GRGCY↓C ER0281 110Eco31I (BsaI) FastDigest® Eco31I Eco31I GGTCTC(1/5)↓ ER0291/2 110

Eco32I (EcoRV) FastDigest® EcoRV (Eco32I) EcoRV GAT↓ATC ER0301/2/3/5 110

Eco47I (AvaII) FastDigest® AvaII (Eco47I) AvaII G↓GWCC ER0311/2 111

Eco47III (AfeI) FastDigest® AfeI (Eco47III) Eco47III AGC↓GCT ER0321/2 111

Eco52I (EagI) FastDigest® EagI (Eco52I) XmaIII C↓GGCCG ER0331/2 111

Eco57I (AcuI) FastDigest® AcuI (Eco57I) Eco57I CTGAAG(16/14)↓ ER0341/2 112

Eco57MI Eco57MI CTGRAG(16/14)↓ ER1671 112Eco72I (PmlI) FastDigest® PmlI (Eco72I) PmaCI CAC↓GTG ER0361 113

Eco81I (Bsu36I) FastDigest® Bsu36I (Eco81I) SauI CC↓TNAGG ER0371/2 113

Eco88I (AvaI) FastDigest® AvaI (Eco88I) AvaI C↓YCGRG ER0381 113

Eco91I (BstEII) FastDigest® Eco91I BstEII G↓GTNACC ER0391/2 114

Eco105I (SnaBI) FastDigest® SnaBI (Eco105I) SnaBI TAC↓GTA ER0401/2 114

Eco130I (StyI) FastDigest® StyI (Eco130I) StyI C↓CWWGG ER0411 114

Eco147I (StuI) FastDigest® StuI (Eco147I) StuI AGG↓CCT ER0421/2 115

EcoO109I (DraII) FastDigest® EcoO109I DraII RG↓GNCCY ER0261 115

EcoRI FastDigest® EcoRI EcoRI G↓AATTC ER0271/2/3/5 116

EcoRII EcoRII ↓CCWGG ER1921 116EheI (SfoI) FastDigest® EheI NarI (GG↓CGCC) GGC↓GCC ER0441 117

Esp3I (BsmBI) FastDigest® BsmBI (Esp3I) Esp3I CGTCTC(1/5)↓ ER0451/2 117

FaqI (BsmFI) FastDigest® BsmFI (FaqI) FinI GGGAC(10/14)↓ ER1811 118

FspAI FastDigest® FspAI FspAI RTGC↓GCAY ER1661/2 118

FspBI (BfaI) FastDigest® BfaI (FspBI) MaeI C↓TAG ER1761/2 119

GsuI (BpmI) FastDigest® BpmI (GsuI) GsuI CTGGAG(16/14)↓ ER0461/2 119

HhaI FastDigest® HhaI HhaI GCG↓C ER1851 120

Hin1I (BsaHI) FastDigest® BsaHI (Hin1I) AcyI GR↓CGYC ER0471 120

Hin1II (NlaIII ) FastDigest® NlaIII (Hin1II) NlaIII CATG↓ ER1831 120

Hin4I Hin4I ↓(8/13-14)GAY(N)5VTC(13-14/8)↓ ER1601/2 121Hin6I (HinP1I) FastDigest® HinP1I (Hin6I) HhaI (GCG↓C) G↓CGC ER0481 121


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HincII (HindII) FastDigest® HincII HindII GTY↓RAC ER0491/2 121

HindIII FastDigest® HindIII HindIII A↓AGCTT ER0501/2/3/5 122

HinfI FastDigest® HinfI HinfI G↓ANTC ER0801/2/3 122

HpaII FastDigest® HpaII HpaII C↓CGG ER0511/2 122

HphI HphI GGTGA(8/7)↓ ER1101/2 123Hpy8I (MjaIV) FastDigest® Hpy8I MjaIV GTN↓NAC ER1571/2 123

HpyF3I (DdeI) FastDigest® DdeI (HpyF3I) DdeI C↓TNAG ER1881/2 123

HpyF10VI (MwoI) FastDigest® HpyF10VI MwoI GCNNNNN↓NNGC ER1731/2 124

KpnI FastDigest® KpnI KpnI GGTAC↓C ER0521/2/3 124

Kpn2I (BspEI) FastDigest® Kpn2I BspMII T↓CCGGA ER0531/2 124

KspAI (HpaI) FastDigest® HpaI (KspAI) HpaI GTT↓AAC ER1031/2 125

LguI (SapI) FastDigest® SapI (LguI) SapI GCTCTTC(1/4)↓ ER1931/2 125

Lsp1109I (BbvI) FastDigest® BbvI (Lsp1109I) BbvI GCAGC(8/12)↓ ER2071 125

LweI (SfaNI) FastDigest® SfaNI (BmsI) SfaNI GCATC(5/9)↓ ER1621/2 126

MauBI FastDigest® MauBI MauBI CG↓CGCGCG ER2081 126

MbiI (BsrBI) FastDigest® BsrBI (MbiI) BsrBI CCGCTC(-3/-3)↓ ER1271 126

MboI FastDigest® MboI MboI ↓GATC ER0811/2 127

MboII FastDigest® MboII MboII GAAGA(8/7)↓ ER0821/2 127

MlsI (MscI) FastDigest® MscI (MlsI) BalI TGG↓CCA ER1211/2 128

MluI FastDigest® MluI MluI A↓CGCGT ER0561/2 128

MnlI FastDigest® MnlI MnlI CCTC(7/6)↓ ER1071/2 128

Mph1103I (NsiI) FastDigest® NsiI (Mph1103I) AvaIII ATGCA↓T ER0731/2 129

MreI (Sse232I) FastDigest® MreI Sse232I CG↓CCGGCG ER2021 129

MspI (HpaII) FastDigest® MspI HpaII C↓CGG ER0541/2 129

MssI (PmeI) FastDigest® MssI PmeI GTTT↓AAAC ER1341/2 130

MunI (MfeI) FastDigest® MfeI (MunI) MfeI C↓AATTG ER0751/2 130

MvaI (BstNI) FastDigest® MvaI EcoRII (↓CCWGG) CC↓WGG ER0551 130

Mva1269I (BsmI) FastDigest® Mva1269I BsmI GAATGC(1/-1)↓ ER0961/2 131

NcoI FastDigest® NcoI NcoI C↓CATGG ER0571/2/5 131

NdeI FastDigest® NdeI NdeI CA↓TATG ER0581/2/5 132

NheI FastDigest® NheI NheI G↓CTAGC ER0971/2/5 132

NmuCI (Tsp45I) FastDigest® NmuCI Tsp45I ↓GTSAC ER1511 133

NotI FastDigest® NotI NotI GC↓GGCCGC ER0591/2/3/5 133

NsbI (FspI) FastDigest® FspI (NsbI) MstI TGC↓GCA ER1221 133

OliI (AleI) FastDigest® AleI (OliI) OliI CACNN↓NNGTG ER1631/2 134

PacI FastDigest® PacI PacI TTAAT↓TAA ER2201 134

PaeI (SphI) FastDigest® SphI (PaeI) SphI GCATG↓C ER0601/2 134

PagI (BspHI) FastDigest® BspHI (PagI) BspHI T↓CATGA ER1281/2 135

PasI PasI CC↓CWGGG ER1861 135PauI (BssHII) FastDigest® BssHII (PteI) BsePI G↓CGCGC ER1091/2 135

PdiI (NaeI) FastDigest® NaeI (PdiI) NaeI GCC↓GGC ER1521/2 136

PdmI (XmnI) FastDigest® PdmI XmnI GAANN↓NNTTC ER1531/2 136

PfeI (TfiI) FastDigest® TfiI (PfeI) TfiI G↓AWTC ER1781 136

Pfl23II (BsiWI) FastDigest® BsiWI (Pfl23II) SplI C↓GTACG ER0851 137

PfoI FastDigest® PfoI PfoI T↓CCNGGA ER1751 137

PpiI PpiI ↓(7/12)GAAC(N)5CTC(13/8)↓ ER1541 138Ppu21I (BsaAI) FastDigest® BsaAI (Ppu21I) BsaAI YAC↓GTR ER1971 138

PscI (PciI) BspLU11I A↓CATGT ER1871/2 138Psp5II (PpuMI) FastDigest® PpuMI (Psp5II) PpuMI RG↓GWCCY ER0761 139

Psp1406I (AclI) FastDigest® AclI (Psp1406I) AclI AA↓CGTT ER0941/2 139

PstI FastDigest® PstI PstI CTGCA↓G ER0611/2/5 140

PsuI (BstYI) FastDigest® PsuI XhoII R↓GATCY ER1551 140

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PsyI (Tth111I) FastDigest® PsyI Tth111I GACN↓NNGTC ER1331 140

PvuI FastDigest® PvuI PvuI CGAT↓CG ER0621/2 141

PvuII FastDigest® PvuII PvuII CAG↓CTG ER0631/3/5 141

RsaI FastDigest® RsaI RsaI GT↓AC ER1121/2 141

RseI (MslI) FastDigest® MslI (RseI) MslI CAYNN↓NNRTG ER2001/2 142

SacI FastDigest® SacI SacI GAGCT↓C ER1131/2/5 142

SalI FastDigest® SalI SalI G↓TCGAC ER0641/2/5 143

SatI (Fnu4HI) FastDigest® Fnu4HI (SatI) Fnu4HI GC↓NGC ER1641/2 143

ScaI FastDigest® ScaI ScaI AGT↓ACT ER0431/2 144

SchI (MlyI) FastDigest® MlyI (SchI) PleI (GAGTC(4/5)↓) GAGTC(5/5)↓ ER1371 144

SdaI (SbfI) FastDigest® SbfI (SdaI) Sse8387I CCTGCA↓GG ER1191/2 144

SduI (Bsp1286I) FastDigest® Bsp1286I (SduI) SduI GDGCH↓C ER0651 145

SfaAI (AsiSI) FastDigest® AsiSI (SfaAI) SgfI GCGAT↓CGC ER2091 145

SfiI FastDigest® SfiI SfiI GGCCNNNN↓NGGCC ER1821 146

SgeI SgeI m5CNNG(9/13)↓ ER2211 146SgrDI SgrDI CG↓TCGACG ER2031 147SgsI (AscI) FastDigest® AscI (SgsI) AscI GG↓CGCGCC ER1891/2 147

SmaI FastDigest® SmaI SmaI CCC↓GGG ER0661/2/3/5 147

SmiI (SwaI) FastDigest® SwaI (SmiI) SwaI ATTT↓AAAT ER1241 148

SmoI (SmlI) SmlI C↓TYRAG ER1981 148SmuI (FauI) FauI CCCGC(4/6)↓ ER1691 148SsiI (AciI) FastDigest® AciI (SsiI) AciI CCGC(-3/-1)↓ ER1791 149

SspI FastDigest® SspI SspI AAT↓ATT ER0771/2 149

SspDI (KasI) NarI (GG↓CGCC) G↓GCGCC ER2191 150TaaI (HpyCH4III) FastDigest® TaaI Tsp4CI ACN↓GT ER1361/2 150

TaiI (MaeII) FastDigest® TaiI MaeII (A↓CGT) ACGT↓ ER1141/2 150

TaqI FastDigest® TaqI TaqI T↓CGA ER0671/2 151

TasI (Tsp509I) FastDigest® Tsp509I (TasI) TspEI ↓AATT ER1351/2 151

TatI FastDigest® TatI TatI W↓GTACW ER1291 151

TauI FastDigest® TauI TauI GCSG↓C ER1651/2 152

Tru1I (MseI) FastDigest® Tru1I MseI T↓TAA ER0981/2/3 152

TscAI (TspRI) FastDigest® TspRI (TscAI) TspRI CASTG(2/-7)↓ ER2101 153

TsoI TsoI TARCCA(11/9)↓ ER1991 153TstI TstI ↓(8/13)CAC(N)6TCC(12/7)↓ ER1911 154Van91I (PflMI) FastDigest® PflMI (Van91I) PflMI CCANNNN↓NTGG ER0711/2 154

VspI (AseI) FastDigest® AseI (VspI) VspI AT↓TAAT ER0911/2 154

XagI (EcoNI) FastDigest® EcoNI (XagI) EcoNI CCTNN↓NNNAGG ER1301 155

XapI (ApoI) FastDigest® XapI ApoI R↓AATTY ER1381 155

XbaI FastDigest® XbaI XbaI T↓CTAGA ER0681/2/3/5 155

XceI (NspI) FastDigest® NspI (XceI) NspI RCATG↓Y ER1471/2 156

XhoI FastDigest® XhoI XhoI C↓TCGAG ER0691/2/3/5 156

XmaJI (AvrII) FastDigest® AvrII (XmaJI) AvrII C↓CTAGG ER1561/2 156

XmiI (AccI) FastDigest® AccI (XmiI) AccI GT↓MKAC ER1481/2 157

Nicking enzymes

Nb.Bpu10I Nb.Bpu10ICCTNA GC GGANT↑CG

ER1681 157

Nt.Bpu10I Nt.Bpu10ICC↓TNAGCGG ANTCG

ER2011 158

Nb.Mva1269I Nb.BsmIGAATG CCTTAC↑G

ER2051 158

Homing enzyme

I-SceI I-SceITAGGG ATAA↓CAGGGTAATATCCC↑TATT GTCCCATTA

ER1771 159

Alphabetic list of commercially available restriction enzymes see p.207.

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Product Description

5’...T T A↓T A A ...3’

3’...A A T↑A T T ...5’

#ER2061 200 uSupplied with:10X Buffer Tango™ 1 ml

Also available asFastDigest® PsiI (AanI)

Concentration10 u/µl

Conditions for 100% Activity 1X Buffer Tango™ at 37°C.

Storage BufferAanI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with AanI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.


Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.

AanI (PsiI) B G O R Tango 2X Tango 50-100 50-100 0-20 0-20 100 20-50

Activity in Five Buffer System, %

5’...C A C C T G C (N)4↓ ...3’

3’...G T G G A C G (N)8↑ ...5’

#ER1581 25 uSupplied with:10X Buffer AarI 1 ml10X Buffer Tango™ 1 ml50X oligonucleotide 25µl

#ER1582 125 uSupplied with:10X Buffer AarI 1 ml10X Buffer Tango™ 1 ml50X oligonucleotide 2x25 µl


Conditions for 100% Activity 1X Buffer AarI at 37°C.Oligonucleotide 0.5 µM.

Storage BufferAarI is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with AarI, more than 95% of the DNA fragments can be ligated and recut.


Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for digestion of 1 µg of agarose-embedded λ DNA in 16 hours.

AarI B G O R Tango 2X Tango NR NR 0-20 0-20 NR 50-100


Note• For cleavage with AarI at least two copies of

its recognition sequence are required.• Inclusion of 0.5 µM oligonucleotide with the

AarI recognition sequence in the reaction mixture significantly improves cleavage of

DNAs, especially of those with a single AarI site. Still, a complete cleavage of some substrates with AarI is difficult to achieve.

• Greater than 10-fold overdigestion with AarI may result in star activity.

• AarI may remain associated with the cleaved

DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

5’...G A C N N N N↓N N G T C ...3’

3’...C T G N N↑N N N N C A G ...5’

#ER1721 300 uSupplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® DrdI (AasI)


Conditions for 100% Activity 1X Buffer B at 37°C.

Storage BufferAasI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with AasI, more than 95% of the DNA fragments can be ligated and recut.


Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1µg of agarose-embed-ded λ DNA in 16 hours.

NoteGreater than 10-fold overdigestion with AasI may result in star activity.

* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).

AasI (DrdI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 20-50 0-20 0-20 50-100* 0-20

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5’...G↓ G T A C C...3’

3’...C C A T G↑ G...5’

#ER0901 1000 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml

#ER0902 5000 uSupplied with:10X Buffer O 2x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® Acc65I


Conditions for 100% Activity 1X Buffer O at 37°C.

Storage BufferAcc65I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Acc65I, approximately 95% of the DNA fragments can be ligated and recut.



Acc65I (Asp718I) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 20-50 20-50 50-100

NoteAcc65I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

AccI Fermentas enzymes FastDigest® AccI (XmiI) and XmiI (AccI)

AccII Fermentas enzymes FastDigest® Bsh1236I and Bsh1236I (BstUI)

AccIII Fermentas enzymes FastDigest® Kpn2I and Kpn2I (BspEI) (different sensitivity to methylation)

AccB7I Fermentas enzymes FastDigest® PflMI (Van91I) and Van91I (PflMI)

AciI Fermentas enzymes FastDigest® AciI (SsiI) and SsiI (AciI)

AclI Fermentas enzymes FastDigest® AcII (Psp1406I) and Psp1406I (AclI)

AcsI Fermentas enzymes FastDigest® XapI and XapI (ApoI)

AcuI Fermentas enzymes FastDigest® AcuI (Eco57I) and Eco57I (AcuI)

AcyI Fermentas enzymes FastDigest® BsaHI (Hin1I) and Hin1I (BsaHI)

5’...G A C G T↓ C...3’

3’...C↑ T G C A G...5’

#ER0991 300 u#ER0992 1500 u

Both supplied with:10X Buffer Tango™ 1 ml

Also available asFastDigest® AatII



Storage BufferAatII is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with AatII, more than 95% of the DNA fragments can be ligated and recut.



NoteActivity decreases if reaction buffer pH is not between 7.5 and 8.0 at 25°C.

AatII Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 100 20-50

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AdeI (DraIII) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 100 20-50 100 100* 20-50* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).

5’...C A C N N N↓G T G...3’

3’...G T G↑N N N C A C...5’

#ER1231 500 uSupplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® DraIII (AdeI)


Conditions for 100% Activity 1X Buffer G at 37°C.

Storage BufferAdeI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with AdeI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.

Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI may overlap – effect not determined.

Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1µg of agarose-embedded λ DNA in 16 hours.

NoteGreater than 20-fold overdigestion with AdeI may result in star activity.

5’...C A C↓G T C...3’

3’...G T G↑C A G...5’

#ER1941 200 uSupplied with:10X Buffer AjiI 1 ml10X Buffer Tango™ 1 ml


Conditions for 100% Activity 1X Buffer AjiI at 37°C.

Storage BufferAjiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with AjiI, approxi-mately 80% of the DNA fragments can be ligated. No more than 50% of these can be recut due to the asymmetric recognition sequence of AjiI. The remaining uncleaved ligation products may be cut by AatII and Eco72I (PmlI).



AjiI (BmgBI) B G O R Tango 2X Tango NR NR 20-50* NR NR 20-50** Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).


NoteLow salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.

AfaI Fermentas enzymes FastDigest® Csp6I and Csp6I (CviQI) (different cleavage position); FastDigest® RsaI and RsaI

AfeI Fermentas enzymes FastDigest® AfeI (Eco47III) and Eco47III (AfeI)

AflII Fermentas enzymes FastDigest® AflII (BspTI) and BspTI (AflII)

AgeI Fermentas enzymes FastDigest® AgeI (BshTI) and BshTI (AgeI)

AhaIII Fermentas enzymes FastDigest® DraI and DraI

AhdI Fermentas enzymes FastDigest® Eam1105I and Eam1105I (AhdI)

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AleI Fermentas enzymes FastDigest® AleI (OliI) and OliI (AleI)

5’...↓10(N)GCA(N)6TGC(N)12↓ ...3’

3’...↑12(N)CGT(N)6ACG(N)10↑...5’

#ER1801 50 uSupplied with:10X Buffer R 1 ml50X SAM 0.1 ml10X BufferTango™ 1 ml


Conditions for 100% Activity1X Buffer R at 37°C.SAM 0.01 mM.

Storage BufferAlfI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 20-fold overdigestion with AlfI, approximately 70% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.



AlfI Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM

0-20 0-20 0-20 100 0-20 20-50

Note• AlfI requires S-adenosylmethionine for activity.• Complete cleavage of some substrates with

AlfI is difficult to achieve.• AlfI concentration is determined by the

maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.

5’...↓ 7(N)GAA(N)7TTGG(N)11 ↓ ...3’

3’...↑12(N)CTT(N)7AACC(N)6 ↑...5’

#ER1951 100 uSupplied with:10X Buffer R 1 ml50X SAM 0.1 ml10X BufferTango™ 1 ml

Also available asFastDigest® AjuI


Conditions for 100% Activity1X Buffer R at 37°C.SAM 0.01 mM.

Storage BufferAjuI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with AjuI, more than 90% of the DNA fragments can be ligated, but none of these can be recut due to the methyla-tion of the recognition sequence by this enzyme.



AjuI Activity in Five Buffer System, %

Note• AjuI requires S-adenosylmethionine for activity.• Complete cleavage of some substrates with

AjuI is difficult to achieve.• AjuI concentration is determined by the


• AjuI produces double-strand cuts on both sides of the interrupted recognition site. In certain sequence contexts, the cleavage position may be shifted by one base pair. However, the cleavage position indicated above will predominate.

B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM

0-20 50-100 20-50 100 50-100 50-100

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AlwI Fermentas enzyme BspPI (AlwI)

5’...↓ 7(N)GAAC(N)6TCC(N)12-13↓...3’

3’...↑12-13(N)CTTG(N)6AGG(N)7 ↑...5’

#ER1491 100 uSupplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml


Conditions for 100% Activity1X Buffer R at 30°C.

Storage BufferAloI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with AloI, approxi-mately 70% of the DNA fragments can be ligated and more than 80% of these can be recut.

Methylation EffectsDam: may overlap – effect not determined.Dcm, EcoKI, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).

AloI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 100 20-50 100

Note• Incubation at 37°C results in 20% activity.• AloI produces double-strand cuts on both

sides from the interrupted recognition site. Its unique feature is a degenerate cleavage point on the 3’ side of the recognition sequence (12 or 13 nt away).

• The presence of S-adenosylmethionine in the reaction mixture results in incomplete cleavage with AloI.

• Greater than 10-fold overdigestion with AloI may result in star activity.

• AloI may remain associated with the cleaved DNA. This may cause DNA band shifting

during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

5’...G W G C W↓C...3’

3’...C↑W C G W G...5’


Also available asFastDigest® Alw21I



Storage BufferAlw21I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Alw21I, more than 95% of the DNA fragments can be ligated and recut.


Alw21I (BsiHKAI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 20-50 50-100

5’...A G↓C T...3’

3’...T C↑G A...5’


#ER0012 3000 uSupplied with:10X Buffer Tango™ 2x1 ml

Also available asFastDigest® AluI


Conditions for 100% Activity1X Buffer Tango™ at 37°C.

Storage BufferAluI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with AluI, approxi-mately 95% of the DNA fragments can be ligated and recut.


AluI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 0-20 0-20 0-20 100 20-50

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5’...G T C T C(N)1↓...3’

3’...C A G A G(N)5↑...5’


Also available asFastDigest® Alw26I



Storage BufferAlw26I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Alw26I, more than 95% of the DNA fragments can be ligated and recut.


Alw26I (BsmAI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 0-20 0-20 100 100

5’...G↓T G C A C...3’

3’...C A C G T↑G...5’


Also available asFastDigest® ApaLI (Alw44I)



Storage BufferAlw44I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Alw44I, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.



Alw44I (ApaLI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 0-20 50-100 100 50-100

ApaI Activity in Five Buffer System, % B G O R Tango 2X Tango 100 20-50 0-20 0-20 20-50 0-20

5’...G G G C C↓C...3’

3’...C↑C C G G G...5’

#ER1411 3000 u#ER1415 5000 u

Both supplied with:10X Buffer B 2x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® ApaI



Storage Buffer ApaI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with ApaI, more than 95% of the DNA fragments can be ligated and recut.



Note• Incubation at 30°C results in a 2-fold

increase in activity.• ApaI is inhibited by salt concentrations

above 50 mM.• ApaI cleavage is impaired by overlapping

dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

AlwNI Fermentas enzymes FastDigest® AIwNI (CaiI) and CaiI (AlwNI)

Aor13HI Fermentas enzymes FastDigest® Kpn2I and Kpn2I (BspEI)

Aor51HI Fermentas enzymes FastDigest® AfeI (Eco47III) and Eco47III (AfeI)

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BamHI Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50* 100 20-50 50-100* 100* 50-100* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).

5’...G↓G A T C C...3’

3’...C C T A G↑G...5’

#ER0051 4000 uSupplied with:10X Buffer BamHI 2x1 ml10X Buffer Tango™ 1 ml

#ER0055 10000 uSupplied with:10X Buffer BamHI 4x1 ml10X Buffer Tango™ 1 ml

#ER0052 5x4000 u#ER0053 HC, 20000 u

Both supplied with:10X Buffer BamHI 4x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® BamHI

Concentration10 u/µl50 u/µl, HC

Conditions for 100% Activity 1X Buffer BamHI at 37°C.

Storage BufferBamHI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BamHI, more than 95% of the DNA fragments can be ligated and recut.




ApaLI Fermentas enzymes FastDigest® ApaLI (Alw44I) and Alw44I (ApaLI) (different sensitivity to methylation)

ApoI Fermentas enzymes FastDigest® XapI and XapI (ApoI)

AscI Fermentas enzymes FastDigest® AscI (SgsI) and SgsI (AscI)

AseI Fermentas enzymes FastDigest® AseI (VspI) and VspI (AseI)

AsiSI Fermentas enzymes FastDigest® AsiSI (SfaAI) and SfaAI (AsiSI)

AspI Fermentas enzymes FastDigest® PsyI and PsyI (Tth111I)

Asp700I Fermentas enzymes FastDigest® PdmI and PdmI (XmnI)

Asp718I Fermentas enzymes FastDigest® Acc65I and Acc65I (Asp718I); FastDigest® KpnI and KpnI (different cleavage position and different sensitivity to methylation)

AspEI Fermentas enzymes FastDigest® Eam1105I and Eam1105I (AhdI)

AspHI Fermentas enzymes FastDigest® Alw21I and Alw21I (BsiHKAI)

AsuI Fermentas enzymes FastDigest® Sau96I (Cfr13I) and Cfr13I (Sau96I)

AsuII Fermentas enzymes FastDigest® Bsp119I and Bsp119I (BstBI)

AvaI Fermentas enzymes FastDigest® AvaI (Eco88I) and Eco88I (AvaI)

AvaII Fermentas enzymes FastDigest® AvaII (Eco47I) and Eco47I (AvaII)

AvaIII Fermentas enzymes FastDigest® NsiI (Mph1103I) and Mph1103I (NsiI)

AviII Fermentas enzymes FastDigest® FspI (NsbI) and NsbI (FspI)

AvrII Fermentas enzymes FastDigest® AvrII (XmaJI) and XmaJI (AvrII)

BaeGI Fermentas enzymes FastDigest® Bme1580I (BseSI) and BseSI (Bme1580I)

BalI Fermentas enzymes FastDigest® MscI (MlsI) and MlsI (MscI)

BanI Fermentas enzymes FastDigest® BanI (BshNI) and BshNI (BanI)

BanII Fermentas enzyme Eco24I (BanII)

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5’...C↓A C G A G...3’

3’...G T G C T↑C...5’




Storage BufferBauI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BauI, more than 95% of the DNA fragments can be ligated and recut.



BauI (BssSI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 0-20 50-100 100 50-100

BclI Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 20-50 20-50 100* 100* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).

5’...T↓G A T C A...3’

3’...A C T A G↑T...5’


#ER0725 3000 u#ER0722 5000 u

Both supplied with:10X Buffer G 2x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® BclI



Storage BufferBclI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with BclI, more than 95% of the DNA fragments can be ligated and recut.


Note• Incubation at 37°C results in 50% activity.• Greater than 15-fold overdigestion with BclI

may result in star activity.• BclI is blocked by dam methylation. To avoid

dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

BbeI Fermentas enzymes FastDigest® EheI and EheI (SfoI) (different cleavage position); Fermentas enzyme SspDI (KasI) (different cleavage position)

BbrPI Fermentas enzymes FastDigest® PmlI (Eco72I) and Eco72I (PmlI)

BbsI Fermentas enzymes FastDigest® BbsI (BpiI) and BpiI (BbsI)

BbuI Fermentas enzymes FastDigest® SphI (PaeI) and PaeI (SphI)

BbvI Fermentas enzymes FastDigest® BbvI (Lsp1109I) and Lsp1109I (BbvI); FastDigest® BseXI and BseXI (BbvI)

BbvII Fermentas enzymes FastDigest® BbsI (BpiI) and BpiI (BbsI)

BciVI Fermentas enzyme BfuI (BciVI)

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5’...C C↓S G G...3’

3’...G G S↑C C...5’


Also available asFastDigest® NciI (BcnI)



Storage BufferBcnI is supplied in: 10 mM potassium phosphate (pH 7.5 at 25°C), 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with BcnI, more than 80% and more than 95% of these can be recut.


BcnI (NciI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 50-100 50-100 100 50-100

5’...A↓C T A G T...3’

3’...T G A T C↑A...5’

#ER1251 400 u#ER1252 2000 u


Also available asFastDigest® SpeI (BcuI)



Storage Buffer BcuI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BcuI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.


Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded Adenovirus-2 DNA in 16 hours.

BcuI (SpeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 0-20

5’...↓10(N)TGA(N)6TCA(N)12↓ ...3’

3’...↑12(N)ACT(N)6AGT(N)10↑...5’

#ER1961 50 uSupplied with:10X Buffer G 1 ml50X SAM 0.1 ml10X BufferTango™ 1 ml


Conditions for 100% Activity1X Buffer G at 30°C.SAM 0.05 mM.

Storage BufferBdaI is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with BdaI, more than 70% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.


BdaI Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM

NR 100 0-20 20-50 50-100* 50-100* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).

Note• Incubation at 37°C results in 30% activity.• Requires S-adenosylmethionine for activity.

Complete cleavage of some substrates with BdaI is difficult to achieve.

• BdaI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.

• BdaI produces double-strand cuts on both sides of the interrupted recognition site. In certain sequence contexts, the cleavage position may be shifted by one base pair. However, the cleavage position indicated above will predominate.

• Greater than 40-fold overdigestion with BdaI may result in star activity.

• BdaI may remain associated with the cleaved

DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample prepara-tion or heat the digested DNA in the presence of SDS prior to electrophoresis.

• BdaI is blocked by overlapping dam methy-lation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

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5’...C↓T R Y A G...3’

3’...G A Y R T↑C...5’

#ER1161 200 u#ER1162 1000 u


Also available asFastDigest® SfcI (BfmI)



Storage Buffer BfmI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BfmI, more than 95% of the DNA fragments can be ligated and recut.


BfmI (SfcI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 0-20 0-20 100 20-50


BfaI Fermentas enzymes FastDigest® BfaI (FspBI) and FspBI (BfaI)

5’...A C T G G G(N)5↓...3’

3’...T G A C C C(N)4↑...5’

#ER1591 50 uSupplied with:10X Buffer Tango™ 1 ml3X BfiI Stop Solution 0.5 ml



BfiI (BmrI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 0-20 0-20 100 0-20

Note• BfiI restriction enzyme cleaves DNA specifically

both in the presence and absence of Mg2+ ions in the reaction mixture (Sapranauskas, R., et al., J.Biol.Chem., 275, 30878-30885, 2000).

• Chelating of Mg2+ ions by EDTA does not inhibit BfiI activity and may cause non-specific products. The non-specific cleavage increases at temperatures over 37°C.

• Greater than 40-fold overdigestion with BfiI may result in star activity.

Termination of Digestion ReactionDo not attempt to stop the digestion reaction by adding EDTA (see Note). Use Stop Solution containing SDS or the supplied BfiI Stop Solu-tion to terminate the reaction by incubating at 65°C for 10 min. If the digested DNA is to be used in down-stream manipulations, inactivate BfiI by incubating at 65°C for 20 min.

3X BfiI Stop Solution (supplied)0.6% SDS, 0.05% bromophenol blue and 50% glycerol.

Storage BufferBfiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with BfiI, more than 40% of the DNA fragments can be ligated and more than 90% of these can be recut.


BfrI Fermentas enzymes FastDigest® AflII (BspTI) and BspTI (AflII)

BfrBI Fermentas enzymes FastDigest® NsiI (Mph1103I) and Mph1103I (NsiI) (different cleavage position)

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5’...G T A T C C(N)6↓...3’

3’...C A T A G G(N)5↑...5’

#ER1501 100 uSupplied with:10X Buffer BfuI 1 ml10X Buffer Tango™ 1 ml


Conditions for 100% Activity 1X Buffer BfuI at 37°C.

Storage Buffer BfuI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 300 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 5-fold overdigestion with BfuI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.

Methylation EffectsDam, Dcm, EcoKI, EcoBI, CpG – no effect.


BfuI (BciVI) B G O R Tango 2X Tango NR NR 0-20 0-20 NR 50-100** Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).


NoteGreater than 5-fold overdigestion with BfuI may result in star activity.

BfuAI Fermentas enzymes FastDigest® BspMI (BveI) and BveI (BspMI)

BfuCI Fermentas enzymes FastDigest® Sau3AI (Bsp143I) and Bsp143I (Sau3AI); FastDigest® DpnI and DpnI (different cleavage position and different sensitivity to methylation); FastDigest® MboI and MboI (different sensitivity to methylation)

5’...G C C N N N N↓N G G C...3’

3’...C G G N↑N N N N C C G...5’


#ER0072 5x2000 uSupplied with:10X Buffer O 2x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® BglI



Storage BufferBglI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BglI, more than 95% of the DNA fragments can be ligated and recut.



BglI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 100 100 0-20 100

5’...A↓G A T C T...3’

3’...T C T A G↑A...5’

#ER0081 500 u#ER0082 2500 u

Both supplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® BglII


Conditions for 100% Activity1X Buffer O at 37°C.

Storage BufferBglII is supplied in: 10 mM Tris-HCl (pH 8.2 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BglII, more than 95% of the DNA fragments can be ligated and recut.



BglII Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 0-20 100

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5’...G A C N N↓N N G T C...3’

3’...C T G N N↑N N C A G...5’


Also available asFastDigest® PshAI (BoxI)



Storage BufferBoxI is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BoxI, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.


Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded λ DNA in 16 hours.

BoxI (PshAI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 20-50 100 20-50

NoteIncubation at 30°C results in a 2.5-fold increase in activity.

5’...C C↓N G G...3’

3’...G G N↑C C...5’

#ER1421 500 u#ER1422 2500 u


Also available asFastDigest® ScrFI (Bme1390I)



Storage BufferBme1390I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Bme1390I, more than 80% of the DNA fragments can be ligate-dand more than 90% of these can be recut.Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm, CpG may overlap – blocked (p.177, 179).

Bme1390I (ScrFI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 100 50-100 50-100 50-100

NoteBme1390I is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

BinI Fermentas enzyme BspPI (AlwI)

BlnI Fermentas enzymes FastDigest® AvrII (XmaJI) and XmaJI (AvrII)

BlpI Fermentas enzymes FastDigest® BlpI (Bpu1102I) and Bpu1102I (BlpI)

Bme1580I Fermentas enzymes FastDigest® Bme1580I (BseSI) and BseSI (Bme1580I)

BmgBI Fermentas enzyme AjiI (BmgBI)

BmrI Fermentas enzyme BfiI (BmrI)

BmtI Fermentas enzymes FastDigest® BmtI (BspOI) and BspOI (BmtI); FastDigest® NheI and NheI (different cleavage position)

BmyI Fermentas enzymes FastDigest® Bsp1286I (SduI) and SduI (Bsp1286I)

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BpiI (BbsI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 50-100 50-100 50-100 50-100

5’...G A A G A C(N)2↓...3’

3’...C T T C T G(N)6↑...5’

#ER1011 200 u#ER1012 1000 u

Both supplied with:10X Buffer G 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® BbsI (BpiI)



Storage BufferBpiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BpiI, more than 95% of the DNA fragments can be ligated and recut.



BpmI Fermentas enzymes FastDigest® BpmI (GsuI) and GsuI (BpmI)

5’...↓ 8(N)G A G(N)5C T C(N)13↓...3’

3’...↑13(N)C T C(N)5G A G(N) 8↑...5’

#ER1311 100 uSupplied with:10X Buffer Tango™ 1 ml50X SAM 0.1 ml

#ER1312 500 uSupplied with:10X Buffer Tango™ 1 ml50X SAM 2x0.1 ml

Also available asFastDigest® BpII


Conditions for 100% Activity 1X Buffer Tango™ at 37°C.SAM 0.05 mM.

Storage BufferBplI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with BplI, more than 70% of the DNA fragments can be ligated, but none of these can be recut due to the methyla-tion of the recognition sequence by this enzyme.



Note• BplI requires only Mg2+ for its activity, but

is stimulated by S-adenosylmethionine. 0.05 mM S-adenosylmethionine gives more than a 100-fold increase in BplI activity. Still, complete cleavage of some substrates with BplI is difficult to achieve.

• BplI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.

BplI Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM

0-20 20-50 0-20 0-20 100 20-50

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BpuAI Fermentas enzymes FastDigest® BbsI (BpiI) and BpiI (BbsI)

BsaI Fermentas enzymes FastDigest® Eco31I and Eco31I (BsaI)

BsaAI Fermentas enzymes FastDigest® BsaAI (Ppu21I) and Ppu21I (BsaAI)

BsaBI Fermentas enzymes FastDigest® BsaBI (BseJI) and BseJI (BsaBI)

BsaHI Fermentas enzymes FastDigest® BsaHI (Hin1I) and Hin1I (BsaHI)

BsaJI Fermentas enzymes FastDigest® BsaJI (BseDI) and BseDI (BsaJI)

BsaMI Fermentas enzymes FastDigest® Mva1269I and Mva1269I (BsmI)

BseAI Fermentas enzymes FastDigest® Kpn2I and Kpn2I (BspEI)

5’...G C↓T N A G C...3’

3’...C G A N T↑C G...5’

#ER0091 200 u#ER0092 1000 u


Also available asFastDigest® BlpI (Bpu1102I)



Storage BufferBpu1102I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Bpu1102I, more than 80% of the DNA fragments can be ligated. More than 95% of these can be recut.



Bpu1102I (BlpI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 20-50 100 20-50

5’...C C↓T N A G C...3’

3’...G G A N T↑C G...5’

#ER1181 200 uSupplied with:10X Buffer Bpu10I 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® Bpu10I


Conditions for 100% Activity 1X Buffer Bpu10I at 37°C.

Storage BufferBpu10I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Bpu10I, more than 90% of the DNA fragments can be ligated. No more than 50% of these can be recut due to asymmetric recognition sequence of Bpu10I.


Note• For cleavage with Bpu10I at least two copies

of its recognition sequence are required.• A large excess of enzyme (>4 u/1 µg DNA)

may result in incomplete DNA cleavage. There-fore, we recommend increasing the incubation time instead of using an excess of Bpu10I.

• Low salt, high glycerol (>5%) concentra-tions, pH >7.0 or a large excess of enzyme may result in star activity.

Bpu10I Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50* 50-100* 100* 50-100* 100** Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).

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BseDI (BsaJI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 100 50-100

5’...C↓C N N G G...3’

3’...G G N N C↑C...5’

#ER1081 300 u#ER1082 1500 u


Also available asFastDigest® BsaJI (BseDI)



Storage Buffer BseDI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BseDI, more than 95% of the DNA fragments can be ligated and recut.


NoteIncubation at 37°C results in 10% activity.

5’...G G A T G N N↓...3’

3’...C C T A C↑N N ...5’

#ER0871 500 u#ER0872 2500 u


Also available asFastDigest® BseGI



Storage Buffer BseGI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BseGI, more than 95% of the DNA fragments can be ligated and recut.


BseGI (BtsCI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 20-50 20-50 100 20-50


5’...G A T N N↓N N A T C...3’

3’...C T A N N↑N N T A G...5’


Also available asFastDigest® BsaBI (BseJI)



Storage Buffer BseJI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with BseJI, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam: may overlap – blocked (p.176).Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.

Note• Incubation at 37°C results in less than 10%

activity.• Greater than 20-fold overdigestion with

BseJI may result in star activity.• BseJI is blocked by overlapping dam

methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

BseJI (BsaBI) Activity in Five Buffer System, % B G O R Tango 2X Tango NR 100* 100 NR NR 100** Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).

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BseLI (BslI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 50-100 20-50 100 50-100

5’...C C N N N N N↓N N G G...3’

3’...G G N N↑N N N N N C C...5’


Also available asFastDigest® BslI (BseLI)



Storage BufferBseLI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BseLI, more than 95% of the DNA fragments can be ligated and recut.


Note• Incubation at 37°C results in 40% activity.• BseLI cleavage is impaired by overlapping dcm

methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

BseMI (BsrDI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 0-20 100 50-100 50-100

5’...G C A A T G N N↓...3’

3’...C G T T A C↑N N ...5’

#ER1261 100 u#ER1262 500 u

Both supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® BsrDI (BseMI)


Conditions for 100% Activity 1X Buffer R at 55°C.

Storage BufferBseMI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BseMI, more than 95% of the DNA fragments can be ligated and approximately 80% of these can be recut.



5’...C T C A G(N)10↓...3’

3’...G A G T C(N) 8↑...5’


Also available asFastDigest® BspCNI (BseMII)



Storage BufferBseMII is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with BseMII, more than 90% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme (see Note).


Note• Incubation at 37°C results in 30% activity.• Requires S-adenosylmethionine for activity.

Sinefungin can replace SAM in the restriction reaction. In this case DNA is not methylated and more than 95% of the ligated BseMII fragments can be recut by this enzyme.

BseMII (BspCNI) Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM

50-100 50-100 50-100 50-100 100 50-100

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BseNI (BsrI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 20-50 0-20 0-20 50-100 20-50

5’...A C T G G N↓...3’

3’...T G A C↑C N ...5’


#ER0882 5000 uSupplied with:10X Buffer B 2x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® BseNI



Storage Buffer BseNI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BseNI, more than 95% of the DNA fragments can be ligated and recut.


NoteIncubation at 37°C results in less than 10% activity.

BsePI Fermentas enzymes FastDigest® BssHII (PteI) and PauI (BssHII)

BseSI (Bme1580I) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 0-20 20-50 50-100 0-20

5’...G K G C M↓C...3’

3’...C↑M C G K G...5’


Also available asFastDigest® Bme1580I (BseSI)



Storage BufferBseSI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BseSI, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam, Dcm, CpG, EcoBI – no effect.EcoKI: may overlap – cleavage impaired (p.181).


Note• Incubation at 37°C results in 20% activity.• Low salt, high glycerol (>5%) concentra-

tions, pH >8.0 or a large excess of enzyme may result in star activity.

5’...G C A G C(N) 8↓...3’

3’...C G T C G(N)12↑...5’

#ER1451 100 u#ER1452 500 u

Both supplied with:10X Buffer BseXI 1 ml

Also available asFastDigest® BseXI


Conditions for 100% Activity 1X Buffer BseXI at 65°C.

Storage BufferBseXI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with BseXI, more than 95% of the DNA fragments can be ligated recut.


Note• Incubation at 37°C results in 10% activity.• Greater than 40-fold overdigestion with

BseXI may result in star activity.• BseXI may remain associated with the cleaved


BseXI (BbvI) Activity in Five Buffer System, % B G O R Tango 2X Tango NR NR NR NR NR NR

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BshNI (BanI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 0-20 100

5’...G↓G Y R C C...3’

3’...C C R Y G↑G...5’


Also available asFastDigest® BanI (BshNI)



Storage BufferBshNI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BshNI, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam, EcoBI – no effect.Dcm, CpG: may overlap – cleavage impaired (p.177, 179).EcoKI: may overlap – effect not determined.


NoteBshNI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

5’...C G↓C G...3’

3’...G C↑G C...5’

#ER0921 500 u#ER0922 2500 u


Also available asFastDigest® Bsh1236I



Storage BufferBsh1236I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Bsh1236I, more than 95% of the DNA fragments can be ligated and recut.


Bsh1236I (BstUI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 50-100 100 20-50 50-100

BseYI Fermentas enzyme FastDigest® PspFI (different cleavage position)

5’...C G R Y↓C G...3’

3’...G C↑Y R G C...5’


Also available asFastDigest® BsiEI (Bsh1285I)



Storage BufferBsh1285I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 150 mM KCl, 1 mM DTT, 1 mM EDTA, 0.15% Triton X-100, 0.5 mg ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Bsh1285I, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam: may overlap – cleavage impaired (p.176).Dcm, EcoKI, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).


Bsh1285I (BsiEI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 20-50 0-20 0-20 20-50

NoteBsh1285I cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

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BshTI (AgeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 20-50 20-50

5’...A↓C C G G T...3’

3’...T G G C C↑A...5’

#ER1461 200 u#ER1462 1000 u


Also available asFastDigest® AgeI (BshTI)



Storage BufferBshTI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BshTI, more than 95% of the DNA fragments can be ligated and recut.




BsiEI Fermentas enzymes FastDigest® BsiEI (Bsh1285I) and Bsh1285I (BsiEI)

BsiHKAI Fermentas enzymes FastDigest® Alw21I and Alw21I (BsiHKAI)

BsiWI Fermentas enzymes FastDigest® BsiWI (Pfl23II) and Pfl23II (BsiWI)

BsiYI Fermentas enzymes FastDigest® BslI (BseLI) and BseLI (BslI)

BslI Fermentas enzymes FastDigest® BslI (BseLI) and BseLI (BslI)

BsmI Fermentas enzymes FastDigest® Mva1269I and Mva1269I (BsmI)

BsmAI Fermentas enzymes FastDigest® Alw26I and Alw26I (BsmAI)

BsmBI Fermentas enzymes FastDigest® BsmBI (Esp3I) and Esp3I (BsmBI)

BsmFI Fermentas enzymes FastDigest® BsmFI (FaqI) and FaqI (BsmFI)

BsoBI Fermentas enzymes FastDigest® AvaI (Eco88I) and Eco88I (AvaI) (different sensitivity to methylation)

5’...T C G↓C G A...3’

3’...A G C↑G C T...5’


Also available asFastDigest® NruI (RruI)



Storage BufferBsp68I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with Bsp68I, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.



NoteGreater than 15-fold overdigestion with Bsp68I may result in star activity.

Bsp68I (NruI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 20-50 50-100

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Bsp1286I Fermentas enzymes FastDigest® Bsp1286I (SduI) and SduI (Bsp1286I)

5’...T T↓C G A A...3’

3’...A A G C↑T T...5’


Also available asFastDigest® Bsp119I



Storage BufferBsp119I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Bsp119I, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam, Dcm, EcoBI – no effect.CpG: completely overlaps – blocked (p.178).EcoKI: may overlap – effect not determined.


Bsp119I (BstBI) B G O R Tango 2X Tango 20-50 0-20 0-20 0-20 100 100


Bsp120I (PspOMI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 20-50 0-20 20-50 50-100 0-20

5’...G↓G G C C C...3’

3’...C C C G G↑G...5’






Ligation and RecleavageAfter 50-fold overdigestion with Bsp120I, more than 95% of the DNA fragments can be ligated and recut.



NoteBsp120I is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

5’...↓G A T C ...3’

3’... C T A G↑...5’

#ER0781 300 u#ER0782 1500 u

Both supplied with:10X Buffer Bsp143I 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® Sau3Al (Bsp143I)


Conditions for 100% Activity1X Buffer Bsp143I at 37°C.


Ligation and RecleavageAfter 50-fold overdigestion with Bsp143I more than 95% of the DNA fragments can be ligated and recut.


NoteDpnI, Bsp143I and MboI all recognize the same sequence but have different methylation sensitivities (pp.175-181) and cleavage sites.

Bsp143I (Sau3AI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 0-20 0-20 50-100 20-50

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5’...T↓G T A C A...3’

3’...A C A T G↑T...5’

#ER0931 300 u#ER0932 1500 u






Ligation and RecleavageAfter 50-fold overdigestion with Bsp1407I more than 95% of the DNA fragments can be ligated and recut.



Bsp1407I (BsrGI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 0-20 20-50 100 50-100

BspCNI Fermentas enzymes FastDigest® BspCNI (BseMII) and BseMII (BspCNI)

BspDI Fermentas enzymes FastDigest® ClaI (Bsu15I) and Bsu15I (ClaI)

BspEI Fermentas enzymes FastDigest® Kpn2I and Kpn2I (BspEI)

BspHI Fermentas enzymes FastDigest® BspHI (PagI) and PagI (BspHI)

BspLI (NlaIV) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 20-50

5’...G G N↓N C C...3’

3’...C C N↑N G G...5’

#ER1151 200 u#ER1152 1000 u


Also available asFastDigest® NlaIV (BspLI)



Storage BufferBspLI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BspLI, approxi-mately 90% of the DNA fragments can be ligated and more than 95% of these can be recut.


NoteBspLI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

BspLU11I Fermentas enzyme PscI (PciI)

BspMI Fermentas enzymes FastDigest® BspMI (BveI) and BveI (BspMI)

BspMII Fermentas enzymes FastDigest® Kpn2I and Kpn2I (BspEI)

5’...G C T A G↓C...3’

3’...C↑G A T C G...5’


Also available asFastDigest® BmtI (BspOI)



Storage BufferBspOI is supplied in: 10 mM Tris-HCl (pH 8.2 at 25°C), 500 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.15% Triton X-100, 0.5 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BspOI, more than 95% of the DNA fragments can be ligated and recut.


Digestion of Agarose-embedded DNAMinimum 20 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded lambda DNA in 16 hours.

BspOI (BmtI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 100 0-20 20-50

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5’...G G A T C(N)4↓...3’

3’...C C T A G(N)5↑...5’

#ER1321 100 u#ER1322 500 u




Storage BufferBspPI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with BspPI, approxi-mately 70% of the DNA fragments can be ligated and more than 50% of these can be recut.

Methylation EffectsDam: completely overlaps – blocked (p.176).Dcm, CpG, EcoKI, EcoBI – no effect.

BspPI (AlwI) Activity in Five Buffer System, %Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 0-20 0-20 100 0-20

Note• Incubation at 37°C results in 30% activity.• BspPI is blocked by overlapping dam methy-

lation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

5’...C↓T T A A G...3’

3’...G A A T T↑C...5’


Also available asFastDigest® AflII (BspTI)



Storage BufferBspTI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BspTI, more than 95% of the DNA fragments can be ligated and more than 95% of these can be recut.



BspTI (AflII) B G O R Tango 2X Tango 0-20 0-20 100 20-50 0-20 50-100


BspQI Fermentas enzymes FastDigest® SapI (LguI) and LguI (SapI)

BspT107I Fermentas enzymes FastDigest® BanI (BshNI) and BshNI (BanI)

BsrI Fermentas enzymes FastDigest® BseNI and BseNI (BsrI)

BsrBI Fermentas enzymes FastDigest® BsrBI (MbiI) and MbiI (BsrBI)

BsrDI Fermentas enzymes FastDigest® BsrDI (BseMI) and BseMI (BsrDI)

BsrFI Fermentas enzymes FastDigest® BsrFI (Cfr10I) and Cfr10I (BsrFI)

BsrGI Fermentas enzymes FastDigest® Bsp1407I and Bsp1407I (BsrGI)

BsrSI Fermentas enzymes FastDigest® BseNI and BseNI (BsrI)

BssHII Fermentas enzyme FastDigest® BssHII (PteI) and PauI (BssHII)

BssKI Fermentas enzymes FastDigest® ScrFI (Bme1390I) and Bme1390I (ScrFI) (different cleavage position)

BssSI Fermentas enzyme BauI (BssSI)

BspT104I Fermentas enzymes FastDigest® Bsp119I and Bsp119I (BstBI)

Bst98I Fermentas enzymes FastDigest® AflII (BspTI) and BspTI (AflII)

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5’...G T A↓T A C...3’

3’...C A T↑A T G...5’


Also available asFastDigest® BstZ17I (Bst1107I)



Storage BufferBst1107I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Bst1107I, more than 90% of the DNA fragments can be ligated and recut.



Bst1107I (BstZ17I) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 100 100 20-50 100

BstBI Fermentas enzymes FastDigest® Bsp119I and Bsp119I (BstBI)

BstEII Fermentas enzymes FastDigest® Eco91I and Eco91I (BstEII)

BstF5I Fermentas enzymes FastDigest® BseGI and BseGI (BtsCI); Fermentas enzyme FastDigest® FokI (different cleavage position)

BstNI Fermentas enzymes FastDigest® MvaI and MvaI (BstNI); EcoRII (different cleavage position and different sensitivity to methylation)

BstOI Fermentas enzymes FastDigest® MvaI and MvaI (BstNI); EcoRII (different cleavage position and different sensitivity to methylation)

BstPI Fermentas enzymes FastDigest® Eco91I and Eco91I (BstEII)

BstUI Fermentas enzymes FastDigest® Bsh1236I and Bsh1236I (BstUI)

BstXI Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 100 50-100 50-100 100

5’...C C A N N N N N↓N T G G...3’

3’...G G T N↑N N N N N A C C...5’

#ER1021 500 u#ER1022 2500 u


Also available asFastDigest® BstXI



Storage BufferBstXI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BstXI, approxi-mately 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam, CpG, EcoKI, EcoBI – no effect.Dcm: may overlap – cleavage impaired (p.177).


Note• Incubation at 37°C results in 50% activity. • Greater than 15-fold overdigestion with

BstXI may result in star activity.• BstXI cleavage is impaired by overlapping dcm


BstYI Fermentas enzymes FastDigest® PsuI and PsuI (BstYI)

BstZI Fermentas enzymes FastDigest® EagI (Eco52I) and Eco52I (EagI)

BstZ17I Fermentas enzymes FastDigest® BstZ17I (Bst1107I) and Bst1107I (BstZ17I)

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Bsu36I Fermentas enzymes FastDigest® Bsu36I (Eco81I) and Eco81I (Bsu36I)

5’...G G↓C C...3’

3’...C C↑G G...5’

#ER0151 3000 uSupplied with:10X Buffer R 2x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® HaeIII (BsuRI)



Storage BufferBsuRI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BsuRI, more than 95% of the DNA fragments can be ligated and recut.


BsuRI (HaeIII) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 50-100 100 50-100 100

Bsu15I (ClaI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 20-50 20-50 100 20-50

5’...A T↓C G A T...3’

3’...T A G C↑T A...5’

#ER0141 600 u#ER0145 1000 u



Also available asFastDigest® ClaI (Bsu15I)



Storage BufferBsu15I is supplied in: 10 mM Tris-HCl (pH 8.0 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Bsu15I, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam: may overlap – blocked (p.176).Dcm, EcoKI – no effect.CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.


NoteBsu15I is blocked by overlapping dam methy-lation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

BtrI Fermentas enzyme AjiI (BmgBI)

BtsCI Fermentas enzymes FastDigest® BseGI and BseGI (BtsCI); Fermentas enzyme FastDigest® FokI (different cleavage position)

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NoteCaiI is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

5’...C A G N N N↓C T G...3’

3’...G T C↑N N N G A C...5’


Also available asFastDigest® AlwNI (CaiI)



Storage BufferCaiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with CaiI, more than 95% of the DNA fragments can be ligated and recut.



CaiI (AlwNI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 20-50 50-100 100 50-100

CauII Fermentas enzymes FastDigest® NciI (BcnI) and BcnI (NciI)

CelII Fermentas enzymes FastDigest® BlpI (Bpu1102I) and Bpu1102I (BlpI)

CfoI Fermentas enzymes FastDigest® HinP1I (Hin6I) and Hin6I (HinP1I) (different cleavage position); FastDigest® HhaI and HhaI

5’...A C C T G C (N)4↓ ...3’

3’...T G G A C G (N)8↑ ...5’

#ER1741 250 uSupplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml50X oligonucleotide 2X25 µl

Also available asFastDigest® BspMI (BveI)


Conditions for 100% Activity 1X Buffer O at 37°C.Oligonucleotide 0.5 µM.

Storage BufferBveI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 150 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with BveI, more than 90% of the DNA fragments can be ligated and recut.


substrates with BveI is difficult to achieve.• BveI concentration is determined by the



Note• At least two copies of BveI recognition site

are required for efficient cleavage.• Inclusion of 0.5 µM oligonucleotide with the

BveI recognition sequence in the reaction mixture significantly improves cleavage of plasmid DNAs, especially of those with a single BveI site. Still, complete cleavage of some

• BveI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electro-phoresis.

BveI (BspMI) B G O R Tango 2X Tango 0-20 20-50 100 20-50 50-100 100


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NoteCfrI is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

5’...Y↓G G C C R...3’

3’...R C C G G↑Y...5’




Storage BufferCfrI is supplied in: 10 mM Tris-HCl (pH 8.5 at 25°C), 500 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with CfrI, more than 95% of the DNA fragments can be ligated and recut.



CfrI (EaeI) Activity in Five Buffer System, %


B G O R Tango 2X Tango 50-100* 50-100 0-20 0-20 100 0-20

NoteTo achieve complete digestion of substrate with Cfr9I, the concentration of DNA should be no less than 50 µg/ml in the reaction buffer.

5’...C↓C C G G G...3’

3’...G G G C C↑C...5’

#ER0171 300 u#ER0172 1500 u

Both supplied with:10X Buffer Cfr9I 1 ml10X Buffer Tango™ 1 ml


Conditions for 100% Activity1X Buffer Cfr9I at 37°C.

Storage BufferCfr9I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 250 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Cfr9I, more than 95% of the DNA fragments can be ligated and recut.



Cfr9I (XmaI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 0-20 20-50 0-20

Note• Greater than 5-fold overdigestion with

Cfr10I may result in star activity.• For cleavage with Cfr10I at least two copies

of its recognition sequence are required.

5’...R↓C C G G Y...3’

3’...Y G G C C↑R...5’

#ER0181 200 uSupplied with:10X Buffer Cfr10I 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® BsrFI (Cfr10I)


Conditions for 100% Activity1X Buffer Cfr10I at 37°C.

Storage BufferCfr10I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and Recutting After 5-fold overdigestion with Cfr10I, more than 95% of the DNA fragments can be ligated and recut.



Cfr10I (BsrFI) Activity in Five Buffer System, %


B G O R Tango 2X Tango 0-20 20-50 20-50 50-100* 20-50 50-100

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ClaI Fermentas enzymes FastDigest® ClaI (Bsu15I) and Bsu15I (ClaI)

NoteCfr13I is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

5’...G↓G N C C...3’

3’...C C N G↑G...5’


Also available asFastDigest® Sau96I (Cfr13I)



Storage BufferCfr13I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.



Cfr13I (Sau96I) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 20-50 100 20-50

5’...C C G C↓G G...3’

3’...G G↑C G C C...5’

#ER0201 1200 u#ER0205 2000 u

Both supplied with:10X Buffer B 1 ml10X Buffer Tango™ 1 ml

#ER0202 5x1200 uSupplied with:10X Buffer B 2x1 ml10X Buffer Tango™ 1 ml



Storage BufferCfr42I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.




Note• Certain sites in lambda DNA are difficult to

cleave with Cfr42I, the same as with its proto-type SacII. At least two copies of Cfr42I recogni-tion site are required for efficient cleavage.

• Cfr42I activity is affected by high salt con-centration. Trace amounts of sodium chloride remaining in the substrate DNA after completion of upstream applications may inhibit enzyme activity and result in impaired DNA cleavage.

Cfr42I (SacII) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 0-20 50-100 0-20

5’...C G↓G W C C G...3’

3’...G C C W G↑G C...5’

#ER0741 200 u#ER0742 1000 u


Also available asFastDigest® RsrII (CpoI)



Storage BufferCpoI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with CpoI, more than 95% of the DNA fragments can be ligated and recut.


CpoI (RsrII) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 50-100 20-50 100 50-100

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CspI Fermentas enzymes FastDigest® RsrII (CpoI) and CpoI (RsrII)

5’...G↓T A C...3’

3’...C A T↑G...5’


Also available asFastDigest® Csp6I



Storage BufferCsp6I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Csp6I, more than 95% of the DNA fragments can be ligated and recut.


Csp6I (CviQI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 0-20 50-100 0-20

Csp45I Fermentas enzymes FastDigest® Bsp119I and Bsp119I (BstBI)

CviAII Fermentas enzymes FastDigest® NlaIII (Hin1II) and Hin1II (NlaIII) (different cleavage position)

CviQI Fermentas enzymes FastDigest® Csp6I and Csp6I (CviQI)

DdeI Fermentas enzymes FastDigest® DdeI (HpyF3I) and HpyF3I (DdeI)

Note• For cleavage with CseI at least two copies of its

recognition sequence are required.• Low salt, high glycerol (>5%) concen-

trations, pH >8.0 or a large excess of enzyme may result in star activity.

• CseI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

5’...G A C G C (N) 5↓...3’

3’...C T G C G (N)10↑...5’


Also available asFastDigest® HgaI (CseI)



Storage BufferCseI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with CseI, more than 95% of the DNA fragments can be ligated and recut.


CseI (HgaI) Activity in Five Buffer System, %


B G O R Tango 2X Tango NR 50-100* 50-100 100 100* 50-100

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DpnII Fermentas enzymes FastDigest® DpnI and DpnI (different cleavage position and different sensitivity to methylation); FastDigest® Sau3AI (Bsp143I) and Bsp143I (Sau3AI) (different sensitivity to methylation); FastDigest® MboI and MboI

5’...T T T↓A A A...3’

3’...A A A↑T T T...5’


#ER0223 HC, 7500 uSupplied with:10X Buffer Tango™ 2x1 ml

Also available asFastDigest® DraI,

Concentration10 u/µl,50 u/µl, HC


Storage BufferDraI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with DraI, more than 95% of the DNA fragments can be ligated and recut.



DraI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 20-50 100 50-100

DraII Fermentas enzymes FastDigest® Eco0109I and EcoO109I

DraIII Fermentas enzymes FastDigest® DraIII (AdeI) and AdeI (DraIII)

DrdI Fermentas enzymes FastDigest® DrdI (AasI) and AasI (DrdI)

EaeI Fermentas enzyme CfrI (EaeI)

EagI Fermentas enzymes FastDigest® EagI (Eco52I) and Eco52I (EagI)



Storage BufferDpnI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 400 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with DpnI, more than 70% of the DNA fragments can be ligated and more than 95% of these can be recut.

Methylation EffectsDam: does not cut dam– DNA (p.176).Dcm, EcoKI, CpG – no effect.EcoBI: may overlap – effect not determined.

Note• DpnI requires the presence of N6-methyladenine

within the recognition sequence to cleave DNA.• DNA purified from a dam+ strain will be a

substrate for DpnI.• DpnI will only cleave fully-adenomethylated

dam sites. Hemi-adenomethylated dam sites DpnI cleaves 60X more slowly.

• DpnI, Bsp143I and MboI all recognize the same sequence but have different methy-lation sensitivities (pp.175-181) and cleavage sites.

DpnI Activity in Five Buffer System, % B G O R Tango 2X Tango 100 100 50-100 50-100 100 50-100

5’...G m6A↓ T C...3’

3’...C T↑m6A G...5’

#ER1701 500 u#ER1705 1000 u#ER1702 2500 u

All supplied with:10X Buffer Tango™ 1 ml

Also available asFastDigest® DpnI

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EarI Fermentas enzymes FastDigest® EarI (Eam1104I) and Eam1104I (EarI)


5’...G A C N N N↓N N G T C...3’

3’...C T G N N↑N N N C A G...5’

#ER0241 1000 uSupplied with:10X Buffer Eam1105I 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® Eam1105I


Conditions for 100% Activity 1X Buffer Eam1105I at 37°C.

Storage BufferEam1105I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with Eam1105I, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.



Eam1105I (AhdI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 0-20 0-20 50-100 20-50

5’...G A G↓C T C...3’

3’...C T C↑G A G...5’

#ER0251 1500 uSupplied with:10X Buffer Ecl136II 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® Ecl136II


Conditions for 100% Activity 1X Buffer Ecl136II at 37°C.

Storage BufferEcl136II is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Ecl136II, more than 90% of the DNA fragments can be ligated and recut.



Ecl136II (EcoICRI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 50-100 0-20

NoteCertain sites in λ DNA are difficult to cleave with Eam1104I, the same as with its prototype Ksp632I.

5’...C T C T T C(N)1↓...3’

3’...G A G A A G(N)4↑...5’

#ER0231 300 u#ER0232 1500 u


Also available asFastDigest® EarI (Eam1104I)



Storage BufferEam1104I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Eam1104I, more than 95% of the DNA fragments can be ligated and recut.



Eam1104I (EarI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 0-20 100 0-20

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5’...G R G C Y↓C...3’

3’...C↑Y C G R G...5’




Storage BufferEco24I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Eco24I, more than 95% of the DNA fragments can be ligated and recut.



Eco24I (BanII) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 0-20

EclHKI Fermentas enzymes FastDigest® Eam1105I and Eam1105I (AhdI)

EclXI Fermentas enzymes FastDigest® EagI (Eco52I) and Eco52I (EagI)

5’...G G T C T C(N)1↓...3’

3’...C C A G A G(N)5↑...5’


#ER0292 5000 uSupplied with:10X Buffer G 2x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® Eco31I


Conditions for 100% Activity1X Buffer G at 37°C.


Ligation and RecleavageAfter 50-fold overdigestion with Eco31I, more than 95% of DNA fragments can be ligated and recut.

Methylation EffectsDam, EcoKI – no effect.Dcm, CpG: may overlap – cleavage impaired (177, 179).EcoBI: may overlap – effect not determined.


Note• Eco31I cleavage is impaired by overlapping

dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).


Eco31I (BsaI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 0-20 0-20 50-100 20-50

5’...G A T↓A T C...3’

3’...C T A↑T A G...5’

#ER0301 2000 uSupplied with:10X Buffer R 1ml10X Buffer Tango™ 1ml

#ER0305 4000 u#ER0302 5x2000 u#ER0303 HC, 10000 u

All supplied with:10X Buffer R 2x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® EcoRV (Eco32I)



Storage BufferEco32I is supplied in: 25 mM Tris-HCl (pH 7.5 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.




Eco32I (EcoRV) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 50-100 100 20-50 100

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NoteEco47I is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

5’...G↓G W C C...3’

3’...C C W G↑G...5’



Also available asFastDigest® AvaII (Eco47I)



Storage BufferEco47I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.



Eco47I (AvaII) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 50-100 100 50-100 50-100

5’...A G C↓G C T...3’

3’...T C G↑C G A...5’

#ER0321 200 u#ER0322 1000 u


Also available asFastDigest® AfeI (Eco47III)



Storage BufferEco47III is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Eco47III, more than 80% of DNA fragments can be ligated and more than 90% of these can be recut.



Eco47III (AfeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 100 50-100 100

5’...C↓G G C C G...3’

3’...G C C G G↑C...5’

#ER0331 500 u#ER0332 2500 u

Both supplied with:10X Buffer Eco52I 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® EagI (Eco52I)


Conditions for 100% Activity 1X Buffer Eco52I at 37°C.

Storage BufferEco52I is supplied in: 10 mM Tris-HCl (pH 8.2 at 25°C), 500 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.




Eco52I (EagI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 20-50 0-20 20-50

Eco53kI Fermentas enzymes FastDigest® Ecl136II and Ecl136II (EcoICRI); Fermentas enzymes FastDigest® SacI and SacI (different cleavage position)

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Ligation and RecleavageAfter 10-fold overdigestion with Eco57I, approximately 70% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.


5’...C T G A A G(N)16↓...3’

3’...G A C T T C(N)14↑...5’

#ER0341 200 uSupplied with:10X Buffer G 1 ml50X SAM 0.1 ml10X Buffer Tango™ 1 ml

#ER0342 1000 uSupplied with:10X Buffer G 1 ml50X SAM 2x0.1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® AcuI (Eco57I)


Conditions for 100% Activity 1X Buffer G at 37°C. SAM 0.01 mM.

Storage Buffer Eco57I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT and 50% (v/v) glycerol.

Note• Eco57I requires only Mg2+ for its activity, but is

stimulated by S-adenosylmethionine. 0.01 mM S-adenosylmethionine gives a 100-fold increase in Eco57I activity. Still, complete cleavage of some substrates with Eco57I is difficult to achieve.

• For cleavage with Eco57I at least two copies of its recognition sequence are required.

• Eco57I concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.


• Eco57I may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

Eco57I (AcuI) B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM

100 100 20-50 20-50 50-100 50-100


5’...C T G R A G(N)16↓...3’

3’...G A C Y T C(N)14↑...5’

#ER1671 50 uSupplied with:10X Buffer B 1 ml50X SAM 0.1 ml10X Buffer Tango™ 1 ml


Conditions for 100% Activity 1X Buffer B at 37°C.SAM 0.01 mM.

Storage Buffer Eco57MI is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with Eco57MI, approximately 90% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.


Note• Eco57MI requires only Mg2+ for its activity,

but is stimulated by S-adenosylmethionine. 0.01 mM S-adenosylmethionine gives more than a 100-fold increase in Eco57MI activity. Still, complete cleavage of some substrates with Eco57MI is difficult to achieve.

• Eco57MI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.

• Eco57MI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA

Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electro-phoresis.

• Eco57MI is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Eco57MI Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM

100 50-100 0-20 20-50 50-100 0-20

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NoteGreater than 10-fold overdigestion with Eco72I may result in star activity.

5’...C A C↓G T G...3’

3’...G T G↑C A C...5’


Also available asFastDigest® PmlI (Eco72I)




Ligation and RecleavageAfter 10-fold overdigestion with Eco72I, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.



Eco72I (PmlI) Activity in Five Buffer System, % B G O R Tango 2X Tango NR NR 0-20 0-20 100 20-50

5’...C C↓T N A G G...3’

3’...G G A N T↑C C...5’

#ER0371 500 u#ER0372 2500 u


Also available asFastDigest® Bsu36l (Eco81I)




Ligation and RecleavageAfter 10-fold overdigestion with Eco81I, more than 80% of the DNA fragments can be ligated in a reaction mixture containing 20-40 u of T4 DNA Ligase/1 µg of fragments and 10% PEG. More than 95% of these can be recut.



Eco81I (Bsu36I) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 0-20 0-20 100 0-20

5’...C↓Y C G R G...3’

3’...G R G C Y↑C...5’

#ER0381 1000 uSupplied with: 10X Buffer Tango™ 1 ml

Also available asFastDigest® AvaI (Eco88I)







Eco88I (AvaI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 0-20 100 20-50

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5’...G↓G T N A C C...3’

3’...C C A N T G↑G...5’



Also available asFastDigest® Eco91I







Eco91I (BstEII) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 100 50-100 50-100 100

NoteGreater than 15-fold overdigestion with Eco105I may result in star activity.

5’...T A C↓G T A...3’

3’...A T G↑C A T...5’



Also available asFastDigest® SnaBI (Eco105I)



Storage BufferEco105I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with Eco105I, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.



Eco105I (SnaBI)* Star activity appears at a greater than 5-fold overdigestion (5 u x 1 h).

Activity in Five Buffer System, % B G O R Tango 2X Tango 100* 50-100 0-20 0-20 100 0-20

5’...C↓C W W G G...3’

3’...G G W W C↑C...5’


Also available asFastDigest® StyI (Eco130I)







Eco130I (StyI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 50-100 100

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NoteEco147I is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

5’...A G G↓C C T...3’

3’...T C C↑G G A...5’



Also available asFastDigest® StuI (Eco147I)







Eco147I (StuI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 20-50 20-50 50-100 0-20

NoteEcoO109I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

5’...R G↓G N C C Y...3’

3’...Y C C N G↑G R...5’


Also available asFastDigest® EcoO109I



Storage BufferEcoO109I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with EcoO109I, more than 95% of the DNA fragments can be ligated and recut.


Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme is required for complete digestion of 1 µg of agarose-embed-ded λ DNA in 16 hours.

EcoO109I (DraII) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 20-50 20-50 100 100

EcoICRI Fermentas enzymes FastDigest® Ecl136II and Ecl136II (EcoICRI); FastDigest® SacI and SacI (different cleavage position)

EcoNI Fermentas enzymes FastDigest® EcoNI (XagI) and XagI (EcoNI)

EcoO65I Fermentas enzymes FastDigest® Eco91I and Eco91I (BstEII)

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NoteLow salt concentration, large excess of the enzyme, pH >8.0, or the replacement of Mg2+ by Mn2+ may result in star activity.

5’...G↓A A T T C...3’

3’...C T T A A↑G...5’

#ER0271 5000 uSupplied with:10X Buffer EcoRI 2x1 ml10X Buffer Tango™ 1 ml

#ER0275 10000 uSupplied with:10X Buffer EcoRI 4x1 ml10X Buffer Tango™ 1 ml

#ER0272 5x5000 u#ER0273 HC, 25000 u

All supplied with:10X Buffer EcoRI 5x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® EcoRI


Conditions for 100% Activity1X Buffer EcoRI at 37°C.

Storage BufferEcoRI is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 300 mM NaCl, 1 mM EDTA, 1 mM DTT,0.2 mg/ml BSA, 0.15% Triton X-100 and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with EcoRI, more than 95% of the DNA fragments can be ligated and recut.



EcoRI Activity in Five Buffer System, %


B G O R Tango 2X Tango 0-20 NR 100 100* NR 100

5’...↓C C W G G ...3’

3’... G G W C C↑...5’




Storage BufferEcoRII is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with EcoRII, more than 90% of the DNA fragments can be ligated and recut.

Methylation EffectsDam, CpG, EcoKI, EcoBI – no effect.Dcm: completely overlaps – blocked (p.177).

Note• At least two copies of EcoRII recognition

site are required for efficient cleavage. For cleavage of DNA substrates with only one copy of recognition site MvaI (#ER0551), neoschizomer of EcoRII, is recommended.

• EcoRII may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

• EcoRII is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

EcoRII Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 100 50-100 20-50 50-100

EcoRV Fermentas enzymes FastDigest® EcoRV (Eco32I) and Eco32I (EcoRV)

EcoT14I Fermentas enzymes FastDigest® StyI (Eco130I) and Eco130I (StyI)

EcoT22I Fermentas enzymes FastDigest® NsiI (Mph1103I) and Mph1103I (NsiI)

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EspI Fermentas enzymes FastDigest® BlpI (Bpu1102I) and Bpu1102I (BlpI)

Note• Unlike NarI, EheI completely digests λ and

pBR322 DNAs.• Low salt, high glycerol (>5%) concentra-


5’...G G C↓G C C...3’

3’...C C G↑C G G...5’


Also available asFastDigest® EheI



Storage BufferEheI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with EheI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.



EheI (SfoI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 0-20 0-20 100 20-50

5’...C G T C T C(N)1↓...3’

3’...G C A G A G(N)5↑...5’

#ER0451 200 u#ER0452 1000 u


Also available asFastDigest® BsmBI (Esp3I)


Conditions for 100% Activity1X Buffer Tango™ at 37°C.DTT (#R0861/2) 1 mM.

Storage BufferEsp3I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Esp3I, more than 95% of the DNA fragments can be ligated and recut.



NoteThe enzyme requires DTT (#R0861/2). Freshly made DTT should be added to the reaction buffer.

Esp3I (BsmBI) Activity in Five Buffer System, % B+DTT G+DTT O+DTT R+DTT Tango+DTT 2X Tango+DTT

100 20-50 0-20 0-20 100 0-20

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5’...R T G C↓G C A Y...3’

3’...Y A C G↑C G T R...5’

#ER1661 100 u#ER1662 500 u

Both supplied with: 10X Buffer O 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® FspAI



Storage BufferFspAI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with FspAI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.


FspAI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 50-100 0-20 50-100

FatI Fermentas enzymes FastDigest® NlaIII (Hin1II) and Hin1II (different cleavage position)

FauI Fermentas enzyme SmuI (FauI)

FbaI Fermentas enzymes FastDigest® BclI and BclI

FinI Fermentas enzymes FastDigest® BsmFI (FaqI) and Faq (BsmFI)

FnuDII Fermentas enzymes FastDigest® Bsh1236I and Bsh1236I (BstUI)

Fnu4HI Fermentas enzymes FastDigest® Fnu4HI (SatI) and SatI (Fnu4HI)

FokI Fermentas enzymes FastDigest® FokI; FastDigest® BseGI and BseGI (BtsCI) (different cleavage position)

FspI Fermentas enzymes FastDigest® FspI (NsbI) and NsbI (FspI)

5’...G G G A C(N)10↓...3’

3’...C C C T G(N)14↑...5’


Also available asFastDigest® BsmFI (FaqI)



Storage BufferFaqI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with FaqI, more than 90% of the DNA fragments can be ligated, but none of these can be recut due to the methyla-tion of the recognition sequence by this enzyme.


Note• FaqI requires only Mg2+ for its activity, but

is stimulated by S-adenosylmethionine. 0.05 mM S-adenosylmethionine gives more than a 2-fold increase in FaqI activity. Still, complete cleavage of some substrates is difficult to achieve.

• For cleavage with FaqI at least two copies of its recognition sequence are required.

• FaqI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.

• FaqI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

FaqI (BsmFI) Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM

20-50 20-50 0-20 0-20 100 20-50

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5’...C↓T A G...3’

3’...G A T↑C...5’

#ER1761 500 u#ER1762 2500 u


Also available asFastDigest® BfaI (FspBI)



Storage BufferFspBI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with FspBI, more than 80% of the DNA fragments can be ligated and more than 95% of these can be recut.



FspBI (BfaI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 100 0-20

HaeIII Fermentas enzymes FastDigest® HaeIII (BsuRI) and BsuRI (HaeIII)

HaeII Fermentas enzyme FastDigest® HaeII (BfoI)

HapII Fermentas enzymes FastDigest® HpaII and HpaII; FastDigest® MspI and MspI (HpaII) (different sensitivity to methylation)

HgaI Fermentas enzymes FastDigest® HgaI (CseI) and CseI (HgaI)

HgiAI Fermentas enzymes FastDigest® Alw21I and Alw21I (BsiHKAI)

HgiCI Fermentas enzymes FastDigest® BanI (BshNI) and BshNI (BanI)

HgiJII Fermentas enzyme Eco24I

5’...C T G G A G(N)16↓...3’

3’...G A C C T C(N)14↑...5’

#ER0461 100 u#ER0462 500 u


Also available asFastDigest® BpmI (GsuI)


Conditions for 100% Activity1X Buffer B at 30°C.

Storage BufferGsuI is supplied in: 10 mM potassium phosphate (pH 7.5 at 25°C), 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with GsuI, approxi-mately 90% of the DNA fragments can be ligated and recut.


Note• Incubation at 37°C results in 70% activity.• GsuI requires only Mg2+ for its activity, but

is stimulated by S-adenosylmethionine. 0.01 mM S-adenosylmethionine gives a 2-fold increase in activity.

• GsuI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

• GsuI is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

GsuI (BpmI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 20-50 20-50 100 50-100

GsaI Fermentas enzyme FastDigest® PspFI

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5’...G C G↓C...3’

3’...C↑G C G...5’


Also available asFastDigest® HhaI



Storage BufferHhaI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with HhaI, more than 95% of the DNA fragments can be ligated and recut.


HhaI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 20-50 100 20-50

NoteHin1I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

5’...G R↓C G Y C...3’

3’...C Y G C↑R G...5’


Also available asFastDigest® BsaHI (Hin1I)



Storage BufferHin1I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Hin1I, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam, EcoKI – no effect.Dcm: may overlap – cleavage impaired (p.177).CpG: completely overlaps – blocked (p.178).EcoBI: may overlap – effect not determined.


Hin1I (BsaHI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 20-50 20-50 20-50 20-50

NoteMore stable when stored at -70°C. At -20°C the half-life of Hin1II is 6 months.

5’... C A T G↓...3’

3’...↑G T A C ...5’


Also available asFastDigest® NlaIII (Hin1II)



Storage BufferHin1II is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 500 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.15% Triton X-100, 0.5 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Hin1II, more than 95% of the DNA fragments can be ligated and recut.


Hin1II (NlaIII) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 20-50 50-100 50-100 50-100

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HinP1I Fermentas enzymes FastDigest® HhaI and HhaI (different cleavage position); FastDigest® HinP1I (Hin6I) and Hin6I (HinP1I)

5’...↓8 (N)GAY(N)5VTC(N)13-14↓...3’

3’...↑13-14(N)CTR(N)5BAG(N)8 ↑...5’





Ligation and RecleavageAfter 10-fold overdigestion with Hin4I, more than 95% of the DNA fragments can be ligated and only 50% of these can be recut due to the methylation of the recognition sequence by restriction enzyme.

Methylation EffectsDam: may overlap – blocked (p.176).Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – effect not determined.

Note• Hin4I requires only Mg2+ for its activity,

but is stimulated by S-adenosylmethionine. 0.05 mM S-adenosylmethionine gives more than a 10-fold increase in Hin4I activity. Still, complete cleavage of some substrates with Hin4I is difficult to achieve.

• Hin4I concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.

• Hin4I produces double-strand cuts on both sides from the interrupted recognition site.

Its unique feature is a degenerate cleavage point on the 3’ side of the recognition sequence (13 or 14 nt away).

• Hin4I is blocked by overlapping dam methy-lation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Hin4I Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM

20-50 20-50 0-20 0-20 100 0-20

5’...G↓C G C...3’

3’...C G C↑G...5’


Also available asFastDigest® HinP1I (Hin6I)




Ligation and RecleavageAfter 50-fold overdigestion with Hin6I, more than 95% of the DNA fragments can be ligated and recut.


Hin6I (HinP1I) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 20-50 100 50-100

HindII Fermentas enzymes FastDigest® HincII and HincII (HindII)

5’...G T Y↓R A C...3’

3’...C A R↑Y T G...5’

#ER0491 500 u#ER0492 2500 u


Also available asFastDigest® HincII



Storage BufferHincII is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with HincII, more than 95% of the DNA fragments can be ligated and recut.


HincII (HindII) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 50-100 100 50-100

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5’...A↓A G C T T...3’

3’...T T C G A↑A...5’



#ER0502 5x5000 u#ER0503 HC, 25000 u

Both supplied with:10X Buffer R 2x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® HindIII



Storage BufferHindIII is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 250 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with HindIII, more than 95% of the DNA fragments can be ligated and recut.



HindIII Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 0-20 100 50-100 50-100

5’...G↓A N T C...3’

3’...C T N A↑G...5’


#ER0802 5x2000 u#ER0803 HC, 10000 u

Both supplied with:10X Buffer R 2x1 ml10X Buffer Tango™ 1 ml

FastDigest® HinfI



Storage BufferHinfI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with HinfI, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – blocked (p.181).

HinfI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 100 50-100 50-100

HpaI Fermentas enzymes FastDigest® HpaI (KspAI) and KspAI (HpaI)

5’...C↓C G G...3’

3’...G G C↑C...5’



Also available asFastDigest® HpaII



Storage BufferHpaII is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with HpaII, more than 95% of the DNA fragments can be ligated and recut.


HpaII Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 20-50

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Note• HphI is blocked by overlapping dam methy-


• High glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.

5’...G G T G A(N)8↓...3’

3’...C C A C T(N)7↑...5’

#ER1101 300 u#ER1102 1500 u




Storage BufferHphI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with HphI, more than 70% of the DNA fragments can be ligated and more than 90% of these can be recut.

Methylation EffectsDam, EcoBI: may overlap – blocked (p.176, 181).Dcm, CpG, EcoKI – no effect.

HphI Activity in Five Buffer System, % B G O R Tango 2X Tango 100 0-20 0-20 0-20 20-50 0-20

5’...G T N↓N A C...3’

3’...C A N↑N T G...5’

#ER1571 200 u#ER1572 1000 u


Also available asFastDigest® Hpy8I



Storage BufferHpy8I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Hpy8I, more than 95% of the DNA fragments can be ligated and recut.


Hpy8I (MjaIV) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 50-100

HpyCH4III Fermentas enzymes FastDigest® TaaI and TaaI (HpyCH4III)

HpyCH4IV Fermentas enzymes FastDigest® TaiI and TaiI (MaeII) (different cleavage position)

5’...C↓T N A G...3’

3’...G A N T↑C...5’

#ER1881 500 u#ER1882 2500 u


Also available asFastDigest® DdeI (HpyF3I)



Storage BufferHpyF3I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with HpyF3I, more than 95% of the DNA fragments can be ligated and recut.


HpyF3I (DdeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 20-50 20-50 100 50-100

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5’...G C N N N N N↓N N G C...3’

3’...C G N N↑N N N N N C G...5’

#ER1731 300 u#ER1732 1500 u


Also available asFastDigest® HpyF10VI



Storage BufferHpyF10VI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with HpyF10VI, more than 95% of the DNA fragments can be ligated and recut.


HpyF10VI (MwoI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 0-20 100 50-100

Hsp92I Fermentas enzymes FastDigest® BsaHI (Hin1I) and Hin1I (BsaHI)

Hsp92II Fermentas enzymes FastDigest® NlaIII (Hin1II) and Hin1II (NlaIII)

ItaI Fermentas enzymes FastDigest® Fnu4HI (SatI) and SatI (Fnu4HI)

KasI Fermentas enzymes FastDigest® EheI and EheI (SfoI) (different cleavage position) Fermentas enzyme SspDI (KasI)

NoteHigh glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.

5’...G G T A C↓C...3’

3’...C↑C A T G G...5’

#ER0521 4000 uSupplied with:10X Buffer KpnI 2x1 ml10X Buffer Tango™ 1 ml

#ER0522 5x4000 u#ER0523 HC, 20000 u

Both supplied with:10X Buffer KpnI 4x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® KpnI


Conditions for 100% Activity1X Buffer KpnI at 37°C.

Storage BufferKpnI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with KpnI, more than 95% of the DNA fragments can be ligated and recut.



KpnI Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 0-20 0-20 0-20 20-50 0-20




Storage BufferKpn2I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Kpn2I, more than 95% of the DNA fragments can be ligated and recut.



5’...T↓C C G G A...3’

3’...A G G C C↑T...5’

#ER0531 500 u#ER0532 2500 u


Also available asFastDigest® Kpn2I

Kpn2I (BspEI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 50-100

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Note• Greater than 10-fold overdigestion with

Lsp1109I may result in star activity.• Lsp1109I may remain associated with the

cleaved DNA. This may cause DNA band shift-ing during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

5’...G C A G C(N) 8↓...3’

3’...C G T C G(N)12↑...5’

#ER2071 200 uSupplied with:10X Buffer Lsp1109I 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® BbvI (Lsp1109I)


Conditions for 100% Activity1X Buffer Lsp1109I at 37°C.

Storage BufferLsp1109I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with Lsp1109I, more than 95% of the DNA fragments can be ligated and recut.


KspI Fermentas enzyme Cfr42I (SacII)

Ksp632I Fermentas enzymes FastDigest® EarI (Eam1104I) and Eam1104I (EarI)

NoteHigh glycerol (>5%) concentration, pH >8.0 or a large excess of enzyme may result in star activity.

5’...G T T↓A A C...3’

3’...C A A↑T T G...5’

#ER1031 500 u#ER1032 2500 u


Also available asFastDigest® HpaI (KspAI)



Storage BufferKspAI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with KspAI, more than 90% of the DNA fragments can be ligated and recut.

Methylation EffectsDam, Dcm, EcoBI – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI: may overlap – blocked (p.181).


KspAI (HpaI) Activity in Five Buffer System, %


B G O R Tango 2X Tango 100 50-100* 20-50 20-50 100* 50-100


5’...G C T C T T C(N)1↓...3’

3’...C G A G A A G(N)4↑...5’

#ER1931 100 u#ER1932 500 u


Also available asFastDigest® SapI (LguI)



Storage BufferLguI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with LguI, more than 90% of the DNA fragments can be ligated and recut.



LguI (SapI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 20-50 20-50 100 20-50

Lsp1109I (BbvI) Activity in Five Buffer System, %


B G O R Tango 2X Tango 0-20 20-50* 50-100* 100* 20-50* 20-50*

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MaeI Fermentas enzymes FastDigest® BfaI (FspBI) and FspBI (BfaI)

MaeII Fermentas enzymes FastDigest® TaiI and TaiI (MaeII) (different cleavage position)

MamI Fermentas enzymes FastDigest® BsaBI (BseJI) and BseJI (BsaBI)

Note• At least two copies of LweI recognition site

are required for efficient cleavage.• LweI may remain associated with the cleaved


5’...G C A T C(N)5↓...3’

3’...C G T A G(N)9↑...5’

#ER1621 100 u#ER1622 500 u


Also available asFastDigest® SfaNI (BmsI)



Storage BufferLweI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with LweI, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam, Dcm, EcoKI– no effect.CpG: may overlap – cleavage impaired (p.179).EcoBI: may overlap – effect not determined.

LweI (SfaNI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 20-50 100 20-50

5’...C G↓C G C G C G...3’

3’...G C G C G C↑G C...5’

#ER2081 100 uSupplied with:10X Buffer Tango™ 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® MauBI



Storage BufferMauBI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with MauBI, more than 90% of the DNA fragments can be ligated and recut.



MauBI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 0-20 100 0-20


5’...C C G↓C T C...3’

3’...G G C↑G A G...5’


Also available asFastDigest® BsrBI (MbiI)



Storage BufferMbiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with MbiI, approxi-mately 80% of the DNA fragments can be ligated. No more than 50% of these can be recut due to the asymmetric recognition sequence of MbiI.

Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: completely overlaps – cleavage impaired (p.178).EcoBI: may overlap – effect not determined.


MbiI (BsrBI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 20-50 20-50 100 20-50

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Note• MboI is blocked by overlapping dam methy-


• MboI, Bsp143I and DpnI all recognize the same sequence but have different methy-lation sensitivities (pp.175-181) and cleavage sites.

5’...↓G A T C ...3’

3’... C T A G↑...5’

#ER0811 300 u#ER0812 1500 u


Also available asFastDigest® MboI



Storage BufferMboI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with MboI, more than 95% of the DNA fragments can be ligated and recut.


MboI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 50-100 100 50-100 100

5’...G A A G A(N)8↓...3’

3’...C T T C T(N)7↑...5’

#ER0821 300 u#ER0822 1500 u


Also available asFastDigest® MboII



Storage BufferMboII is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 5-fold overdigestion with MboII, more than 80% of the DNA fragments can be ligated and more than 80% of these can be recut.


Note• MboII is blocked by overlapping dam methy-


• Greater than 15-fold overdigestion with MboII may result in star activity.

• MboII produces DNA fragments that have a single-base 3’-extension which are more difficult to ligate than blunt-ended fragments.

• MboII may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample prepara-tion or heat the digested DNA in the presence of SDS prior to electrophoresis.

MboII Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 20-50 0-20 50-100 20-50

McrI Fermentas enzymes FastDigest® BsiEI (Bsh1285I) and Bsh1285I (BsiEI)

MfeI Fermentas enzymes FastDigest® MfeI (MunI) and MunI (MfeI)

MflI Fermentas enzymes FastDigest® PsuI and PsuI (BstYI)

MjaIV Fermentas enzymes FastDigest® Hpy8I and Hpy8I (MjaIV)

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NoteMlsI is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

5’...T G G↓C C A...3’

3’...A C C↑G G T...5’

#ER1211 200 u#ER1212 1000 u


Also available asFastDigest® MscI (MlsI)

Concentration5 u/µ


Storage BufferMlsI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with MlsI, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.



MlsI (MscI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 0-20 100 20-50 50-100

5’...A↓C G C G T...3’

3’...T G C G C↑A...5’



Also available asFastDigest® MluI



Storage BufferMluI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with MluI, more than 95% of the DNA fragments can be ligated and recut.



MluI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 100 20-50 50-100

MluNI Fermentas enzymes FastDigest® MscI (MlsI) and MlsI (MscI)

MlyI Fermentas enzymes FastDigest® MlyI (SchI) and SchI (MlyI)

Note• MnlI produces DNA fragments that have a

single-base 3’-extension which are more difficult to ligate than blunt-ended fragments.

• MnlI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

5’...C C T C(N)7↓...3’

3’...G G A G(N)6↑...5’

#ER1071 300 u#ER1072 1500 u


Also available asFastDigest® MnlI



Storage BufferMnlI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with MnlI, approxi-mately 90% of the DNA fragments can be ligated and more than 90% of these can be recut.


MnlI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 20-50 20-50 20-50 20-50

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5’...A T G C A↓T...3’

3’...T↑A C G T A...5’



Also available asFastDigest® NsiI (Mph1103I)



Storage BufferMph1103I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Mph1103I, more than 95% of the DNA fragments can be ligated and recut.


Mph1103I (NsiI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 20-50 100 50-100 50-100

5’...C G↓C C G G C G...3’

3’...G C G G C C↑G C...5’


Also available asFastDigest® MreI



Storage BufferMreI is supplied in: 10 mM potassium phosphate (pH 7.0 at 25°C), 200 mM KCl, 0.1 mM EDTA, 0.01% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with MreI, more than 95% of the DNA fragments can be ligated and recut.


MreI (Sse232I) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 0-20 0-20 50-100 0-20

MroI Fermentas enzymes FastDigest® Kpn2I and Kpn2I (BspEI)

MscI Fermentas enzymes FastDigest® MscI (MlsI) and MlsI (MscI)

MseI Fermentas enzymes FastDigest® Tru1I and Tru1I (MseI); Fermentas enzyme FastDigest® MseI (SaqAI)

MslI Fermentas enzymes FastDigest® MslI (RseI) and RseI (MslI)

NoteMspI is an isoschizomer of HpaII. When the external C in the sequence CCGG is methy-lated, MspI and HpaII cannot cleave. However, unlike HpaII, MspI can cleave the sequence when the internal C residue is methylated.

5’...C↓C G G...3’

3’...G G C↑C...5’

#ER0541 3000 uSupplied with:10X Buffer Tango™ 2X1 ml

#ER0542 5x3000 uSupplied with:10X Buffer Tango™ 2x1 ml

Also available asFastDigest® MspI



Storage BufferMspI is supplied in: 10 mM potassium phosphate (pH 7.5 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with MspI, more than 95% of the DNA fragments can be ligated and recut.


MspI (HpaII) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 0-20 100 50-100

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5’...C↓A A T T G...3’

3’...G T T A A↑C...5’

#ER0751 300 u#ER0752 1500 u


Also available asFastDigest® MfeI (MunI)



Storage BufferMunI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with MunI, more than 95% of the DNA fragments can be ligated and recut.



MunI (MfeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 100 0-20 0-20 100 0-20

5’...G T T T↓A A A C...3’

3’...C A A A↑T T T G...5’

#ER1341 250 u#ER1342 1250 u


Also available asFastDigest® MssI



Storage BufferMssI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with MssI, more than 90% of the DNA fragments can be ligated and more than 90% of these can be recut.



MssI (PmeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 0-20 0-20 0-20 20-50 0-20

MstI Fermentas enzymes FastDigest® FspI (NsbI) and NsbI (FspI)

Note• Low salt, high glycerol (>5%) concentrations

or a large excess of enzyme may result in star activity.

• Unlike its neoschizomer EcoRII, MvaI does not require multiple copies of recognition site for efficient cleavage.

5’...C C↓W G G...3’

3’...G G W↑C C...5’


Also available asFastDigest® MvaI



Storage BufferMvaI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 400 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with MvaI, more than 90% of the DNA fragments can be ligated and more than 90% of these can be recut.


MvaI (BstNI) Activity in Five Buffer System, %

* Star activity appears at a greater than 5-fold overdigestion (5u x 1h).

B G O R Tango 2X Tango 20-50 20-50 50-100 100 20-50* 100

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5’...G A A T G C N↓...3’

3’...C T T A C↑G N ...5’

#ER0961 200 u#ER0962 1000 u


Also available asFastDigest® Mva1269I



Storage BufferMva1269I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Mva1269I, more than 95% of the DNA fragments can be ligated and recut.



Mva1269I (BsmI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 100 0-20 50-100

MvnI Fermentas enzymes FastDigest® Bsh1236I and Bsh1236I (BstUI)

MwoI Fermentas enzymes FastDigest® HpyF10VI and HpyF10VI (MwoI)

NaeI Fermentas enzymes FastDigest® NaeI (PdiI) and PdiI (NaeI)

NarI Fermentas enzymes FastDigest® EheI and EheI (SfoI) (different cleavage position); Fermentas enzyme SspDI (KasI) (different cleavage position)

NciI Fermentas enzymes FastDigest® NciI (BcnI) and BcnI (NciI)


5’...C↓C A T G G...3’

3’...G G T A C↑C...5’

#ER0571 500 u#ER0575 1000 u#ER0572 2500 u


Also available asFastDigest® NcoI



Storage BufferNcoI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with NcoI, more than 95% of the DNA fragments can be ligated and recut.



NcoI Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 20-50 50-100 100 100

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5’...C A↓T A T G...3’

3’...G T A T↑A C...5’

#ER0581 500 u#ER0582 2500 u


#ER0585 4000 uBoth supplied with:10X Buffer O 2x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® NdeI,



Storage BufferNdeI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with NdeI, more than 95% of the DNA fragments can be ligated and recut.



NdeI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 50-100 0-20 50-100

NdeII Fermentas enzymes FastDigest® Sau3AI (Bsp143I) and Bsp143I (Sau3AI) (different sensitivity to methylation); FastDigest® DpnI and DpnI (different cleavage position and different sensitivity to methylation); FastDigest® MboI and MboI

NgoMIV Fermentas enzymes FastDigest® NaeI (PdiI) and PdiI (NaeI) (different cleavage position)

Note• Low salt, high glycerol (>5%) concentra-


• NheI is inhibited by salt concentrations above 100 mM.

• Supercoiled plasmids may require up to 10-fold more NheI for complete digestion than linear DNAs (e.g. 10 units are required to cleave 1 µg of pBR322 DNA).

5’...G↓C T A G C...3’

3’...C G A T C↑G...5’

#ER0971 500 u#ER0975 1000 u#ER0972 2500 u


Also available asFastDigest® NheI



Storage BufferNheI is supplied in: 10 mM Tris-HCl (pH 8.0 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and Recleavage After 50-fold overdigestion with NheI, more than 95% of DNA fragments can be ligated and recut.



NheI Activity in Five Buffer System, % B G O R Tango 2X Tango 100 20-50 0-20 0-20 100 0-20

NlaIII Fermentas enzymes FastDigest® NlaIII (Hin1II) and Hin1II (NlaIII)

NlaIV Fermentas enzymes FastDigest® NlaIV (BspLI) and BspLI (NlaIV)

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5’...↓G T S A C ...3’

3’... C A S T G↑...5’


Also available asFastDigest® NmuCI



Storage BufferNmuCI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with NmuCI, more than 95% of the DNA fragments can be ligated and recut.


NmuCI (Tsp45I) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 100 20-50 50-100

5’...G C↓G G C C G C...3’

3’...C G C C G G↑C G...5’

#ER0591 300 u#ER0595 500 u#ER0592 1500 u#ER0593 HC, 1500 u

All supplied with:10X Buffer O 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® NotI



Storage BufferNotI is supplied in: 20 mM Tris-HCl (pH 7.8 at 25°C), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.02% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with NotI, more than 95% of DNA fragments can be ligated and recut.


Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded Adenovirus-2 DNA in 16 hours.

NoteSupercoiled plasmids may require up to 5-fold more NotI for complete digestion than linear DNAs.

NotI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 20-50 0-20 20-50

NruI Fermentas enzymes FastDigest® NruI (RruI) and Bsp68I (NruI)

5’...T G C↓G C A...3’

3’...A C G↑C G T...5’


Also available asFastDigest® FspI (NsbI)



Storage BufferNsbI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with NsbI, more than 80% of the DNA fragments can be ligated and more tha 95% of these can be recut.



NsbI (FspI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 0-20 20-50 100 20-50

NsiI Fermentas enzymes FastDigest® Nsil (Mph1103I) and Mph1103I (NsiI)

NspI Fermentas enzymes FastDigest® NspI (XceI) and XceI (NspI)

NspV Fermentas enzymes FastDigest® Bsp119I and Bsp119I (BstBI)

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5’...C A C N N↓N N G T G...3’

3’...G T G N N↑N N C A C...5’

#ER1631 200 u#ER1632 1000 u


Also available asFastDigest® AleI (OliI)



Storage BufferOliI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with OliI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.

Methylation EffectsDam, Dcm – no effect.CpG: may overlap – cleavage impaired (p.179).EcoKI, EcoBI: may overlap– blocked (p.181).


OliI (AleI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 100 0-20 50-100

5’...G C A T G↓C...3’

3’...C↑G T A C G...5’

#ER0601 500 u#ER0602 2500 u


Also available asFastDigest® SphI (PaeI)



Storage BufferPaeI is supplied in: 10 mM potassium-phosphate (pH 7.0 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.15% Triton X-100, 0.5 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with PaeI, more than 95% of the DNA fragments can be ligated and recut.



PaeI (SphI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 0-20 50-100 0-20

PaeR7I Fermentas enzymes FastDigest® XhoI and XhoI

PacI Activity in Five Buffer System, %

5’...T T A A T↓T A A...3’

3’...A A T↑T A A T T...5’

#ER2201 250 u#ER2202 1250 u

Supplied with:10X Buffer PacI 1 ml

Also available asFastDigest® PacI


Conditions for 100% Activity1X Buffer PacI at 37°C.

Storage BufferPacI is supplied in: 10 mM potassium phosphate (pH 7.5 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with PacI, more than 95% of the DNA fragments can be ligated and recut.


Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded pJET1 DNA with inserted PacI recognition sequence in 16 hours.

B G O R Tango 2X Tango 20-50 20-50 0-20 0-20 0-20 0-20

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5’...C C↓C W G G G...3’

3’...G G G W C↑C C...5’

#ER1861 200 uSupplied with:10X Buffer PasI 1 ml


Conditions for 100% Activity1X Buffer PasI at 55°C.

Storage BufferPasI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with PasI, more than 90% of the DNA fragments can be ligated and recut.



Note• Incubation at 37°C results in 30% activity.• Greater than 10-fold overdigestion with PasI

results in star activity.

PasI Activity in Five Buffer System, % B G O R Tango 2X Tango NR NR NR NR NR NR

5’...T↓C A T G A...3’

3’...A G T A C↑T...5’

#ER1281 400 u#ER1282 2000 u

Both supplied with:10X Buffer O 1 ml

Also available asFastDigest® BspHI (PagI)



Storage BufferPagI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with PagI, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam, EcoBI: may overlap – cleavage impaired (p.176, 181).Dcm, CpG, EcoKI – no effect.




• PagI cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

PagI (BspHI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 100 NR NR NR

5’...G↓C G C G C...3’

3’...C G C G C↑G...5’

#ER1091 200 u#ER1092 1000 u


Also available asFastDigest® BssHII (PteI)



Storage BufferPauI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with PauI, more than 95% of the DNA fragments can be ligated and recut.



NoteLow salt, high glycerol (>5%) concentrations or a large excess of enzyme may result in star activity.

PauI (BssHII) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 100 0-20 100

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PciI Fermentas enzyme PscI (PciI)

5’...G A A N N↓N N T T C...3’

3’...C T T N N↑N N A A G...5’

#ER1531 500 u#ER1532 2500 u


Also available asFastDigest® PdmI



Storage BufferPdmI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl,1 mM DTT, 5 mM MgCl2, 0.2 mg/ml BSAand 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with PdmI, more than 95% of the DNA fragments can be ligated and recut.



PdmI (XmnI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 0-20 0-20 100 0-20

5’...G C C↓G G C...3’

3’...C G G↑C C G...5’

#ER1521 200 u#ER1522 1000 u


Also available asFastDigest® NaeI (PdiI)



Storage BufferPdiI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 500 mM KCl,1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA, 0.15% Triton X-100 and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with PdiI, more than 90% of the DNA fragments can be ligated and recut.


Digestion of Agarose-embedded DNAMinimum 20 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded pBR322 DNA in 16 hours.

Note• For cleavage with PdiI at least two copies of its

recognition sequence are required.• Certain sites in pBR322 are difficult to cleave

with PdiI, the same as with its prototype NaeI.

PdiI (NaeI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 100 50-100

5’...G↓A W T C...3’

3’...C T W A↑G...5’


Also available asFastDigest® TfiI (PfeI)



Storage BufferPfeI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 250 mM KCl,1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSAand 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with PfeI, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam, Dcm, EcoKI – no effect.CpG: may overlap – blocked (p.179).EcoBI: may overlap – effect not determined.

PfeI (TfiI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 50-100 20-50 50-100

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5’...T↓C C N G G A...3’

3’...A G G N C C↑T...5’


Also available asFastDigest® PfoI



Storage BufferPfoI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with PfoI, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam: may overlap – cleavage impaired (p.176).Dcm, CpG: may overlap – blocked (p.177, 179).EcoKI, EcoBI – no effect.


NotePfoI cleavage is impaired by overlapping dam methylation and blocked by overlapping dcm methylation. To avoid dam or dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

PfoI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 0-20 100 50-100

PflFI Fermentas enzymes FastDigest® PsyI and PsyI (Tth111I)

PflMI Fermentas enzymes FastDigest® PflMI (Van91I) and Van91I (PflMI)

5’...C↓G T A C G...3’

3’...G C A T G↑C...5’


Also available asFastDigest® BsiWI (Pfl23II)



Storage BufferPfl23II is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with Pfl23II, more than 95% of the DNA fragments can be ligated and recut.



Pfl23II (BsiWI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 20-50 20-50 100 0-20

PhoI Fermentas enzymes FastDigest® HaeIII (BsuRI) and BsuRI (HaeIII)

PinAI Fermentas enzymes FastDigest® AgeI (BshTI) and BshTI (AgeI)

PleI Fermentas enzymes FastDigest® MlyI (SchI) and SchI (MlyI) (different cleavage position)

PmaCI Fermentas enzymes FastDigest® PmlI (Eco72I) and Eco72I (PmlI)

PmeI Fermentas enzymes FastDigest® MssI and MssI (PmeI)

PmlI Fermentas enzymes FastDigest® PmlI (Eco72I) and Eco72I (PmlI)

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5’...↓ 7(N)G A A C(N)5C T C(N)13↓...3’

3’...↑12(N)C T T G(N)5G A G(N) 8 ↑...5’




Storage BufferPpiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with PpiI, more than 90% of the DNA fragments can be ligated and recut.


Note• Incubation at 37°C results in 60% activity.• PpiI cleaves certain DNA sequences at

random 7 or 8 nt away on the top strand of the recognition sequence:

5’...↓7-8(N) G A A C (N)5 C T C (N)13↓...3’ 3’...↑ 12(N) C T T G (N)5 G A G (N)8 ↑...5’• The presence of SAM in a reaction mixture

results in incomplete cleavage with PpiI.• Greater than 10-fold overdigestion with PpiI

may result in star activity.

PpiI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 100 50-100 50-100

PpuMI Fermentas enzymes FastDigest® PpuMI (Psp5II) and Psp5II (PpuMI)

5’...A↓C A T G T...3’

3’...T G T A C↑A...5’

#ER1871 200 u#ER1872 1000 u




Storage BufferPscI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with PscI, more than 95% of the DNA fragments can be ligated and recut.



NotePscI, like its isoschizomers BspLU11I and PciI, is inhibited by nonionic detergents Triton X-100 (>0.002%) and Nonidet P40 (>0.001%).

PscI (PciI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 20-50 0-20 0-20 100 0-20

5’...Y A C↓G T R...3’

3’...R T G↑C A Y...5’

#ER1971 500 uSupplied with:10X Buffer Ppu21I 1 ml

Also available asFastDigest® BsaAI (Ppu21I)


Conditions for 100% Activity1X Buffer Ppu21I at 30°C.

Storage BufferPpu21I is supplied in: 10 mM potassium phosphate (pH 7.4 at 25°C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Ppu21I, more than 90% of the DNA fragments can be ligated and recut.




activity.• Low salt, high glycerol (>5%) concentra-



Ppu21I (BsaAI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100* 100* 20-50 NR NR NR

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5’...R G↓G W C C Y...3’

3’...Y C C W G↑G R...5’


Also available asFastDigest® PpuMI (Psp5II)



Storage BufferPsp5II is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Psp5II, more than 95% of the DNA fragments can be ligated and recut.



NotePsp5II is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Psp5II (PpuMI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 100 20-50 20-50 50-100 100

PshAI Fermentas enzymes FastDigest® PshAI (BoxI) and BoxI (PshAI)

PshBI Fermentas enzymes FastDigest® AseI (VspI) and VspI (AseI)

PsiI Fermentas enzymes FastDigest® PsiI (AanI) and AanI (PsiI)

5’...A A↓C G T T...3’

3’...T T G C↑A A...5’

#ER0941 300 u#ER0942 1500 u


Also available asFastDigest® AclI (Psp1406I)



Storage BufferPsp1406I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Psp1406I, more than 95% of the DNA fragments can be ligated and recut.



Psp1406I (AclI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 20-50 100 0-20

PspGI Fermentas enzymes EcoRII, FastDigest® MvaI and MvaI (BstNI) (different cleavage position and different sensitivity to methylation)

PspOMI Fermentas enzymes FastDigest® ApaI and ApaI (different cleavage position); FastDigest® Bsp120I and Bsp120I (PspOMI)

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5’...C T G C A↓G...3’

3’...G↑A C G T C...5’




Also available asFastDigest® PstI



Storage BufferPstI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with PstI, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam, Dcm,CpG, EcoKI, EcoBI – no effect.


Note• Conditions of high pH, low salt, high glycerol,

8% DMSO can cause star activity (Malyguine, E., et al., Gene, 8, 163-177, 1980).

• Surrounding sequences: the presence of adjacent runs of G-C base pairs confers sig-nificant resistance to cleavage (Armstrong, K. and Bauer, W.R., NAR, 10, 993-1007, 1982).

• 100% dUTP incorporation at the recognition site reduces PstI cleavage to 25% (Glenn, T.C., et al., Biotechniques, 17, 1086-1090, 1994).

• PstI will not cut AGCTGCAG when me-thylated by AluI methyltransferase.

PstI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 100 100 50-100 50-100

5’...G A C N↓N N G T C...3’

3’...C T G N N↑N C A G...5’


Also available asFastDigest® PsyI



Storage BufferPsyI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with PsyI, more than 60% of the DNA fragments can be ligated and more than 95% of these can be recut.



PsyI (Tth111I) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 0-20 50-100 0-20

5’...R↓G A T C Y...3’

3’...Y C T A G↑R...5’


Also available asFastDigest® PsuI



Storage BufferPsuI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with PsuI, more than 95% of the DNA fragments can be ligated and recut.


NoteHigh glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity.

PsuI (BstYI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 20-50 0-20 0-20 50-100 0-20

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5’...C G A T↓C G...3’

3’...G C↑T A G C...5’

#ER0621 300 u#ER0622 1500 u


Also available asFastDigest® PvuI



Storage BufferPvuI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with PvuI, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.



PvuI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 100 50-100 100

RcaI Fermentas enzymes FastDigest® BspHI (PagI) and PagI (BspHI)

5’...C A G↓C T G...3’

3’...G T C↑G A C...5’


#ER0635 5000 u#ER0633 HC, 12500 u

Both supplied with:10X Buffer G 2x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® PvuII



Storage BufferPvuII is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with PvuII, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.



NoteGreater than 15-fold overdigestion with PvuII may result in star activity.

PvuII Activity in Five Buffer System, %


B G O R Tango 2X Tango 50-100* 100 20-50 50-100 20-50* 20-50*

5’...G T↓A C...3’

3’...C A↑T G...5’



Also available asFastDigest® RsaI



Storage BufferRsaI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with RsaI, more than 95% of the DNA fragments can be ligated and recut.


RsaI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 100 0-20

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RsrII Fermentas enzymes FastDigest® RsrII (CpoI) and CpoI (RsrII)

5’...C A Y N N↓N N R T G...3’

3’...G T R N N↑N N Y A C...5’

#ER2001 200 u#ER2002 1000 u


Also available asFastDigest® MslI (RseI)



Storage BufferRseI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with RseI, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam, Dcm, CpG – no effect.EcoKI: may overlap – blocked (p.181).EcoBI: may overlap – effect not determined.


RseI (MslI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 50-100 100 20-50 100

5’...G A G C T↓C...3’

3’...C↑T C G A G...5’

#ER1131 1200 u#ER1135 2000 u

Both supplied with:10X Buffer SacI 1 ml10X Buffer Tango™ 1 ml

#ER1132 5x1200 uSupplied with:10X Buffer SacI 2x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® SacI


Conditions for 100% Activity1X Buffer SacI at 37°C.

Storage BufferSacI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with SacI, more than 95% of the DNA fragments can be ligated and recut.



Note• SacI is sensitive to cytosine methylation at

GAGmCTC but not GAGCTmC and insensi-tive to adenine methylation at GmAGCTC. AluI methyltransferase (AGmCT) can be used to block SacI.

• Supercoiled plasmids may require up to 5-fold more SacI for complete digestion than linear DNA.

• SacI is inhibited by common clinical anticoagulants found in some preparation of anticoagulated peripheral blood and bone marrow. Levels of EDTA and ACD (citric acid-sodium citrate-dextrose) in standard sample preparation have been shown to inhibit SacI. Three times the normal concentration for heparin is required to inhibit SacI. (Coad, J.E., et al., Inhibition of restriction endonu-cleases by common clinical anticoagulants, Anal. Biochem., 205, 368-369,1992).

SacI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 0-20 50-100 20-50

SacII Fermentas enzyme Cfr42I (SacII)

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SapI Fermentas enzymes FastDigest® SapI (LguI) and LguI (SapI)

SanDI Fermentas enzyme FastDigest® SanDI (KflI)

5’...G↓T C G A C...3’

3’...C A G C T↑G...5’

#ER0641 1500 u#ER0645 2000 u



Also available asFastDigest® SalI



Storage BufferSalI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C),100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with SalI, more than 95% of the DNA fragments can be ligated and recut.





• Supercoiled forms of pBR322 and pUC require 10-fold overdigestion with SalI to achieve complete digestion.

• Incubation at 25°C results in 50-75% activity.

SalI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 20-50 0-20 50-100

SauI Fermentas enzymes FastDigest® Bsu36I (Eco81I) and Eco81I (Bsu36I)

Sau96I Fermentas enzymes FastDigest® Sau96I (Cfr13I) and Cfr13I (Sau96I)

Sau3AI Fermentas enzymes FastDigest® Sau3AI (Bsp143I) and Bsp143I (Sau3AI); FastDigest® DpnI and DpnI (different cleavage position and different sensitivity to methylation); FastDigest® MboI and MboI (different sensitivity to methylation)

SbfI Fermentas enzymes FastDigest® SbfI (SdaI) and SdaI (SbfI)

5’...G C↓N G C...3’

3’...C G N↑C G...5’

#ER1641 200 u#ER1642 1000 u


Also available asFastDigest® Fnu4HI (SatI)



Storage BufferSatI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with SatI, more than 60% of the DNA fragments can be ligated and more than 90% of these can be recut.


SatI (Fnu4HI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 20-50 20-50 50-100 20-50

NoteAt least two copies of SatI recognition site are required for efficient cleavage.

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5’...A G T↓A C T...3’

3’...T C A↑T G A...5’

#ER0431 1000 uSupplied with:10X Buffer ScaI 1 ml

#ER0432 5000 uSupplied with:10X Buffer ScaI 2x1 ml

Also available asFastDigest® ScaI


Conditions for 100% Activity1X Buffer ScaI at 37°C.

Storage BufferScaI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with ScaI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.



Note• Conditions of low salt, high enzyme concen-

tration, glycerol concentration > 5% or pH > 8.0 may result in star activity.

• Supercoiled plasmids may require up to 20-fold more ScaI for complete digestion than linear DNAs.

ScaI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 0-20 0-20 0-20

Methylation EffectsDam, Dcm,CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.

5’...G A G T C(N)5↓...3’

3’...C T C A G(N)5↑...5’


Also available asFastDigest® MlyI (SchI)



Storage BufferSchI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with SchI, approxi-mately 90% of DNA fragments can be ligated and more than 90% of these can be recut.

Note• Greater than 10-fold overdigestion with SchI

may result in star activity.• SchI may remain associated with the cleaved


SchI (MlyI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 0-20 0-20 100 0-20

ScrFI Fermentas enzymes FastDigest® ScrFI (Bme1390I) and Bme1390I (ScrFI)

5’...C C T G C A↓G G...3’

3’...G G↑A C G T C C...5’

#ER1191 300 u#ER1192 1500 u

Both supplied with:10X Buffer SdaI 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® SbfI (SdaI)


Conditions for 100% Activity1X Buffer SdaI at 37°C.

Storage BufferSdaI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 5-fold overdigestion with SdaI, more than 95% of the DNA fragments can be ligated and recut.



NoteGreater than 5-fold overdigestion with SdaI may result in star activity.

SdaI (SbfI) Activity in Five Buffer System, % B G O R Tango 2X Tango NR NR 0-20 0-20 NR 20-50

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5’...G C G A T↓C G C...3’

3’...C G C↑T A G C G...5’


Also available asFastDigest® AsiSI (SfaAI)



Storage BufferSfaAI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with SfaAI, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.


Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded pJET1 DNA with inserted SfaAI recognition site in 16 hours.

SfaAI (AsiSI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 0-20 0-20 0-20 100 0-20

SecI Fermentas enzymes FastDigest® BsaJI (BseDI) and BseDI (BsaJI)

SexAI Fermentas enzyme FastDigest® SexAI (CsiI)

5’...G D G C H↓C...3’

3’...C↑H C G D G...5’

#ER0651 500 uSupplied with:10X Buffer SduI 1 ml

Also available asFastDigest® Bsp1286I (SduI)


Conditions for 100% Activity1X Buffer SduI at 37°C.

Storage BufferSduI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with SduI, more than 95% of the DNA fragments can be ligated and recut.



SduI (Bsp1286I) Activity in Five Buffer System, %


B G O R Tango 2X Tango NR 50-100* 50-100 0-20 NR NR

SfaNI Fermentas enzymes FastDigest® SfaNI (BmsI) and LweI (SfaNI)

SfcI Fermentas enzymes FastDigest® SfcI (BfmI) and BfmI (SfcI)

SfeI Fermentas enzymes FastDigest® SfcI (BfmI) and BfmI (SfcI)

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SfoI Fermentas enzymes FastDigest® EheI and EheI (SfoI); Fermentas enzyme FastDigest® SspDI (KasI) (different cleavage position)

SfuI Fermentas enzymes FastDigest® Bsp119I and Bsp119I (BstBI)

SgfI Fermentas enzymes FastDigest® AsiSI (SfaAI) and SfaAI (AsiSI)

5’...G G C C N N N N↓N G G C C...3’

3’...C C G G N↑N N N N C C G G...5’


Also available asFastDigest® SfiI



Storage BufferSfiI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 300 mM NaCl, 10 mM MgCl2, 1 mM DTT, 0.15% Triton X-100, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with SfiI, more than 95% of the DNA fragments can be ligated and recut.


Digestion of Agarose-embedded DNAMinimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embed-ded Adenovirus-2 DNA in 16 hours.

Note• Incubation at 37°C results in 10% activity. • SfiI cleavage is impaired by overlapping dcm


• For cleavage with SfiI at least two copies of its recognition sequence are required. The two sites can be on either the same or different DNA molecules. (Wertzell, L.M. et al., J. Mol. Biol., 248, 581-595, 1995.) Therefore also an oligonucleotide harboring a SfiI recognition site can be supplemented.

SfiI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 20-50 0-20 100 0-20

Note• DNA methylated by Dcm or CpG methyltrans-

ferases will be a substrate for SgeI.• Greater than 3-fold overdigestion with SgeI

may result in nonspecific cleavage.• At least two copies of SgeI recognition site

are required for an efficient cleavage.• Amount of the enzyme required for complete

digestion of methylated DNA depends on the number of SgeI recognition sites. DNA cleavage products generated by target site cleavage facilitate the nonspecific cleavage by SgeI. Therefore, optimization of enzyme amount is recommended for DNA cleavage.

• pBR322 DNA isolated from E.coli dcm+ strain (#SD0041) can be used as a DNA cleavage efficiency control. SgeI cleaves all six dcm-methylated targets on pBR322 DNA.


Conditions for 100% Activity1X Buffer SgeI at 37°C.

Storage BufferSgeI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA and 50% glycerol.

Ligation and RecleavageAfter a 2-fold overdigestion with SgeI, more than 80% of the DNA fragments can be ligated and recut..

Methylation EffectsDam, EcoKI, EcoBI – no effect.Dcm: always cleaves DNA methylated by Dcm methyltransferase (p.177).CpG: cleaves targets overlapping with CpG methylated sequences (p.179).

5’... m5C N N G (N)9 ↓...3’

3’... G N N C (N)13↑...5’** SgeI cleaves DNA targets containing 5-methylcytosine on

one or both DNA strands

#ER2211 250 uSupplied with:10X Buffer SgeI 1 ml

SgeI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 NR NR NR

Digestion of Agarose-embedded DNAA minimum of 3 units of the enzyme is required for complete digestion of 1 µg of agarose-embedded lambda DNA in 16 hours.

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SinI Fermentas enzymes FastDigest® AvaII (Eco47I) and Eco47I (AvaII)

5’...G G↓C G C G C C...3’

3’...C C G C G C↑G G...5’

#ER1891 300 u#ER1892 1500 u


Also available asFastDigest® AscI (SgsI)



Storage BufferSgsI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with SgsI, more than 95% of the DNA fragments can be ligated and recut.



SgsI (AscI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 50-100 100 50-100

5’...C C C↓G G G...3’

3’...G G G↑C C C...5’

#ER0661 1200 u#ER0665 2000 u


#ER0662 5x1200 u#ER0663 HC, 6000 u

Both supplied with:10X Buffer Tango™ 2x1 ml

Also available asFastDigest® SmaI



Storage BufferSmaI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with SmaI, more than 90% of the DNA fragments can be ligated and recut.



Note• Incubation at 37°C results in 50% activity.

SmaI has a half-life of 15 min at 37°C.• Incubation at 25°C results in 100% activity.• SmaI needs K+ to work for activity.

SmaI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 0-20 0-20 0-20 100 0-20

5’...C G↓T C G A C G...3’

3’...G C A G C T↑G C...5’




Storage BufferSgrDI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 30-fold overdigestion with SgrDI, more than 80% of the DNA fragments can be ligated. More than 95% of these can be recut.



NoteLow salt concentration or large excess of the enzyme may result in star activity.

SgrDI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 100 NR 100

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SmlI Fermentas enzyme SmoI (SmlI)

5’...A T T T↓A A A T...3’

3’...T A A A↑T T T A...5’


Also available asFastDigest® SwaI (SmiI)



Storage BufferSmiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with SmiI, more than 80% of the DNA fragments can be ligated and more than 95% of these can be recut.


Digestion of Agarose-embedded DNAMinimum 10 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded Adenovirus-2 DNA in 16 hours.


SmiI (SwaI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 20-50 0-20 20-50

5’...C↓T Y R A G...3’

3’...G A R Y T↑C...5’




Storage BufferSmoI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with SmoI, more than 95% of the DNA fragments can be ligated and recut.

Methylation EffectsDam: may overlap – cleavage impaired (p.176).Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.


Note• Incubation at 37°C results in 10% activity.• SmoI cleavage is impaired by overlapping dam

methylation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).


SmoI (SmlI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 20-50 0-20 20-50 100 20-50

5’...C C C G C(N)4↓...3’

3’...G G G C G(N)6↑...5’




Storage BufferSmuI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with SmuI, more than 95% of the DNA fragments can be ligated and recut.


NoteGreater than 40-fold overdigestion with SmuI may result in star activity.

SmuI (FauI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 0-20 20-50 100 20-50

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5’...C↓C G C...3’

3’...G G C↑G...5’


Also available asFastDigest®AciI (SsiI)



Storage BufferSsiI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with SsiI, approxi-mately 95% of the DNA fragments can be ligated. No more than 50% of these can be recut due to asymmetric recognition sequence of SsiI.



SsiI (AciI) Activity in Five Buffer System, % B G O R Tango 2X Tango NR 20-50 100 50-100 NR 100

5’...A A T↓A T T...3’

3’...T T A↑T A A...5’

#ER0771 500 u#ER0772 2500 u


Also available asFastDigest® SspI



Storage BufferSspI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with SspI, more than 90% of the DNA fragments can be ligated and recut.



NoteGreater than 15-fold overdigestion with SspI may result in star activity.

SspI Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 100 0-20 50-100 100 20-50

SnaI Fermentas enzymes FastDigest® BstZ17I (Bst1107I) and Bst1107I (BstZ17I)

SnaBI Fermentas enzymes FastDigest® SnaBI (Eco105I) and Eco105I (SnaBI)

SpeI Fermentas enzymes FastDigest® SpeI (BcuI) and BcuI (SpeI)

SphI Fermentas enzymes FastDigest® SphI (PaeI) and PaeI (SphI)

SplI Fermentas enzymes FastDigest® BsiWI (Pfl23II) and Pfl23II (BsiWI)

Sse232I Fermentas enzymes FastDigest® MreI and MreI (Sse232I)

Sse8387I Fermentas enzymes FastDigest® SbfI (SdaI) and SdaI (SbfI)

SspBI Fermentas enzymes FastDigest® Bsp1407I and Bsp1407I (BsrGI)

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StuI Fermentas enzymes FastDigest® StuI (Eco147I) and Eco147I (StuI)

StyI Fermentas enzymes FastDigest® StyI (Eco130I) and Eco130I (StyI)

StyD4I Fermentas enzymes FastDigest® ScrFI (Bme1390I) and Bme1390I (ScrFI) (different cleavage position)

SwaI Fermentas enzymes FastDigest® SwaI (SmiI) and SmiI (SwaI)

5’...A C N↓G T...3’

3’...T G↑N C A...5’

#ER1361 200 u#ER1362 1000 u


Also available asFastDigest® TaaI


Conditions for 100% Activity1X Buffer Tango™ at 65°C.To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.

Storage BufferTaaI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and Recleavage After 50-fold overdigestion with TaaI, approxi-mately 90% of the DNA fragments can be ligated and recut.



TaaI (HpyCH4III) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 50-100 100 100

5’...G↓G C G C C...3’

3’...C C G C G↑G...5’




Storage BufferSspDI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter a 50-fold overdigestion with SspDI, more than 95% of the DNA fragments can be ligated and recut.



SspDI (KasI) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 0-20 NR 0-20 100 20-50


Conditions for 100% Activity 1X Buffer R at 65°C.To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.

Storage BufferTaiI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with TaiI, more than 95% of DNA fragments can be ligated and recut.



Tail (MaeII*) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 100 100 50-100

* Unlike MaeII, TaiI produces DNA fragments with a 4-base 3’-extension

5’... A C G T↓...3’

3’...↑T G C A ...5’

#ER1141 400 u#ER1142 2000 u


FastDigest® TaiI

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5’...↓A A T T ...3’

3’... T T A A↑...5’



Also available asFastDigest® Tsp509I (TasI)


Conditions for 100% Activity1X Buffer B at 65°C.To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.

Storage BufferTasI is supplied in:10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with TasI, more than 95% of DNA fragments can be ligated and recut.



TasI (Tsp509I) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 20-50 0-20 20-50 0-20

5’...T↓C G A...3’

3’...A G C↑T...5’

#ER0671 3000 uSupplied with:10X Buffer TaqI 2x1 ml10X Buffer Tango™ 1 ml

#ER0672 5x3000 uSupplied with:10X Buffer TaqI 4x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® TaqI

Concentration10 u/µl,

Conditions for 100% Activity1X Buffer TaqI at 65°C.To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.

Storage BufferTaqI is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with TaqI, more than 95% of DNA fragments can be ligated and recut.


Note• TaqI is blocked by overlapping dam methyla-

tion. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

• Incubation at 37°C results in 10% activity. We recommend using TaqI, HC (#ER0673) at 37°C.

TaqI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 20-50 20-50 20-50 20-50

5’...W↓G T A C W...3’

3’...W C A T G↑W...5’


Also available asFastDigest® TatI



Storage BufferTatI is supplied in:10 mM Tris-HCl (pH 7.5 at 25°C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 2-fold overdigestion with TatI, more than 95% of the DNA fragments can be ligated and recut.


Note• Incubation at 37°C results in 20% activity.• Greater than 5-fold overdigestion with TatI

may result in star activity.


TatI Activity in Five Buffer System, % B G O R Tango 2X Tango NR 50-100* 20-50 20-50 100* 0-20

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TfiI Fermentas enzymes FastDigest® TfiI (PfeI) and PfeI (TfiI)

TliI Fermentas enzymes FastDigest® XhoI and XhoI

5’...T↓T A A ...3’

3’...A A T↑T...5’

#ER0981 300 u#ER0982 1500 u#ER0983 HC, 1500 u

All supplied with:10X Buffer R 1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® Tru1I


Conditions for 100% Activity1X Buffer R at 65°C.To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.

Storage BufferTru1I is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Tru1I, more than 90% of DNA fragments can be ligated and recut.


NoteIncubation at 37°C results in 10% activity. We recommend using Tru1I, HC (#ER0983) at 37°C.

Tru1I (MseI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 100 50-100 100

5’...G C S G↓C...3’

3’...C↑G S C G...5’

#ER1651 50 u#ER1652 250 u


Also available asFastDigest® TauI



Storage BufferTauI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with TauI, more than 90% of DNA fragments can be ligated and recut.


Note• Incubation at 37°C results in 30% activity.• TauI may remain associated with the cleaved


TauI Activity in Five Buffer System, % B G O R Tango 2X Tango 100 50-100 0-20 0-20 20-50 0-20

Tru9I Fermentas enzymes FastDigest® Tru1I and Tru1I (MseI); Fermentas enzyme FastDigest® MseI (SaqAI)

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5’...T A R C C A (N)11↓...3’

3’...A T Y G G T (N) 9↑...5’

#ER1991 50 uSupplied with:10X Buffer G 1 ml50X SAM 0.1 ml10X Buffer Tango™ 1 ml


Conditions for 100% Activity1X Buffer G at 55°C.SAM 0.05 mM.

Storage Buffer TsoI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with TsoI, approxi-mately 80% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.


Note• Incubation at 37° results in 10% activity.• TsoI requires only Mg2+ for its activity, but

is stimulated by S-adenosylmethionine. 0.05 mM S-adenosylmethionine gives a 2-fold increase in TsoI activity. Still, com-plete cleavage of some substrates with TsoI is difficult to achieve.

• TsoI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.


• TsoI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

TsoI Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2X Tango+SAM

NR 100 50-100 0-20 50-100 20-50

Tsp4CI Fermentas enzymes FastDigest® TaaI and TaaI (HpyCH4III)

Tsp45I Fermentas enzymes FastDigest® NmuCI and NmuCI (Tsp45I)

Tsp509I Fermentas enzymes FastDigest® Tsp509I (TasI) and TasI (Tsp509I)

TspMI Fermentas enzymes Cfr9I (XmaI); FastDigest® SmaI and SmaI (different cleavage position)

TspRI Fermentas enzymes FastDigest® TspRI (TscAI) and TscAI (TspRI)

5’... N N C A S T G N N↓...3’

3’...↑N N G T S A C N N ...5’


Also available asFastDigest® TspRI (TscAI)



Storage BufferTscAI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with TscAI, more than 95% of the DNA fragments can be ligated and recut.



activity.• Low salt, high glycerol (>5%) concentra-


• TscAI produces DNA fragments with a 9-base 3’-extension. To avoid atypical DNA band patterns, use 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

TscAI (TspRI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 20-50 100 20-50

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Tth111I Fermentas enzymes FastDigest® PsyI and PsyI (Tth111I)

5’...↓ 8(N)CAC(N)6TCC(N)12↓ ...3’

3’...↑13(N)GTG(N)6AGG(N) 7↑...5’




Storage BufferTstI is supplied in:10 mM Tris-HCl (pH 7.5 at 25°C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with TstI, more than 80% of DNA fragments can be ligated and recut.

Methylation EffectsDam: may overlap – blocked (p.176).Dcm, CpG, EcoKI – no effect.EcoBI: may overlap – effect not determined.


Note• TstI is blocked by overlapping dam methyla-

tion. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

• The presence of S-adenosylmethionine in a reaction mixture results in incomplete

cleavage with TstI.• Greater than 10-fold overdigestion with TstI

may result in star activity.• TstI may remain associated with the cleaved

DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical

DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

TstI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 0-20 100 0-20 100

VpaK11BI Fermentas enzymes FastDigest® AvaII (Eco47I) and Eco47I (AvaII)

5’...A T↓T A A T...3’

3’...T A A T↑T A...5’



Also available asFastDigest® AseI (VspI)



Storage BufferVspI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with VspI, more than 95% of DNA fragments can be ligated and recut.



VspI (AseI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 100 20-50 100 100

5’...C C A N N N N↓N T G G...3’

3’...G G T N↑N N N N A C C...5’

#ER0711 400 u#ER0712 2000 u


Also available asFastDigest® PflMI (Van91I)



Storage BufferVan91I is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with Van91I, more than 90% of DNA fragments can be ligated and recut.



NoteVan91I is blocked by overlapping dcm methy-lation. To avoid dcm methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

Van91I (PflMI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 50-100 100 20-50 50-100

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5’...R↓A A T T Y...3’

3’...Y T T A A↑R...5’


Also available asFastDigest® XapI



Storage BufferXapI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 10-fold overdigestion with XapI, more than 95% of the DNA fragments can be ligated and recut.



NoteGreater than 15-fold overdigestion with XapI may result in star activity.

XapI (ApoI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 100 0-20 0-20 100 20-50

5’...T C T A G A...3’

3’...A G A T C T...5’


#ER0685 3000 u#ER0682 5x1500 u#ER0683 HC, 7500 u

All supplied with:10X Buffer Tango™ 2x1 ml

Also available asFastDigest® XbaI



Storage BufferXbaI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with XbaI, more than 95% of DNA fragments can be ligated and recut.



NoteXbaI is blocked by overlapping dam methy-lation. To avoid dam methylation, use a dam–, dcm– strain such as GM2163 (#M0099).

XbaI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 20-50 0-20 100 50-100

5’...C C T N N↓N N N A G G...3’

3’...G G A N N N↑N N T C C...5’


Also available asFastDigest® EcoNI (XagI)



Storage BufferXagI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with XagI, approxi-mately 80% of DNA fragments can be ligated and more than 95% of these fragments can be recut.



XagI (EcoNI) Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 50-100 100 20-50 50-100

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5’...R C A T G↓Y...3’

3’...Y↑G T A C R...5’

#ER1471 500 u#ER1472 2500 u


Also available asFastDigest® NspI (XceI)



Storage BufferXceI is supplied in:10 mM Tris-HCl (pH 7.5 at 25°C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with XceI, more than 95% of the DNA fragments can be ligated and recut.


XceI (NspI) Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 0-20 0-20 0-20 100 0-20

5’...C↓T C G A G...3’

3’...G A G C T↑C...5’


#ER0695 5000 u#ER0692 5x2000 u#ER0693 HC, 10000 u

All supplied with:10X Buffer R 2x1 ml10X Buffer Tango™ 1 ml

Also available asFastDigest® XhoI



Storage BufferXhoI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with XhoI, more than 95% of the DNA fragments can be ligated and recut.



NoteSupercoiled plasmids may require up to 5-fold more XhoI for complete digestion than linear DNA.

XhoI Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 50-100 50-100 100 20-50 100

XhoII Fermentas enzymes FastDigest® PsuI and PsuI (BstYI)

XmaI Fermentas enzymes Cfr9I (XmaI); FastDigest® SmaI and SmaI (different cleavage position)

XmaIII Fermentas enzymes FastDigest® EagI (Eco52I) and Eco52I (EagI)

XmaCI Fermentas enzymes Cfr9I (XmaI); FastDigest® SmaI and SmaI (different cleavage position)

5’...C↓C T A G G...3’

3’...G G A T C↑C...5’

#ER1561 200 u#ER1562 1000 u


Also available asFastDigest® AvrII (XmaJI)



Storage BufferXmaJI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with XmaJI, more than 95% of the DNA fragments can be ligated and recut.



XmaJI (AvrII) Activity in Five Buffer System, % B G O R Tango 2X Tango 20-50 50-100 50-100 50-100 100 50-100

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XmnI Fermentas enzymes FastDigest® PdmI and PdmI (XmnI)

XspI Fermentas enzymes FastDigest® BfaI (FspBI) and FspBI (BfaI)

ZraI Fermentas enzymes FastDigest® AatII and AatII (different cleavage position)

Nicking Enzymes

5’...G T↓M K A C...3’

3’...C A K M↑T G...5’

#ER1481 400 u#ER1482 2000 u


Also available asFastDigest® AccI (XmiI)



Storage BufferXmiI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 50% (v/v) glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with XmiI, more than 95% of the DNA fragments can be ligated and recut.



XmiI (AccI) Activity in Five Buffer System, % B G O R Tango 2X Tango 100 0-20 0-20 0-20 50-100 20-50

5’...C C T N A G C...3’

3’...G G A N T↑C G...5’



Conditions for 100% Activity1X Buffer R: 10 mM Tris-HCl (pH 8.5 at 37°C), 10 mM MgCl2, 100 mM KCl, 0.1 mg/ml BSA.Incubate at 37°C.

Storage BufferNb.Bpu10I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA, 50% glycerol.

Nicking and Cleavage• Incubation of 10 u of enzyme with 1 µg pUC19

DNA (lacking the recognition sequence of Bpu10I) for 1 h at 37°C in 50 µl reaction buffer results in <10% conversion to circular form.

• Incubation of 1 u of enzyme with 1 µg pBR322 DNA for 1 h at 37°C in 50 µl reaction buffer results in <5% conversion to linear form.

Applications• Production of single-stranded circular DNA

from supercoiled double-stranded plasmids in vitro with subsequent use in DNA sequenc-ing, site-specific mutagenesis, etc.

• Creation of nested deletions. • Vector preparation for ligation independent

cloning method.• Preparations of covalently closed, double-

stranded linear DNA molecules.

NoteNb.Bpu10I may remain associated with the cleaved DNA. This may cause DNA band shift-ing during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

Nb.Bpu10I Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 20-50 100 20-50 50-100

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5’...G A A T G C...3’

3’...C T T A C↑G...5’



Conditions for 100% Activity1X Buffer O:50 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 100 mM NaCl and 0.1 mg/ml BSA.Incubate at 37°C.

Storage BufferNb.Mva1269I is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA, 50% glycerol.

Nicking and Cleavage• Incubation of 10 u of enzyme with 1 µg

pUC19 DNA (lacking the recognition sequence of Mva1269I) for 16 h at 37°C in 50 µl reaction buffer results in <3% conversion to circular form.

• Incubation of 10 u of enzyme with 1 µg pBR322 DNA for 16 h at 37°C in 50 µl reaction buffer results in <1% conversion to linear form.






Nb.Mva1269I Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 20-50 100 100 20-50 50-100

5’...C C↓T N A G C...3’

3’...G G A N T C G...5’



Conditions for 100% Activity 1X Buffer R: 10 mM Tris-HCl (pH 8.5 at 37°C), 10 mM MgCl2, 100 mM KCl, 0.1 mg/ml BSA. Incubate at 37°C.

Storage BufferNt.Bpu10I is supplied in: 10 mM Tris-HCl (pH 7.5 at 25°C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA, 50% glycerol.

Nicking and Cleavage• Incubation of 20 u of enzyme with 1 µg pUC19

DNA (lacking the recognition sequence of Bpu10I) for 1 h at 37°C in 50 µl reaction buffer results in <3% conversion to circular form.

• No detectable conversion of pBR322 DNA (1 µg) to linear form was observed after incu-bation with 20 u of Nt.Bpu10I for 1 h at 37°C in 50 µl reaction buffer.






NoteNt.Bpu10I may remain associated with the cleaved DNA. This may cause DNA band shift-ing during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

Nt.Bpu10I Activity in Five Buffer System, % B G O R Tango 2X Tango 0-20 0-20 100 100 0-20 50-100

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I-SceI is a site-specific homing enzyme encoded by a mitochondrial intron of Saccharomyces cerevisiae (1, 2). Intron-encoded endonucleases are proteins that promote the first step in mobility of the intron at the DNA level. They recognize and cleave an intronless allele of their cognate gene to insert a copy of the intron by a double-strand-break repair mechanism that results in the recipient allele also becoming intron-plus (3-5). Homing endonucleases recognize long, 14-40 base pairs sequences and are, therefore, extremely rare-cutting enzymes. They allow the introduction of a single or several double-strand breaks into complex genomes. This capability makes these enzymes powerful tools in high-resolution physical mapping, genome organization analysis, gene cloning, site-directed recombination and double-strand-break repair studies in diverse biological systems (4, 6).

Homing Enzyme

References1. Colleaux, L., et al., Recognition and cleavage site of the

intron-encoded omega transposase, Proc. Natl. Acad.Sci. U.S.A.,85, 6022-6026, 1988.

2. Monteihet, C., et al., Purification and characterization of the in vitro activity of I-SceI, a novel and highly specific endonuclease encoded by a group I intron, Nucleic Acids Res., 18, 1407-1413, 1990.

3. Dujon, B., Group I introns as mobile genetic elements: facts and mechanistic speculations – review, Gene, 82, 91-114, 1989.

4. Belfort, M., Roberts R.J., Homing endonucleases: keeping the house in order, Nucleic Acids Res., 25, 3379-3388, 1997.

5. Chevalier, B.S., Stoddard, B.L., Homing endonucleases:structural and functional insight into the catalysis of intron/intein mobility, Nucleic Acids Res., 29, 3757-3774, 2001.

6. Jasin, M., Genetic manipulation of genomes with rare-cutting endonucleases, Trends in Genetics, 12, 224-228, 1996.

Digestion of the Agarose-embedded DNA with I-SceI1. Immerse an agarose plug in 50-100 µl of the 1X Tango™ buffer without Mg-acetate* (supplied with the

enzyme). The volume of the buffer should be sufficient to completely cover the plug.2. Add 20 u of the enzyme.3. Incubate 2 hours on ice.4. Add 1/10 volume of the 100 mM Mg-acetate solution (supplied with the enzyme).5. Incubate at 37°C for 1 hour. Note* Diffusion of the enzyme in the absence of Mg-acetate prior to digestion is necessary, because I-SceI is unstable in the

presence of Mg2+ ions.

5’...T A G G G A T A A↓C A G G G T A A T...3’

3’...A T C C C↑T A T T G T C C C A T T A...5’

#ER1771 250 uSupplied with:10X Buffer Tango™ 1 ml10X Buffer Tango™ (without Mg-acetate) 1 ml100mM Mg-acetate 1 ml


Conditions for 100% Activity1X Buffer Tango™:33 mM Tris-acetate (pH 7.9 at 37°C), 10 mM Mg-acetate, 66 mM K-acetate and 0.1 mg/ml BSA. Incubate at 37°C.

Storage BufferI-SceI is supplied in:10 mM Tris-HCl (pH 7.4 at 25°C), 500 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% glycerol.

Ligation and RecleavageAfter 50-fold overdigestion with I-SceI, more than 95% of the DNA fragments can be ligated and recut.


Digestion of Agarose-embedded DNAMinimum 20 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded pUC-I-SceI DNA in 1 hour (see the protocol below).

Note• Homing enzymes do not have stringently

defined recognition sequences. They can tolerate minor sequence changes, which only partially affect the cleavage reaction.

• I-SceI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electro-phoresis.

• Assayed using pUC-I-SceI DNA.

I-SceI Activity in Five Buffer System, % B G O R Tango 2X Tango 50-100 50-100 50-100 50-100 100 50-100

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Protocols and Recommendations

1.5. DNA digestionWe recommend digesting 0.2-1.5 µg DNA with a 2-fold to 10-fold excess of enzyme in a total volume of 20 µl . A typical restriction enzyme digestion protocol is below.1. Add the following reaction components in

the order indicated:Water, nuclease-free (#R0581) 16-16.5 µl 10X recommended buffer for restriction enzyme 2 µl Substrate DNA 1 µl (~1 µg)Restriction enzyme 0.5-1 µl (5-10 u)Total volume 20 µl

2. Mix gently and spin down briefly.3. Incubate at the optimal reaction tempera-

ture for 1-16 hours.Note• The digestion reaction may be scaled either up or down.• Some enzymes require additional components to obtain

the stated activity. In these cases, add the required addi-tive and adjust the volume of water appropriately.

• See Tables 1.5 and 1.6 for restriction enzyme buffer composition and reaction conditions, respectively.

1.6. Digestion of PCR productsThe most convenient option for digestion of PCR-amplified DNA is the addition of a restric-tion enzyme directly to the reaction tube after completion of PCR. The majority of Fermentas restriction enzymes are active in Fermentas PCR buffers. However, digestion of PCR products in the am-plification mixture is often inefficient. Therefore, PCR reaction mixture should not make more than 1/3 volume of digestion reaction mixture to avoid inhibitory effects. 1. Add the following reaction components in

the order indicated:PCR reaction mixture 10 µl (~0.1-0.5 µg

of DNA)

Water, nuclease-free (#R0581) 16-17 µl

10X recommended buffer for restriction enzyme 2 µl *

Restriction enzyme 1-2 µl (10-20 u)

Total volume 30 µl

* Only 2 µl of 10X reaction buffer is required for unpurified PCR product in a 30 µl reaction volume.

2. Mix gently and spin down briefly.3. Incubate at the optimal reaction tempera-

ture for 1-16 hours.Note• For cloning applications, purification of PCR products prior

to digestion is necessary to remove the active thermophilic DNA polymerase present in the PCR mixture. DNA polyme-rases may alter the ends of the cleaved DNA and reduce the yield of ligation.

• If the restriction enzyme requires special additives (e.g., SAM), reduce the amount of water appropriately.

• If cleavage of the PCR product is inefficient purify the PCR product with the GeneJET™ PCR Purification Kit (#K0702) prior to digestion.

• After digestion, gel-purify the PCR product with the GeneJET™ Gel Extraction Kit (#K0692) or Silica Bead DNA Gel Extraction Kit (#K0513) to remove short DNA fragments which compete with the insert in a ligation reaction.

1.7. Setting up double digestion

1.7.1. Double digestion with FastDi-gest® enzymesFor protocols and recommendations for FastDi-gest® restriction enzymes see p.70.

1.7.2. DoubleDigest™ EngineDoubleDigest™ engine (www.fermentas/doubl-edigest.com) is a web tool for finding informa-tion on buffer and reaction conditions for double digests. Simply select two restriction enzymes required for digestion, submit the query and follow the recommendations.

1.7.3. Double digestion in the univer-sal Tango™ BufferUse Table 1.8 (p.168) to set up the optimal condi-tions for your double digestion reactions in the universal Tango™ buffer.1. Determine the concentration of Tango™ buffer

recommended for each restriction enzyme.2. If the recommended concentration of Tango™

buffer is the same for both enzymes, the digests can be performed at the same time.

3. If the two restriction enzymes require different concentrations of Tango™ buffer, perform the digestions sequentially. Set-up the first digestion reaction (including both enzymes) in 1X Tango™ buffer, then add an additional aliquot of the 10X Tango™ buffer (1/8 of initial reaction volume) to attain a final 2X Tango™ buffer concentration to optimize conditions for the second enzyme.

NoteIf both 1X and the 2X concentrations of Tango™ buffer are suitable for the double digest, use the 2X concentrated buffer to reduce probability of star activity.

1.7.4. Double digestion in Five Buffer SystemTable 1.6 (p.163) presents the activities of Fermentas restriction enzymes in the Five Buffer System.1. Determine which color-coded buffers are

recommended for each enzyme.2. If possible, use the buffer in which both

enzymes have 100% activity.3. If this is not possible, choose the buffer in

which both enzymes maintain at least 20% of their activity. Increase the amount of the enzymes in your digest according to their activity in that buffer.

NoteFor enzymes that are prone to relaxation of target specificity use a buffer in which they do not exhibit star activity.

1.7.5. Digestion with enzymes having different temperature optimumsFor double digestion with enzymes working at different temperatures perform sequential DNA cleavage. The optimal reaction temperature for each restriction enzyme is indicated both in the product description and the Certificate of Analysis. Information about the activity of mesophilic and thermophilic restriction enzymes at 37°C is given in Table 1.7 on p.167.

1.7.6. Sequential digestionIf a double digest is not possible due to buffer incompatibility or star activity, perform sequential digestions in the optimal buffer for each enzyme.1. Digest the DNA with the first restriction

enzyme in its optimal buffer.2. Purify the digested DNA by spin column or

chloroform extraction and ethanol precipitation.3. Digest the DNA with the second restriction

enzyme in its optimal buffer.

1.8. Stability during prolonged incubationThe stability of restriction enzymes in a reaction mixture depends on the nature of the enzyme, the buffer composition and the incubation temperature.If a restriction enzyme retains its activity in the reaction mixture for more than one hour, DNA can be digested with less enzyme in a pro-longed incubation period. The exact quantities of enzymes sufficient for overnight digestion are listed in the Table 1.6 on p.163.

1.9. Dilution of restriction enzymesDilution Buffer for Restriction Enzymes (#B19) is available from Fermentas for applications that require diluted enzymes.Enzymes diluted in this buffer retain 50-100% activity after storage for one month at -20°C.

1.10. Partial digestion of DNACertain cloning experiments may require incom-plete DNA cleavage. Such partial digestion of the DNA can be achieved by using the following conditions: • suboptimal concentration of the restriction

enzyme in the reaction mixture, • short incubation time, • incubation at a suboptimal temperature. For certain targets, partial cleavage of the desired DNA site is inefficient due to site prefer-ences of restriction enzymes (see Site Prefer-ences by Restriction Endonucleases on p.173).


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1.11. Digestion of agarose-embedded DNA

1. Embed the substrate DNA in 1% low melting temperature agarose, 1 µg / 30 µl.

2. Prepare ~30 µl agarose plugs with a GelSy-ringe® or similar agarose dispensing system.

3. Equilibrate the plug in 100 µl of the appropri-ate 1X restriction enzyme buffer for 15 min.

4. Place the plug in 100 µl of fresh 1X buffer containing the restriction enzyme (see Table 1.9 on p.170 for recommended quantities of enzymes).

5. Incubate at 30-37°C for mesophilic enzymes or at 50-55°C for thermophilic enzymes for 4-16 hours.

1.12. Inactivation of restriction enzymesInactivation of restriction enzymes following a digestion reaction is often required for down-stream applications. Thermal inactivation is a convenient method used to terminate enzyme activity. The majority of restriction enzymes can be heat-inactivated at 65°C or 80°C in 20 min. Information on susceptibility of Fermentas restriction enzymes to thermal inactivation is listed in the Table 1.6 on p.163), as well as in product descriptions and certificates of analysis.All known restriction enzymes with exception of BfiI, BmuI and BmrI require Mg2+ for DNA cleavage. Thus, addition of EDTA (20 mM final concentration) to the reaction mixture is an alternative method that can be used to halt digestion. EDTA is generally not compat-ible with most of downstream applications, therefore purification of the digested DNA using GeneJET™ PCR Purification Kit (#K0702) or by chloroform extraction (see p.358) is recom-mended.

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an oligonucleotide (also supplied with the enzyme), and Esp3I requires DTT.The following enzymes require unique buffers for optimal digestion: AarI, AjiI (BmgBI), BamHI, BfuI (BciVI), Bpu10I, BseXI (BbvI), Bsp143I (Sau3AI), Cfr9I (XmaI), Cfr10I (BsrFI), Eam1105I (AhdI), Ecl136II (EcoICRI), Eco52I (EagI), EcoRI, KpnI, Lsp1109I (BbvI), PacI, PasI, Ppu21I (BsaAI), SacI, ScaI, SdaI (SbfI), SduI (Bsp1286I), SgeI and TaqI. The compositions of these unique buffers are listed in Table 1.5 as well as in of restriction enzymes Certificates of Analysis.

All Fermentas restriction enzyme buffers should be stored at -20°C.

Reaction Conditions

Reaction BuffersOur Five Buffer System ensures the optimum reaction conditions for each restriction enzyme. This system consists of 10X B (blue), G (green), O (orange), R (red) and Tango™ (yellow) buffers (Table 1.6). All restriction enzymes are supplied in color-coded tubes to indicate the recom-mended reaction buffer. The recommended buffer and/or the universal Tango™ buffer, witch has been designed for double digestions of DNA, are supplied with each enzyme. Restriction enzyme buffers are also available separately. see Table 1.5 below for ordering information. For more information on double digestion see p.160 or visit the Fermentas DoubleDigest™ engine at www.fermentas.com/doubledigest.

To ensure consistent enzyme performance, Fermentas restriction enzyme buffers contain BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations. Multiple freeze-thaw cycles of the buffers will not cause BSA precipitation.

Fermentas restriction enzymes exhibit 100% of their certified activity in the recommended buffer. However, some enzymes require additives to achieve 100% activity. For example, AjuI, AlfI, BdaI, BplI, BseMII (BspCNI), Eco57I (AcuI), Eco57MI, FaqI (BsmFI), Hin4I, TsoI require S-adenosylmethionine, which is supplied with the enzyme, while AarI and BveI (BspMI) require

Table 1.5. Reaction buffers for restriction enzymes.

10X bufferCat.

#

1X buffer compositionpH, at

37°CQuantity,

ml

Tris-HCl, mM

Tris-acetate,

mM

Bis-Tris Propane-HCl,

mM

MgCl2,mM

NaCl, mM

KCl, mM

Mg-acetate,

mM

K-acetate, mM

Sodium glutamate,

mM

Triton X-100,

%

EDTA, mM

DTT, mM

Gly cerol,

%

BSA, mg/ml

Five Buffer System

10X Buffer B BB5 5x1 10 10 0.1 7.5

10X Buffer G BG5 5x1 10 10 50 0.1 7.5

10X Buffer O BO5 5x1 50 10 100 0.1 7.5

10X Buffer R BR5 5x1 10 10 100 0.1 8.5

10X Buffer Tango™ BY5 5x1 33 10 66 0.1 7.9

Buffer Set for Restriction Enzymes B30

1 ml of each buffer

Unique buffers

10X Buffer AarI, AjiI, Bpu10I, ScaI, PasI B27 1 10 10 100 0.1 6.5

10X Buffer BamHI, Lsp1109I, SgeI B57 5x1 10 5 100 0.02 0.1 8.0

10X Buffer BfuI B59 1 50 15 100 0.1 7.9

10X Buffer BseXI B31 1 50 2 100 0.1 7.5

10X Buffer Bsp143I B13 1 33 10 66 0.02 0.1 7.9

10X Buffer Cfr9I B02 1 10 5 200 0.1 7.2

10X Buffer Cfr10I B04 1 10 5 100 0.02 0.1 8.0

10X Buffer Eam1105I B25 1 10 5 100 0.1 7.5

10X Buffer Ecl136II, PacI,SacI B26 1 10 10 0.1 6.5

10X Buffer Eco52I B22 1 10 3 100 0.1 8.5

10X Buffer EcoRI B12 5x1 50 10 100 0.02 0.1 7.5

10X Buffer KpnI B29 1 10 10 0.02 0.1 7.5

10X Buffer SdaI B24 1 37 15 150 0.1 7.0

10X Buffer SduI, Ppu21I B23 1 10 3 150 0.1 7.2

10X Buffer TaqI B28 1 10 5 100 0.1 8.0

Dilution Buffer for Restriction Enzymes B19 5x1 ml 10 100 1 1 50 0.2 7.4

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Recommended Reaction Conditions

Table 1.6. Reaction conditions for restriction enzymes.

Enzyme

Units for overnight

incubation, u/µg DNA


in 20 min

Reco- mmended buffer for

100% activity

Enzyme activity in Fermentas buffers, %Tango™ buffer

for double digestion

B (blue) 1X

G (green) 1X

O (orange) 1X

R (red) 1X

Tango™ (yellow)1X 2X

AanI (PsiI) 0.5 65° C Tango™ 50-100 50-100 0-20 0-20 100 20-50 1X or 2X

AarI 1.0 65° CUnique (+oligo)

NR NR0-20

(+oligo)0-20

(+oligo)NR

50-100 (+oligo)

2X (+oligo)

AasI (DrdI) 0.1 80° C B 100 20-50 0-20 0-20 50-100* 0-20 1X*

AatII 0.3 65° C Tango™ 50-100 20-50 0-20 0-20 100 20-50 1X or 2X

Acc65I (Asp718I) 0.3 65° C O 0-20 20-50 100 20-50 20-50 50-100 1X or 2X

AdeI (DraIII ) 0.5 NO G 0-20 100 20-50 100 100* 20-50 1X* or 2X

AjiI (BmgBI) 0.5 65° C Unique NR NR 20-50* NR NR 20-50* 2X*

AjuI 0.5 65° CR

(+SAM)0-20

(+SAM)50-100 (+SAM)

20-50 (+SAM)

100 (+SAM)

50-100 (+SAM)

50-100 (+SAM)

1X or 2X (+SAM)

AlfI 1.0 65° CR

(+SAM)0-20

(+SAM)0-20

(+SAM)0-20

(+SAM)100

(+SAM)0-20

(+SAM)20-50

(+SAM)2X

(+SAM)

AloI (30°C) 0.1 65° C R 0-20 0-20 0-20 100 20-50 100 1X or 2X

AluI 0.1 65° C Tango™ 50-100 0-20 0-20 0-20 100 20-50 1X or 2X

Alw21I (BsiHKAI) 0.1 65° C O 0-20 20-50 100 50-100 20-50 50-100 1X or 2X

Alw26I (BsmAI) 0.2 65° C Tango™ 50-100 100 0-20 0-20 100 100 1X or 2X

Alw44I (ApaLI) 0.1 65° C Tango™ 50-100 100 0-20 50-100 100 50-100 1X or 2X

ApaI 0.2 65° C B 100 20-50 0-20 0-20 20-50 0-20 1X

BamHI 0.5 80° C (10 u) Unique 20-50* 100 20-50 50-100* 100* 50-100 1X* or 2X

BauI (BssSI) 0.2 65° C Tango™ 0-20 50-100 0-20 50-100 100 50-100 1X or 2X

BclI (55°C) 0.1 80° C (10 u) G 20-50 100 20-50 20-50 100* 100 1X* or 2X

BcnI (NciI) 0.2 65° C Tango™ 20-50 50-100 50-100 50-100 100 50-100 1X or 2X

BcuI (SpeI) 0.5 NO Tango™ 50-100 50-100 0-20 20-50 100 0-20 1X

BdaI (30°C) 1.0 65° CG

(+SAM)NR

100 (+SAM)

0-20 (+SAM)

20-50 (+SAM)

50-100*(+SAM)

50-100 (+SAM)

1X* or 2X(+SAM)

BfiI (BmrI) 1.0 65° C Tango™ 20-50 20-50 0-20 0-20 100 0-20 1X

BfmI (SfcI) 1.0 65° C Tango™ 0-20 50-100 0-20 0-20 100 20-50 1X or 2X

BfuI (BciVI) 1.0 80° C Unique NR NR 0-20 0-20 NR 50-100* 2X*

BglI 0.1 65° C O 0-20 50-100 100 100 0-20 100 2X

BglII 0.1 NO O 0-20 20-50 100 50-100 0-20 100 2X

Bme1390I (ScrFI) 0.2 80° C O 20-50 50-100 100 50-100 50-100 50-100 1X or 2X

BoxI (PshAI) 0.5 80° C Tango™ 0-20 0-20 0-20 20-50 100 20-50 1X or 2X

BpiI (BbsI) 0.3 65° C G 20-50 100 50-100 50-100 50-100 50-100 1X or 2X

BplI 0.3 65° CTango™

(+SAM)0-20

(+SAM)20-50

(+SAM)0-20

(+SAM)0-20

(+SAM)100

(+SAM)20-50

(+SAM)1X

(+SAM)

Bpu10I 0.2 80° C Unique 0-20 20-50* 50-100* 100* 50-100* 100* 1X* or 2X*

Bpu1102I (BlpI) 0.1 80° C Tango™ 50-100 50-100 20-50 20-50 100 20-50 1X or 2X

BseDI (BsaJI) (55°C) 0.2 80° C Tango™ 50-100 20-50 0-20 0-20 100 50-100 1X or 2X

BseGI (BtsCI) (55°C) 0.2 80° C Tango™ 20-50 50-100 20-50 20-50 100 20-50 1X or 2X

BseJI (BsaBI) (65°C) 0.1 NO O NR 100* 100 NR NR 100* 2X*

BseLI (BslI) (55°C) 0.1 NO Tango™ 20-50 100 50-100 20-50 100 50-100 1X or 2X

BseMI (BsrDI) (55°C) 0.3 80° C R 0-20 20-50 0-20 100 50-100 50-100 1X or 2X

BseMII (BspCNI) (55°C)

0.5 80° CTango™

(+SAM)50-100 (+SAM)

50-100 (+SAM)

50-100 (+SAM)

50-100 (+SAM)

100 (+SAM)

50-100 (+SAM)

1X or 2X (+SAM)

BseNI (BsrI) (65°C) 0.1 80° C B 100 20-50 0-20 0-20 50-100 20-50 1X or 2X

BseSI (Bme1580I) (55°C) 0.1 80° C (10 u) G 20-50 100 0-20 20-50 50-100 0-20 1X

BseXI (BbvI) (65°C) 0.3 80° C Unique NR NR NR NR NR NR NR

Bsh1236I (BstUI) 0.1 65°C R 0-20 0-20 50-100 100 20-50 50-100 1X or 2X

Bsh1285I (BsiEI) 0.2 80° C G 20-50 100 20-50 0-20 0-20 20-50 2X


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Enzyme

Units for overnight



in 20 min


100% activity



B (blue) 1X

G (green) 1X

O (orange) 1X

R (red) 1X


BshNI (BanI) 0.3 65° C O 0-20 20-50 100 50-100 0-20 100 2X

BshTI (AgeI) 0.1 80° C O 0-20 20-50 100 50-100 20-50 20-50 1X or 2X

Bsp68I (NruI) 0.2 65° C O 0-20 20-50 100 50-100 20-50 50-100 1X or 2X

Bsp119I (BstBI) 0.1 80° C Tango™ 20-50 0-20 0-20 0-20 100 100 1X or 2X

Bsp120I (PspOMI) 0.1 80° C B 100 20-50 0-20 20-50 50-100 0-20 1X

Bsp143I (Sau3AI) 0.1 65° C Unique 20-50 20-50 0-20 0-20 50-100 20-50 1X or 2X

Bsp1407I (BsrGI) 0.1 65° C Tango™ 0-20 20-50 0-20 20-50 100 50-100 1X or 2X

BspLI (NlaIV) 0.3 65° C Tango™ 50-100 50-100 0-20 20-50 100 20-50 1X or 2X

BspOI (BmtI) 0.2 80° C O 0-20 0-20 100 100 0-20 20-50 2X

BspPI (AlwI) (55°C) 0.5 80° C Tango™ 20-50 20-50 0-20 0-20 100 0-20 1X

BspTI (AfIII ) 0.1 65° C O 0-20 0-20 100 20-50 0-20 50-100 2X

Bst1107I (BstZ17I) 0.1 80° C O 20-50 50-100 100 100 20-50 100 1X or 2X

BstXI (55°C) 0.1 80° C O 20-50 100 100 50-100 50-100 100 1X or 2X

Bsu15I (ClaI) 0.1 65° C Tango™ 20-50 20-50 20-50 20-50 100 20-50 1X or 2X

BsuRI (HaeIII) 0.1 80° C R 20-50 20-50 50-100 100 50-100 100 1X or 2X

BveI (BspMI) 0.2 65° CO

(+oligo)0-20

(+oligo)20-50

(+oligo)100

(+oligo)20-50

(+oligo)50-100 (+oligo)

100 (+oligo)

1X or 2X (+oligo)

CaiI (AlwNI) 0.2 65° C Tango™ 20-50 20-50 20-50 50-100 100 50-100 1X or 2X

CfrI (EaeI) 1.0 65° C Tango™ 50-100* 50-100 0-20 0-20 100 0-20 1X

Cfr9I (XmaI) 0.2 65° C Unique 0-20 0-20 0-20 0-20 20-50 0-20 1X

Cfr10I (BsrFI) 0.1 NO Unique 0-20 20-50 20-50 50-100* 20-50 50-100 1X or 2X

Cfr13I (Sau96I) 0.3 65° C Tango™ 50-100 50-100 20-50 20-50 100 20-50 1X or 2X

Cfr42I (SacII) 0.1 65° C B 100 50-100 0-20 0-20 50-100 0-20 1X

CpoI (RsrII) 0.5 65° C Tango™ 20-50 50-100 50-100 20-50 100 50-100 1X or 2X

CseI (HgaI) 0.5 80° C (10 u) R NR 50-100* 50-100 100 100* 50-100 1X* or 2X

Csp6I (CviQI) 0.1 65° C B 100 50-100 0-20 0-20 50-100 0-20 1X

DpnI 0.1 80° C Tango™ 100 100 50-100 50-100 100 50-100 1X or 2X

DraI 0.1 65° C Tango™ 50-100 50-100 20-50 20-50 100 50-100 1X or 2X

Eam1104I (EarI) 0.5 65° C Tango™ 50-100 50-100 0-20 0-20 100 0-20 1X

Eam1105I (AhdI) 0.1 65° C Unique 20-50 50-100 0-20 0-20 50-100 20-50 1X or 2X

Ecl136II (EcoICRI) 0.2 65° C Unique 50-100 20-50 0-20 0-20 50-100 0-20 1X

Eco24I (BanII) 0.2 65° C Tango™ 50-100 50-100 0-20 20-50 100 0-20 1X

Eco31I (BsaI) 0.3 65° C G 50-100 100 0-20 0-20 50-100 20-50 1X or 2X

Eco32I (EcoRV) 0.1 80° C R 0-20 50-100 50-100 100 20-50 100 1X or 2X

Eco47I (AvaII) 0.3 65° C R 0-20 50-100 50-100 100 50-100 50-100 1X or 2X

Eco47III (AfeI) 0.1 65° C O 0-20 20-50 100 100 50-100 100 1X or 2X

Eco52I (EagI) 0.2 65° C Unique 0-20 0-20 0-20 20-50 0-20 20-50 2X

Eco57I (AcuI) 1.0 65° CG

(+SAM)100

(+SAM)100

(+SAM)20-50

(+SAM)20-50

(+SAM)50-100 (+SAM)

50-100 (+SAM)

1X or 2X (+SAM)

Eco57MI 1.0 65° CB

(+SAM)100

(+SAM)50-100 (+SAM)

0-20 (+SAM)

20-50 (+SAM)

50-100 (+SAM)

0-20 (+SAM)

1X (+SAM)

Eco72I (PmlI) 0.5 65° C Tango™ NR NR 0-20 0-20 100 20-50 1X or 2X

Eco81I (Bsu36I) 0.1 80° C Tango™ 50-100 100 0-20 0-20 100 0-20 1X

Eco88I (AvaI) 0.2 65° C Tango™ 100 50-100 0-20 0-20 100 20-50 1X or 2X

Eco91I (BstEII) 0.1 65° C O 20-50 20-50 100 50-100 50-100 100 1X or 2X

Eco105I (SnaBI) 0.5 65° C Tango™ 100* 50-100 0-20 0-20 100 0-20 1X

Eco130I (StyI) 0.2 65° C O 0-20 20-50 100 50-100 50-100 100 1X or 2X

Eco147I (StuI) 0.1 80° C B 100 50-100 20-50 20-50 50-100 0-20 1X

EcoO109I (DraII) 0.2 65° C Tango™ 50-100 20-50 20-50 20-50 100 100 1X or 2X

EcoRI 0.2 65° C Unique 0-20 NR 100 100* NR 100 2X


165

1




Enzyme

Units for overnight



in 20 min


100% activity



B (blue) 1X

G (green) 1X

O (orange) 1X

R (red) 1X


EcoRII 0.1 80° C O 20-50 50-100 100 50-100 20-50 50-100 1X or 2X

EheI (SfoI) 1.0 65° C Tango™ 20-50 50-100 0-20 0-20 100 20-50 1X or 2X

Esp3I (BsmBI) 0.2 65° CTango™

(+DTT)100

(+DTT)20-50 (+DTT)

0-20 (+DTT)

0-20 (+DTT)

100 (+DTT)

0-20 (+DTT)

1X (+DTT)

FaqI (BsmFI) 1.0 80° CTango™

(+SAM)20-50

(+SAM)20-50

(+SAM)0-20

(+SAM)0-20

(+SAM)100

(+SAM)20-50

(+SAM)1X or 2X (+SAM)

FspAI 0.2 65° C O 0-20 0-20 100 50-100 0-20 50-100 2X

FspBI (BfaI) 0.3 65° C Tango™ 50-100 20-50 0-20 0-20 100 0-20 1X

GsuI (BpmI) (30°C) 1.0 65° C B 100 50-100 20-50 20-50 100 50-100 1X or 2X

HhaI 0.1 NO Tango™ 50-100 50-100 20-50 20-50 100 20-50 1X or 2X

Hin1I (BsaHI) 0.1 65° C G 20-50 100 20-50 20-50 20-50 20-50 1X or 2X

Hin1II (NlaIII ) 0.3 65° C G 50-100 100 20-50 50-100 50-100 50-100 1X or 2X

Hin4I 0.2 65° CTango™ (+SAM)

20-50 (+SAM)

20-50 (+SAM)

0-20 (+SAM)

0-20 (+SAM)

100 (+SAM)

0-20 (+SAM)

1X (+SAM)

Hin6I (HinP1I) 0.1 65° C Tango™ 50-100 50-100 20-50 20-50 100 50-100 1X or 2X

HincII (HindII) 0.1 65° C Tango™ 50-100 50-100 20-50 50-100 100 50-100 1X or 2X

HindIII 0.1 80° C R 0-20 20-50 0-20 100 50-100 50-100 1X or 2X

HinfI 0.1 65° C R 0-20 20-50 50-100 100 50-100 50-100 1X or 2X

HpaII 0.1 65° C Tango™ 50-100 50-100 0-20 20-50 100 20-50 1X or 2X

HphI 0.1 65° C B 100 0-20 0-20 0-20 20-50 0-20 1X

Hpy8I (MjaIV) 0.1 80° C Tango™ 50-100 50-100 0-20 20-50 100 50-100 1X or 2X

HpyF3I (DdeI) 0.2 65° C Tango™ 20-50 20-50 20-50 20-50 100 50-100 1X or 2X

HpyF10VI (MwoI) 0.1 80° C Tango™ 0-20 0-20 0-20 0-20 100 50-100 1X or 2X

KpnI 0.2 80° C Unique 20-50 0-20 0-20 0-20 20-50 0-20 1X

Kpn2I (BspEI) (55°C) 0.2 80° C Tango™ 50-100 50-100 0-20 20-50 100 50-100 1X or 2X

KspAI (HpaI) 0.5 65° C B 100 50-100* 20-50 20-50 100* 50-100 1X* or 2X

LguI (SapI) 0.5 65° C Tango™ 20-50 50-100 20-50 20-50 100 20-50 1X or 2X

Lsp1109I (BbvI) 0.5 65° C Unique 0-20 20-50* 50-100* 100* 20-50* 20-50* 1X* or 2X*

LweI (SfaNI) 0.2 65° C Tango™ 0-20 0-20 0-20 20-50 100 20-50 1X or 2X

MauBI 0.1 65° C Tango™ 0-20 0-20 0-20 0-20 100 0-20 1X

MbiI (BsrBI) 0.2 65° C Tango™ 20-50 100 20-50 20-50 100 20-50 1X or 2X

MboI 0.1 65° C R 50-100 50-100 50-100 100 50-100 100 1X or 2X

MboII 1.0 65° C B 100 50-100 20-50 0-20 50-100 20-50 1X or 2X

MlsI (MscI) 0.5 65° C R 0-20 20-50 0-20 100 20-50 50-100 1X or 2X

MluI 0.1 80° C R 0-20 20-50 50-100 100 20-50 50-100 1X or 2X

MnlI 0.5 65° C G 50-100 100 20-50 20-50 20-50 20-50 1X or 2X

Mph1103I (NsiI) 0.3 65° C R 0-20 50-100 20-50 100 50-100 50-100 1X or 2X

MreI (Sse232I) 0.5 80° C G 20-50 100 0-20 0-20 50-100 0-20 1X

MspI (HpaII) 0.3 80° C Tango™ 50-100 50-100 0-20 0-20 100 50-100 1X or 2X

MssI (PmeI) 0.5 65° C B 100 0-20 0-20 0-20 20-50 0-20 1X

MunI (MfeI) 0.1 65° C G 100 100 0-20 0-20 100 0-20 1X

MvaI (BstNI) 0.1 NO R 20-50 20-50 50-100 100 20-50* 100 1X* or 2X

Mva1269I (BsmI) 0.1 65° C R 0-20 20-50 50-100 100 0-20 50-100 2X

NcoI 0.1 65° C Tango™ 20-50 20-50 20-50 50-100 100 100 1X or 2X

NdeI 0.2 65° C O 0-20 0-20 100 50-100 0-20 50-100 2X

NheI 0.2 65° C Tango™ 100 20-50 0-20 0-20 100 0-20 1X

NmuCI (Tsp45I) 0.1 65° C R 0-20 20-50 50-100 100 20-50 50-100 1X or 2X

NotI 0.1 80° C O 0-20 0-20 100 20-50 0-20 20-50 2X

NsbI (FspI) 0.1 65° C Tango™ 20-50 50-100 0-20 20-50 100 20-50 1X or 2X

OliI (AleI) 0.2 65° C R 0-20 0-20 0-20 100 0-20 50-100 2X


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Enzyme

Units for overnight



in 20 min


100% activity



B (blue) 1X

G (green) 1X

O (orange) 1X

R (red) 1X


PacI 0.3 65° C Unique 20-50 20-50 0-20 0-20 0-20 0-20 NR

PaeI (SphI) 0.1 65° C B 100 50-100 0-20 0-20 50-100 0-20 1X

PagI (BspHI) 0.2 80° C O 0-20 50-100 100 NR NR NR NR

PasI (55°C) 0.2 80° C Unique NR NR NR NR NR NR NR

PauI (BssHII) 0.2 80° C R 0-20 0-20 100 100 0-20 100 2X

PdiI (NaeI) 0.5 65°C Tango™ 50-100 20-50 0-20 0-20 100 50-100 1X or 2X

PdmI (XmnI) 0.5 65° C Tango™ 20-50 50-100 0-20 0-20 100 0-20 1X

PfeI (TfiI) 0.1 65° C O 0-20 20-50 100 50-100 20-50 50-100 1X or 2X

Pfl23II (BsiWI) 0.3 65° C Tango™ 20-50 50-100 20-50 20-50 100 0-20 1X

PfoI 0.1 65° C Tango™ 0-20 20-50 50-100 0-20 100 50-100 1X or 2X

PpiI (30°C) 0.1 65° C R 0-20 0-20 0-20 100 50-100 50-100 1X or 2X

Ppu21I (BsaAI) (30°C) 0.5 65° C Unique 50-100* 100* 20-50 NR NR NR NR

PscI (PciI) 0.2 65° C Tango™ 20-50 20-50 0-20 0-20 100 0-20 1X

Psp5II (PpuMI) 0.2 80° C G 0-20 100 20-50 20-50 50-100 100 1X or 2X

Psp1406I (AclI) 0.5 65° C Tango™ 100 50-100 0-20 20-50 100 0-20 1X

PstI 0.2 NO O 50-100 50-100 100 100 50-100 50-100 1X or 2X

PsuI (BstYI) 0.5 80° C B 100 20-50 0-20 0-20 50-100 0-20 1X

PsyI (Tth111I) 0.1 80° C B 100 50-100 0-20 0-20 50-100 0-20 1X

PvuI 0.2 80° C (10 u) R 0-20 20-50 50-100 100 50-100 100 1X or 2X

PvuII 0.2 NO G 50-100* 100 20-50 50-100 20-50* 20-50* 1X* or 2X*

RsaI 0.2 80° C Tango™ 50-100 20-50 0-20 0-20 100 0-20 1X

RseI (MslI) 0.5 65° C R 0-20 50-100 50-100 100 20-50 100 1X or 2X

SacI 0.2 65° C Unique 50-100 20-50 0-20 0-20 50-100 20-50 1X or 2X

SalI 0.1 65° C O 0-20 0-20 100 20-50 0-20 50-100 2X

SatI (Fnu4HI) 0.1 65° C G 20-50 100 20-50 20-50 50-100 20-50 1X or 2X

ScaI 0.5 80° C (10 u) Unique 0-20 0-20 0-20 0-20 0-20 0-20 NR

SchI (MlyI) 0.2 65° C Tango™ 20-50 50-100 0-20 0-20 100 0-20 1X

SdaI (SbfI) 0.3 80° C Unique NR NR 0-20 0-20 NR 20-50 2X

SduI (Bsp1286I) 0.3 65° C Unique NR 50-100* 50-100 0-20 NR NR NR

SfaAI (AsiSI) 0.2 80° C Tango™ 50-100 0-20 0-20 0-20 100 0-20 1X

SfiI (50°C) 0.2 NO G 50-100 100 20-50 0-20 100 0-20 1X

SgeI NR 65° C Unique 0-20 0-20 0-20 NR NR NR NR

SgrDI 0.3 65° C R 0-20 0-20 0-20 100 NR 100 2X

SgsI (AscI) 0.1 65° C Tango™ 0-20 0-20 0-20 50-100 100 50-100 1X or 2X

SmaI (30°C) 0.2 65° C Tango™ 50-100 0-20 0-20 0-20 100 0-20 1X

SmiI (SwaI) (30°C) 0.1 65° C O 0-20 0-20 100 20-50 0-20 20-50 2X

SmoI (SmlI) (55°C) 0.2 80° C Tango™ 50-100 20-50 0-20 20-50 100 20-50 1X or 2X

SmuI (FauI) 0.2 65° C Tango™ 50-100 50-100 0-20 20-50 100 20-50 1X or 2X

SsiI (AciI) 0.5 65° C O NR 20-50 100 50-100 NR 100 2X

SspI 0.1 65° C G 20-50 100 0-20 50-100 100 20-50 1X or 2X

SspDI (KasI) 0.1 80° C Tango™ 20-50 0-20 NR 0-20 100 20-50 1X or 2X

TaaI (HpyCH4III) (65°C) 0.2 NO Tango™ 0-20 0-20 0-20 50-100 100 100 1X or 2X

TaiI (MaeII) (65°C) 0.3 NO R 50-100 50-100 20-50 100 100 50-100 1X or 2X

TaqI (65°C) 0.3 NO Unique 0-20 20-50 20-50 20-50 20-50 20-50 1X or 2X

TasI (Tsp509I) (65°C) 0.3 NO B 100 50-100 20-50 0-20 20-50 0-20 1X

TatI (65°C) 0.2 NO Tango™ NR 50-100* 20-50 20-50 100* 0-20 1X*

TauI (55°C) 1.0 NO B 100 50-100 0-20 0-20 20-50 0-20 1X

Tru1I (MseI) (65°C) 0.2 NO R 50-100 50-100 20-50 100 50-100 100 1X or 2X

TscAI (TspRI) (65°C) 0.2 NO Tango™ 50-100 50-100 20-50 20-50 100 20-50 1X or 2X


167

1



Enzyme

Units for overnight



in 20 min


100% activity



B (blue) 1X

G (green) 1X

O (orange) 1X

R (red) 1X


TsoI (55°C) 1.0 80° CG

(+SAM)NR

(+SAM)100

(+SAM)50-100 (+SAM)

0-20 (+SAM)

50-100 (+SAM)

20-50 (+SAM)

1X or 2X (+SAM)

TstI 0.1 NO R 0-20 0-20 0-20 100 0-20 100 2X

Van91I (PflMI) 0.1 65° C R 0-20 50-100 50-100 100 20-50 50-100 1X or 2X

VspI (AseI) 0.1 65° C O 0-20 50-100 100 20-50 100 100 1X or 2X

XagI (EcoNI) 0.1 65° C R 0-20 20-50 50-100 100 20-50 50-100 1X or 2X

XapI (ApoI) 0.1 80° C Tango™ 50-100 100 0-20 0-20 100 20-50 1X or 2X

XbaI 0.1 65°C Tango™ 50-100 50-100 20-50 0-20 100 50-100 1X or 2X

XceI (NspI) 0.2 65° C Tango™ 50-100 0-20 0-20 0-20 100 0-20 1X

XhoI 0.1 80° C R 0-20 50-100 50-100 100 20-50 100 1X or 2X

XmaJI (AvrII) 0.2 80° C Tango™ 20-50 50-100 50-100 50-100 100 50-100 1X or 2X

XmiI (AccI) 0.1 65°C B 100 0-20 0-20 0-20 50-100 20-50 1X or 2X

Nicking enzymes

Nb.Bpu10I 0.3 80° C R 0-20 20-50 20-50 100 20-50 50-100 1X or 2X

Nt.Bpu10I 0.3 65° C R 0-20 0-20 100 100 0-20 50-100 2X

Nb.Mva1269I 0.3 80° C O 0-20 20-50 100 100 20-50 50-100 1X or 2X

Homing enzyme

I-SceI 0.5 65° C Tango™ 50-100 50-100 50-100 50-100 100 50-100 1X or 2X


* – star activity appears at a greater than 5-fold overdigestion (5 units x 1 hour). NR – buffer is not recommended, because of high star activity.NO – no thermal inactivation.80°C (10 u) – only small amounts of the restriction enzyme (up to 10 units) can be inactivated at 80°C in 20 min.

EnzymeOptimal

temperatureActivity at 37°C, %

AloI 30 20BclI 55 50BdaI 30 30BseDI (BsaJI) 55 10BseGI (BtsCI) 55 25BseJI (BsaBI) 65 <10BseLI (BslI) 55 40BseMI (BsrDI) 55 20BseMII (BspCNI) 55 30BseNI (BsrI) 65 <10BseSI (Bme1580I) 55 20BseXI (BbvI) 65 10BspPI (AlwI) 55 30BstXI 55 50GsuI (BpmI) 30 70Kpn2I (BspEI) 55 50PasI 55 30

EnzymeOptimal

temperatureActivity at 37°C, %

PpiI 30 60Ppu21I (BsaAI) 30 <30SfiI 50 10SmaI* 30 50SmiI (SwaI) 30 70SmoI (SmlI) 55 10TaaI (HpyCH4III) 65 10TaiI (MaeII) 65 <10TasI (Tsp509I) 65 <10TaqI* 65 10TauI 55 30TatI 65 20Tru1I (MseI)* 65 10TscAI (TspRI) 65 <5TsoI 55 10

* – high concentration enzyme preparations are available for incubation at non-optimal temperature.

Activity of Mesophilic and Thermophilic Enzymes at 37°CTable 1.7. Activity of mesophilic and thermophilic enzymes at 37°C.

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Double Digestion in Universal Tango™ Buffer

Table 1.8. Double digestion in universal Tango™ buffer.

Fermentas enzyme

Buffers for double digestionsRecommended buffer

Tango™

1X 2XAanI (PsiI) Tango™

AarI AarI +oligo NR +oligo

AasI (DrdI) B * NRAatII Tango™

Acc65I (Asp718I) OAdeI (DraIII ) G *AjiI (BmgBI) AjiI NR *AjuI R +SAM +SAM +SAM

AlfI R +SAM NR +SAM

AloI (30°C) RAluI Tango™

Alw21I (BsiHKAI) OAlw26I (BsmAI) Tango™

Alw44I (ApaLI) Tango™

ApaI B NRBamHI BamHI *BauI (BssSI) Tango™

BclI (55°C) G *BcnI (NciI) Tango™

BcuI (SpeI) Tango™ NRBdaI (30°C) G +SAM +SAM * +SAM

BfiI (BmrI) Tango™ NRBfmI (SfcI) Tango™

BfuI (BciVI) BfuI NR *BglI O NRBglII O NRBme1390I (ScrFI) OBoxI (PshAI) Tango™

BpiI (BbsI) GBplI Tango™+SAM +SAM NRBpu10I Bpu10I * *Bpu1102I (BlpI) Tango™

BseDI (BsaJI) (55°C) Tango™

BseGI (BtsCI) (55°C) Tango™

BseJI (BsaBI) (65°C) O NR *BseLI (BslI) (55°C) Tango™

BseMI (BsrDI) (55°C) RBseMII (BspCNI) (55°C) Tango™+SAM +SAM +SAM

BseNI (BsrI) (65°C) BBseSI (Bme1580I) (55°C) G NRBseXI (BbvI) (65°C) BseXI NR NRBsh1236I (BstUI) RBsh1285I (BsiEI) G NRBshNI (BanI) O NRBshTI (AgeI) OBsp68I (NruI) OBsp119I (BstBI) Tango™

Bsp120I (PspOMI) B NRBsp143I (Sau3AI) Bsp143IBsp1407I (BsrGI) Tango™

BspLI (NlaIV) Tango™

BspOI (BmtI) O NRBspPI (AlwI) (55°C) Tango™ NRBspTI (AflII) O NR

Fermentas enzyme


Tango™

1X 2XBst1107I (BstZ17I) OBstXI (55°C) OBsu15I (ClaI) Tango™

BsuRI (HaeIII) RBveI (BspMI) O +oligo +oligo +oligo

CaiI (AlwNI) Tango™

CfrI (EaeI) Tango™ NRCfr9I (XmaI) Cfr9I NRCfr10I (BsrFI) Cfr10ICfr13I (Sau96I) Tango™

Cfr42I (SacII) B NRCpoI (RsrII) Tango™

CseI (HgaI) R *Csp6I (CviQI) B NRDpnI Tango™

DraI Tango™

Eam1104I (EarI) Tango™ NREam1105I (AhdI) Eam1105IEcl136II (EcoICRI) Ecl136II NREco24I (BanII) Tango™ NREco31I (BsaI) GEco32I (EcoRV) REco47I (AvaII) REco47III (AfeI) OEco52I (EagI) Eco52I NREco57I (AcuI) G +SAM +SAM +SAM

Eco57MI B +SAM +SAM NREco72I (PmII) Tango™

Eco81I (Bsu36I) Tango™ NREco88I (AvaI) Tango™

Eco91I (BstEII) OEco105I (SnaBI) Tango™ NREco130I (StyI) OEco147I (StuI) B NREcoO109I (DraII) Tango™

EcoRI EcoRI NREcoRII OEheI (SfoI) Tango™

Esp3I (BsmBI) Tango™+DTT +DTT NRFaqI (BsmFI) Tango™+SAM +SAM +SAM

FspAI O NRFspBI (BfaI) Tango™ NRGsuI (BpmI) (30°C) BHhaI Tango™

Hin1I (BsaHI) GHin1II (NlaIII ) GHin4I Tango™+SAM +SAM NRHin6I (HinP1I) Tango™

HincII (HindII) Tango™

HindIII RHinfI RHpaII Tango™

HphI B NRHpy8I (MjaIV) Tango™


169

1



Table 1.8. Double digestion in universal Tango™ buffer.

Fermentas enzyme


Tango™

1X 2XHpyF3I (DdeI) Tango™

HpyF10VI (MwoI) Tango™

KpnI KpnI NRKpn2I (BspEI) (55°C) Tango™

KspAI (HpaI) B *LguI (SapI) Tango™

Lsp1109I (BbvI) Lsp1109I * *LweI (SfaNI) Tango™

MauBI Tango™ NRMbiI (BsrBI) Tango™

MboI RMboII BMlsI (MscI) RMluI RMnlI GMph1103I (NsiI) RMreI (Sse232I) G NRMspI (HpaII) Tango™

MssI (PmeI) B NRMunI (MfeI) G NRMvaI (BstNI) R *Mva1269I (BsmI) R NRNcoI Tango™

NdeI O NRNheI Tango™ NRNmuCI (Tsp45I) RNotI O NRNsbI (FspI) Tango™

OliI (AleI) R NRPaeI (SphI) B NRPacI PacI NR NRPagI (BspHI) O NR NRPasI (55°C) PasI NR NRPauI (BssHII) R NRPdiI (NaeI) Tango™

PdmI (XmnI) Tango™ NRPfeI (TfiI) OPfl23II (BsiWI) Tango™ NRPfoI Tango™

PpiI (30°C) RPpu21I (BsaAI) (30°C) Ppu21I NR NRPscI (PciI) Tango™ NRPsp5II (PpuMI) GPsp1406I (AciI) Tango™ NRPstI OPsuI (BstYI) B NRPsyI (Tth111I) B NRPvuI RPvuII G * *RsaI Tango™ NRRseI (MslI) RSacI SacISalI O NRSatI (Fnu4HI) GScaI ScaI NR NR

Fermentas enzyme


Tango™

1X 2XSchI (MlyI) Tango™ NRSdaI (SbfI) SdaI NRSduI (Bsp1286I) SduI NR NRSfaAI (AsiSI) Tango™ NRSfiI (50°C) G NRSgeI SgeI NR NRSgrDI R NRSgsI (AscI) Tango™

SmaI (30°C) Tango™ NRSmiI (SwaI) (30°C) O NRSmoI (SmoI) (55°C) Tango™

SmuI (FauI) Tango™

SsiI (AciI) O NRSspI GSspDI (KasI) Tango™

TaaI (HpyCH4III) (65°C) Tango™

TaiI (MaeII) (65°C) RTaqI (65°C) TaqITasI (Tsp509I) (65°C) B NRTauI (55°C) B NRTatI (65°C) Tango™ * NRTru1I (MseI) (65°C) RTscAI (TspRI) (65°C) Tango™

TsoI (55°C) G +SAM +SAM +SAM

TstI R NRVan91I (PflMI) RVspI (AseI) OXagI (EcoNI) RXapI (ApoI) Tango™

XbaI Tango™

XceI (NspI) Tango™ NRXhoI RXmaJI (AvrII) Tango™

XmiI (AccI) BI-SceI Tango™

Nb.Bpu10I RNt.Bpu10I R NRNb.Mva1269I O

* – star activity appears at a greater than 5-fold overdigestion (5 units x 1 hour).

Optimal temperature for the enzymes listed is 37°C unless otherwise indicated in parenthesis.

NR – buffer is not recommended since enzyme activity is less than 20% or star activity is too high.

Cleavage efficiency20-50%50-100%

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Fermentas enzyme

Units of enzyme, needed for DNA cleavage in 16 h

AanI (PsiI) 5AarI 5AasI (DrdI) 5AatII 5Acc65I (Asp718I) 5AdeI (DraIII ) 10AjiI (BmgBI) 5AjuI 5

AlfI 5Alw44I (ApaLI) 5ApaI 5BamHI 5BauI (BssSI) 5BcuI (SpeI) 10BfuI (BciVI) 5BglI 5BglII 5BoxI (PshAI) 10BpiI (BbsI) 5BplI 10Bpu1102I (BlpI) 5BseSI (Bme1580I) 5Bsh1285I (BsiEI) 5BshNI (BanI) 5BshTI (AgeI) 20Bsp68I (NruI) 5Bsp119I (BstBI) 5Bsp120I (PspOMI) 5Bsp1407I (BsrGI) 10BspOI (BmtI) 20BspTI (AflII) 5Bst1107I (BstZ17I) 5BstXI 5Bsu15I (ClaI) 5CaiI (AlwNI) 5CfrI (EaeI) 5Cfr9I (XmaI) 5Cfr10I (BsrFI) 5Cfr42I (SacII) 20DraI 5Eam1104I (EarI) 5Eam1105I (AhdI) 5Ecl136II (EcoICRI) 5Eco24I (BanII) 5Eco31I (BsaI) 5Eco32I (EcoRV) 5Eco47III (AfeI) 5Eco52I (EagI) 5Eco72I (PmlI) 5Eco81I (Bsu36I) 5Eco88I (AvaI) 5Eco91I (BstEII) 5Eco105I (SnaBI) 5Eco130I (StyI) 5Eco147I (StuI) 5

Fermentas enzyme


EcoO109I (DraII) 5EcoRI 5EheI (SfoI) 20Esp3I (BsmBI) 5FspBI (BfaI) 10Hin1I (BsaHI) 5HindIII 5KpnI 5Kpn2I (BspEI) (55°C) 5KspAI (HpaI) 5LguI (SapI) 5MauBI 5MbiI (BsrBI) 5MlsI (MscI) 20MluI 5MssI (PmeI) 5MunI (MfeI) 5Mva1269I (BsmI) 5NcoI 5NdeI 5NheI 5NotI 5NsbI (FspI) 5OliI (AleI) 5PaeI (SphI) 5PacI 5PagI (BspHI) 5PasI (55°C) 10PauI (BssHII) 10PdiI (NaeI) 20PdmI (XmnI) 10Pfl23II (BsiWI) 5PfoI 10Ppu21I (BsaAI) 5PscI (PciI) 5Psp5II (PpuMI) 5Psp1406I (AcII) 10PstI 5PsyI (Tth111I) 5PvuI 5PvuII 5RseI (MslI) 5SacI 5SalI 5ScaI 20SdaI (SbfI) 5SfaAI (AsiSI) 5SfiI (50°C) 5SgeI 3SgrDI 5SgsI (AscI) 5SmaI (30°C) 5SmiI (SwaI) (30°C) 10SmoI (SmlI) (55°C) 5SspI 5

Fermentas enzyme


SspDI (KasI) 5TstI 5Van91I (PflMI) 5VspI (AseI) 5XagI (EcoNI) 5XapI (ApoI) 5XbaI 5XhoI 5XmaJI (AvrII) 10XmiI (AccI) 5I-SceI 20

Table 1.9. Digestion of agarose-embedded DNA.

Digestion of Agarose-Embedded DNA

NoteFor digestion of agarose-embedded DNA protocol see 1.11 on p.161.

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Table 1.10. Cleavage efficiency close to the termini of PCR fragments.

Enzymebp from the recognition site to fragment end

1 2 3 4 5AanI 0 0-20 20-50AarI 20-50 50-100AasI 50-100AatII 0 0-20 20-50 50-100Acc65I 0-20 50-100AdeI 50-100AjiI 50-100AluI 0-20 20-50 50-100Alw21I 50-100Alw26I 50-100Alw44I 0 20-50 50-100ApaI 50-100BamHI 50-100BauI 0-20 20-50 50-100BcnI 20-50 50-100BclI 0 50-100BcuI 50-100BfiI 50-100BfmI 50-100BfuI 50-100BglI 20-50 50-100BglII 0 50-100Bme1390I 20-50 50-100BoxI 0 50-100BpiI 50-100Bpu10I 20-50 50-100Bpu1102I 50-100BseDI 0 50-100BseGI 50-100BseJI 0 50-100BseLI 0 50-100BseMI 0-20 50-100BseMII 50-100BseNI 0 50-100BseSI 50-100BseXI 20-50 50-100Bsh1236I 50-100Bsh1285I 0-20 50-100BshNI 50-100BshTI 20-50 50-100Bsp68I 0 50-100Bsp119I 50-100Bsp120I 20-50 50-100Bsp143I 50-100Bsp1407I 20-50 50-100BspLI 50-100BspOI 0 20-50 50-100BspPI 0 50-100BspTI 0 0-20 50-100Bst1107I 0-20 50-100BstXI 0 50-100Bsu15I 50-100BsuRI 0-20 20-50 50-100BveI 0-20 50-100CaiI 0 0-20 50-100CfrI 0 50-100Cfr9I 20-50 50-100Cfr10I 20-50 50-100Cfr13I 50-100Cfr42I 50-100CpoI 50-100CseI 50-100Csp6I 50-100DpnI 50-100DraI 0 0-20 50-100


1 2 3 4 5Eam1104I 0 50-100Eam1105I 0 50-100Ecl136II 50-100Eco24I 50-100Eco31I 20-50 50-100Eco32I 20-50 50-100Eco47I 50-100Eco47III 0 0-20 50-100Eco52I 0-20 50-100Eco57I 50-100Eco57MI 50-100Eco72I 0-20 50-100Eco81I 50-100Eco88I 50-100Eco91I 20-50 50-100Eco105I 20-50 50-100Eco130I 0 50-100Eco147I 0 50-100EcoO109I 50-100EcoRI 50-100EcoRII 0 0-20 20-50 50-100EheI 20-50 50-100Esp3I 50-100FaqI 0-20 50-100FspAI 0-20 20-50 50-100FspBI 20-50 50-100GsuI 50-100HhaI 50-100Hin1I 50-100Hin1II 0 0-20 50-100Hin6I 0 0-20 50-100HincII 50-100HindIII 0 0-20 50-100HinfI 50-100HpaII 0-20 50-100HphI 0 50-100Hpy8I 0-20 50-100HpyF3I 20-50 50-100HpyF10VI 50-100KpnI 50-100Kpn2I 0 50-100KspAI 20-50 50-100LguI 50-100Lsp1109I 0-20 50-100LweI 20-50 50-100MauBI 0 0-20 20-50MbiI 0 0-20 50-100MboI 50-100MboII 50-100MlsI 0-20 50-100MluI 20-50 50-100MnlI 0 50-100Mph1103I 20-50 50-100MreI 0 20-50 50-100MspI 0-20 50-100MssI 20-50 50-100MunI 20-50 50-100MvaI 0 20-50 50-100Mva1269I 0 50-100NcoI 0 50-100NdeI* 0-20 20-50 50-100NheI 0 20-50 50-100NmuCI 20-50 50-100NotI 20-50 50-100NsbI 0 0-20 50-100


1 2 3 4 5OliI 0-20 20-50 50-100PacI 20-50 50-100PaeI 0 0-20 20-50 50-100PagI 20-50 50-100PasI 50-100PauI 0 50-100PdiI 0-20 50-100PdmI 50-100PfeI 50-100Pfl23II 0-20 20-50 50-100PfoI 0 20-50 50-100Ppu21I 50-100PscI 0 50-100Psp5II 0 50-100Psp1406I 0-20 50-100PstI 0-20 50-100PsuI 0-20 50-100PsyI 0 50-100PvuI 20-50 50-100PvuII 50-100RsaI 50-100RseI 0 0-20 20-50 50-100SacI 50-100SalI 20-50 50-100SatI 0 50-100ScaI 0-20 50-100SchI 50-100SdaI 0-20 50-100SduI 50-100SfaAI 0 0-20 20-50SfiI 50-100SgrDI 0-20 20-50 50-100SgsI 50-100SmaI 50-100SmiI 0-20 50-100SmoI 0 50-100SmuI 50-100SsiI 50-100SspI 0-20 50-100SspDI 20-50 50-100TaaI 20-50 50-100TaiI 50-100TaqI 0 20-50 50-100TasI 0 20-50 50-100TatI 0-20 20-50 50-100TauI 20-50 50-100Tru1I 0 0-20 50-100TscAI 0 50-100TsoI 20-50 50-100Van91I 0 50-100VspI 50-100XagI 20-50 50-100XapI 20-50 50-100XbaI 20-50 50-100XceI 0 50-100XhoI 0-20 50-100XmaJI 50-100XmiI 50-100I-SceI 50-100

Some restriction enzymes cleave DNA poorly when their recognition sites are located close to DNA termini. Table 1.10 lists the activities of Fermentas restriction enzymes when their target sites are located close to the end of a PCR product.Experiments were performed as follows: PCR primers were designed with 1-5 extra

nucleotides at their 5’-end adjacent to the recognition site for the restriction enzyme. The 5’-end was labeled with [32P] by T4 Polynu-cleotide Kinase (#EK0031) and these labeled primers were used in the PCR reaction. PCR products were purified with the Silica Bead DNA Gel Extraction Kit (#K0513) and precipitated with

ethanol. The PCR products (0.5 µg) were incu-bated with 10 units of restriction enzymes in the optimal buffer for 1 hour at the recommended temperature. Reaction products were separated on 10% PAGE and the cleavage efficiency was determined using OptiQuant® Image Analysis software.

Cleavage Efficiency Close to the Termini of PCR Fragments

* – incubation was performed for 16 hours.Cleavage efficiency

0% 20-50% 0-20% 50-100%

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Close vicinity of restriction targets within multiple cloning sites (MSC) of vectors may have negative effect on the efficiency of double digestions within MSC. For efficient double digestion within MSC the first reaction should be performed with a restriction enzyme that cleaves DNA inef-ficiently when enzyme’s target is located close to the end of DNA. The second digestion should

Cleavage of Restriction Targets Located in Close Vicinity within pUC19 Multiple Cloning Site

be performed with a restriction enzyme which tolerates a close proximity to the DNA end.Data presented below were generated using pUC19 plasmid DNA. The plasmid was cleaved with a primary restriction enzyme (the first cut) and end-labeled with [32P] by T4 Polynucle-otide Kinase (#EK0031). The DNA was then digested for one hour with a second restriction

enzyme (the second cut). Reaction products were separated by PAGE, and the amount of the label present on the DNA was determined by autoradiography. A decrease in radioactiv-ity reflects cleavage efficiency of the second restriction enzyme.

Table 1.11. Cleavage of restriction targets located in close vicinity within pUC19 multiple cloning site.

* – only double-stranded portion of DNA is included, not the overhangs.

– simultaneous double digestion may be performed with these enzyme pairs.

– this enzyme should be used for the first cut – this enzyme should be used for the second cut

– not recommended

For reaction conditions see www.fermentas.com/doubledigest

Enzyme pair First cutSecond cut

Efficiency of the 2nd

cut, %

bp from 1st cut*

Acc65I BamHI

Acc65I BamHI 100 4BamHI Acc65I 100 4

Acc65IEcl136II

Acc65I Ecl136II 100 1Ecl136II Acc65I 50-100 3

Acc65IEco24I

Acc65I Eco24I 100 1Eco24I Acc65I 0 1

Acc65IEcoRI

Acc65I EcoRI 100 7EcoRI Acc65I 100 7

Acc65ISacI

Acc65I SacI 50-100 1SacI Acc65I 0-20 1

Acc65IXapI

Acc65I XapI 50-100 7XapI Acc65I 50-100 7

BamHICfr9I

BamHI Cfr9I 50-100 0Cfr9I BamHI 50-100 0

BamHIEco88I

BamHI Eco88I 100 0Eco88I BamHI 50-100 0

BamHIHincII

BamHI HincII 50-100 7HincII BamHI 100 9

BamHIKpnI

BamHI KpnI 100 4KpnI BamHI 100 4

BamHISalI

BamHI SalI 50-100 7SalI BamHI 100 7

BamHIXbaI

BamHI XbaI 20-50 1XbaI BamHI 100 1

Cfr9IEcl136I

Cfr9I Ecl136II 50-100 5Ecl136II Cfr9I 100 7

Cfr9IEco24I

Cfr9I Eco24I 50-100 5Eco24I Cfr9I 100 5

Cfr9ISacI

Cfr9I SacI 50-100 5SacI Cfr9I 50-100 5

Cfr9IXbaI

Cfr9I XbaI 50-100 6XbaI Cfr9I 50-100 6

Ecl136IIEco88I

Ecl136II Eco88I 100 7Eco88I Ecl136II 50-100 5

Ecl136IIEcoRI

Ecl136II EcoRI 100 3EcoRI Ecl136II 100 1

Ecl136IIXapI

Ecl136II XapI 50-100 3XapI Ecl136II 50-100 1

Eco24IEco88I

Eco24I Eco88I 100 5Eco88I Eco24I 50-100 5

Eco24IEcoRI

Eco24I EcoRI 100 1EcoRI Eco24I 100 1

Eco24IKpnI

Eco24I KpnI 50-100 1KpnI Eco24I 0 1

Eco24IXapI

Eco24I XapI 50-100 1XapI Eco24I 20-50 1

Eco88ISacI

Eco88I SacI 100 5SacI Eco88I 50-100 5

Eco88IXbaI

Eco88I XbaI 100 6XbaI Eco88I 100 6



cut, %

bp from 1st cut*

EcoRICfr9I

EcoRI Cfr9I 50-100 11Cfr9I EcoRI 100 11

EcoRIEco88I

EcoRI Eco88I 50-100 11Eco88I EcoRI 100 11

EcoRIKpnI

EcoRI KpnI 100 7KpnI EcoRI 100 7

EcoRISacI

EcoRI SacI 50-100 1SacI EcoRI 20-50 1

HincIIPaeI

HincII PaeI 100 9PaeI HincII 100 7

HincIIPstI

HincII PstI 100 3PstI HincII 20-50 1

HincIISdaI

HincII SdaI 0-20 2SdaI HincII 20-50 1

HincIIXbaI

HincII XbaI 50-100 3XbaI HincII 50-100 1

HindIIIPaeI

HindIII PaeI 50-100 1PaeI HindIII 0 1

HindIIIPstI

HindIII PstI 100 7PstI HindIII 100 7

HindIIISdaI

HindIII SdaI 50-100 6SdaI HindIII 50-100 7

KpnIEcl136II

KpnI Ecl136II 50-100 1Ecl136II KpnI 100 3

KpnISacI

KpnI SacI 50-100 1SacI KpnI 100 1

KpnI XapI

KpnI XapI 100 7XapI KpnI 50-100 7

PaeIPstI

PaeI PstI 0 1PstI PaeI 20-50 1

PaeISalI

PaeI SalI 50-100 7SalI PaeI 100 7

PaeISdaI

PaeI SdaI 0 0SdaI PaeI 20-50 1

PstIBamHI

PstI BamHI 50-100 13BamHI PstI 50-100 13

PstISalI

PstI SalI 0 1SalI PstI 50-100 1

PstIXbaI

PstI XbaI 100 7XbaI PstI 50-100 7

SacISmaI

SacI SmaI 100 5SmaI SacI 50-100 7

SacIXapI

SacI XapI 0-20 1XapI SacI 50-100 1

SalISdaI

SalI SdaI 0 0SdaI SalI 20-50 1

SalIXbaI

SalI XbaI 50-100 1XbaI SalI 0-20 1

SdaIXbaI

SdaI XbaI 100 7XbaI SdaI 50-100 6



cut, %

bp from 1st cut*

SmaIAcc65I

SmaI Acc65I 0-20 1Acc65I SmaI 0 -1

SmaIBamHI

SmaI BamHI 50-100 2BamHI SmaI 0-20 0

SmaIEcl136II

SmaI Ecl136II 50-100 7Ecl136II SmaI 100 7

SmaIEco24I

SmaI Eco24I 50-100 7Eco24I SmaI 100 5

SmaIKpnI

SmaI KpnI 0 1KpnI SmaI 0 -1

SmaIXbaI

SmaI XbaI 50-100 8XbaI SmaI 100 6

pUC19 multiple cloning site

Eco24I Cfr9I HincII PstII XapI Ecl136II Acc65I Eco88I SalI BveI PaeI 396 EcoRI SacI KpnI SmaI BamHI XbaI XmiI SdaI HindIII 452 5’ - C CAG TGA ATT CGA GCT CGG TAC CCG GGG ATC CTC TAG AGT CGA CCT GCA GGC ATG CAA GCT TGG C - 3’ 3’ - G GTC ACT TAA GCT CGA GCC ATG GGC CCC TAG CAG ATC TCA GCT GGA CGT CCG TAC GTT CGA ACC G - 5’

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Site Preferences by Restriction Endonucleases

In 1975, Thomas and Davis discovered that EcoRI cleaves its five recognition sites on λ DNA at rates that differ by an order of magnitude (4). Similar examples have been documented for other restriction enzymes. Factors such as flanking sequences and the number of cleavage sites appear to influence cleavage efficiency (5). There are numerous restriction enzymes (EcoRII, NaeI, NarI, Ksp632I, BspMI, Eco57I, etc.), which never completely digest certain unmethylated target DNAs, even when using an excess of enzyme or prolonged incubation (6-9). Most of these enzymes are members of the expand-ing group of type II restriction enzymes which require simultaneous interaction with two copies of the target site for effective cleavage (10). These enzymes cleave DNA molecules with one recognition site very slowly. In the case of type IIE enzymes (EcoRII, NaeI), one of the target sequences serves as an allosteric effector for effective cleavage of the other recognition site (6, 11-13). Type IIF enzymes, (e.g., SfiI, Cfr10I, NgoMIV, BspMI) and majority of type IIB restriction enzymes (AlfI, AloI, BalI, BcgI, BsaXI, CspCI, FalI, PpiI, PsrI) cleave both recognition sequences in a concerted reaction (14-18). Type IIS enzymes, such as FokI, BpmI, BsgI and MboII, also interact with two copies of their recognition sequence before cleaving DNA by different mechanisms (19).Cleavage of resistant sites can be significantly enhanced by the addition of cleavable DNA, an oligodeoxyribonucleotide containing the recogni-tion site, or spermidine (7, 9, 17, 20, 21). Different restriction enzymes recognizing the same nucleotide sequence (isoschizomers or neoschizomers) often do not effectively cleave the same resistant recognition site (e. g., Fermentas enzymes BveI, Cfr42I, Eam1104I and PdiI and their prototypes BspMI, SacII, Ksp632I and NaeI). However, some isoschizomers or neoschizomers cleave “resistant” sites at the same rate as normal sites. For example, EheI cleaves target DNA more efficiently than its prototype NarI. Thus, one recognition site of NarI on λ DNA and two sites on pBR322 are not cleaved to completion, even after incubation with 50 units of the enzyme for 16 hours. Unlike NarI, Fermentas neoschizomer EheI cleaves λ DNA and pBR322 DNA completely under standard conditions.

Common PropertiesClassification of Restriction Enzymes

Restriction enzymes recognize specific nucleo-tide sequences and cleave DNA molecules either within or outside their recognition site. Digestion results in the generation of DNA with “sticky” (with 5’- or 3’-overhang) or “blunt” ends.

The phenomenon of host specificity was first observed by Luria and Human in the early 1950s (1). Nearly a decade later, Arber and Dussoix predicted its molecular basis (2). They proposed that host specificity is based on a two-enzyme system: a restriction enzyme, which recognizes specific DNA sequences and cleaves foreign DNA upon its entrance into the bacterial cell, and a modification enzyme (methyltransferase), which protects the host DNA from degradation by its own restriction enzyme. Both restriction and modification enzymes recognize the same nucleotide sequence and together they form a restriction-modification (R-M) system.

Restriction-modification systems are wide-spread among bacteria and have also been isolated from phage, Archaea and eukaryotic algae viruses. R-M systems have been classi-fied into four types (I, II, III and IV), depending on the complexity of their structure, cofactor requirements, mode of action and sequence cleaved (3). The best characterized and the most frequently used are type II restriction endonucleases. These enzymes recognize specific 4-8 bp DNA sequences and cleave a DNA sequence either within the recognition site, or at a specified position up to 20 base pairs outside the site.

Often there is more than one enzyme that recognizes a particular nucleotide sequence. According to restriction enzyme nomenclature, an enzyme with a unique specificity which has been discovered first is called the prototype. Subsequently discovered enzymes with the same specificity are termed isoschizomers. An isoschizomer may differ from the prototype enzyme in site preferences, reaction condi-tions, as well as in sensitivity to methylation and exhibition of star activity. Restriction enzymes that recognize the same nucleotide sequence, but cleave DNA at different posi-tions are called neoschizomers.

References1. Luria, S.E., Human, M.L., A nonhereditary, host-induced vari-

ation of bacterial viruses, J. Bacteriol., 64, 557-569, 1952.2. Arber, W., Dussoix, D., Host specifi city of DNA producted by

Escherichia Coli: I. Host controlled modifi cation of bacterio-phage lambda, J. Mol. Biol., 5, 18-36, 1962.

3. Roberts, R.J., et al., A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes, Nucleic Acids Res., 31, 1805-1812, 2003.

4. Thomas M., Davis R.W., Studies on the cleavage of bacte-riophage lambda DNA with EcoRI restriction endonucle-ase, J. Mol. Biol., 91, 315-328, 1975.

5. Sapienza P.J., et al., Thermodynamic and Kinetic Basis for the relaxed DNA Sequence Specificity of “Promiscuous” Mutant EcoRI endonuclease, J. Mol. Biol., 348, 307-324, 2005.

6. Kruger, D.H., et al., EcoRII can be activated to cleave refractory DNA recognition sites, Nucleic Acids Res., 16, 3997-4008, 1988.

7. Oller, A. R., et al., Ability of DNA and spermidine to affect the activity of restriction endonucleases from several bacterial species, Biochemistry, 30, 2543-2549, 1991.

8. Bolton, B.J., et al., Ksp632I: a novel class IIS restriction endonuclease from Kluyvera species 632 with the asym-metric hexanucleotide recognition sequence: 5’-CTCT-TCN↓-3’ 3’-GAGAAGNNNN↓-5’, Gene, 66, 31-43, 1988.

9. Reuter, M., et al., Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases, Anal. Biochem., 209, 232-237, 1993.

10. Halford, S.E., Hopping, jumping and looping by restriction endonucleases, Biochem. Soc. Trans, 29, 363-373, 2001.

11. Gabbara, S., Bhagwat, A.S., Interaction of EcoRII endonu-clease with DNA substrates containing single recognition sites, J.Biol.Chem., 267, 18623-18630, 1992.

12. Yang, C.C., Topal, M.D., Nonidentical DNA-binding sites of endonuclease NaeI recognize different families of sequences flanking the recognition site, Biochemistry, 31, 9657-9664, 1992.

13. Huai, Q., et al., Crystal structure of NaeI – an evolutionary bridge between DNA endonuclease and topoisomerase, EMBO J., 19, 3110-3118, 2000.

14. Wentzell, L.M., et al., The SfiI restriction endonuclease makes a four-strand DNA break at two copies of its recognition sequence, J. Mol. Biol., 248, 581-595, 1995.

15. Siksnys, V., et al., The Cfr10I restriction enzyme is func-tional as a tetramer, J. Mol. Biol., 291, 1105-1118, 1999.

16. Deibert, M., et al., Structure of the tetrameric restriction endonuclease NgoMIV in complex with cleaved DNA, Nat. Struct. Biol., 7, 792-799, 2000.

17. Gormley, N.A., et al., The type IIs restriction endonuclease BspMI is a tetramer that acts concertedly at two copies of an asymmetric DNA sequence, J. Biol. Chem., 277, 4034-4041, 2002.

18. Marshall, J.J., et al., Restriction Endonucleases that Bridge and lexcise two recognition sites from DNA, J. Mol. Biol., 367, 419-431, 2007.

19. Bath, A.J., et al., Many type IIs restriction endonucleases interact with two recognition sites before cleaving DNA, J. Biol. Chem., 277, 4024-4033, 2002.

20. Conrad, M., Topal, M.D., DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme NaeI, Proc. Natl.Acad Sci. USA, 86, 9707-9711, 1989.

21. Grigaite, R.J., et al., AarI, a restriction endonuclease from Arthrobacter aurescens SS2-322, which recognizes the novel non-palindromic sequence 5’-CACCTGC(N)4/8-3’, Nucleic Acids Res., 30, e123, 2002.

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References1. Nasri, M., Thomas, D., Relaxation of recognition sequence

of specific endonuclease HindIII, Nucleic Acids Res., 14, 811-821, 1986.

2. George, J., Chirikjian, J.G., Sequence-specific endonu-clease BamHI: Relaxation of sequence recognition, Proc. Natl. Acad. Sci. USA, 79, 2432-2436, 1982.

3. Kolesnikov, V.A., et al., Relaxed specificity of endonuclease BamHI as determined by identification of recognition sites in SV40 and pBR322 DNAs, FEBS Letters, 132, 101-103, 1981.

4. Polisky, B., et al., Specificity of substrate recognition by the EcoRI restriction endonuclease, Proc. Natl. Acad. Sci. USA, 72, 3310-3314, 1975.

5. Woodbury, C.P., et al., DNA site recognition and reduced specificity of the EcoRI endonuclease, J. Biol. Chem., 255, 11534-11546, 1980.

6. George, J., et al., Sequence-specific endonuclease BamHI, J. Biol. Chem., 255, 6521-6524, 1980.

7. Malyguine, E., et al., Alteration of the specificity of restric-tion endonucleases in the presence of organic solvents, Gene, 8, 163-177, 1980.

8. Hsu, M., Berg, P., Altering the specificity of restriction endonuclease: effect of replacing Mg2+ with Mn2+, Biochemistry, 17, 131-138, 1978.

9. Mayer, H., Optimization of the EcoRI* activity of EcoRI endonuclease, FEBS Letters, 90, 341-344, 1978.

10. Nasri, M., Thomas, D., Alteration of the specificity of PvuII restriction endonuclease, Nucleic Acids Res., 15, 7677-7687, 1987.

11. Bitinaite, J., Schildkraut, I., Self generated DNA termini relax the specificity of SgrAI restriction endonuclease, Proc. Natl. Acad. Sci. USA, 99, 1164-1169, 2002.

Star Activity

Restriction enzymes recognize specific nucleo-tide sequences within DNA molecules. However, their recognition site specificity can be reduced in vitro (1). Under certain conditions, enzymes are able to recognize and cleave nucleotide sequences which differ from the canonical site. At low ionic strength, for example, BamHI (with the recognition sequence GGATCC) is able to cleave the following sequences: NGATCC, GPuATCC and GGNTCC (2, 3). This phenomenon is called “relaxed” or “star” activity (4, 5). For most restriction enzyme applications, star activity is not desirable.

Reports (4, 6-10) on star activity have sug-gested that this phenomenon is the result of:

• prolonged incubation (over digestion), • high enzyme concentration in the reaction

mixture (over digestion), • high glycerol concentration in the reaction

mixture,• presence of organic solvents, such as etha-

nol, dimethyl sulfoxide (DMSO) or dimethyl formamide (DMFA), in the reaction mixture,

• low ionic strength of the reaction buffer,• suboptimal pH values of the reaction buffer,• substitution of Mg2+ for other divalent

cations, such as Mn2+ or Co2+.

In some cases, the termini generated by DNA cleavage with a restriction enzyme at the canonical site have been shown to stimulate enzyme star activity (11).Both star activity and incomplete DNA digestion result in atypical electrophoresis patterns that can be distinguished by careful examination of gel images (see Fig. 1.4). Any tendency of a restriction enzyme to exhibit star activity is indicated both in the product description and in the Certificate of Analysis.

Figure 1.4. Enzyme complete digestion and star activity.1 – Lambda DNA.2 – Lambda DNA incubated 1 hour with 0.15 u of EcoRI (incomplete cleavage).3 – Lambda DNA incubated 1 hour with 0.4 u of EcoRI (incomplete cleavage).4 – Lambda DNA incubated 1 hour with 1 u of EcoRI (complete digestion).5 – Lambda DNA incubated 16 hours with 40 u of EcoRI (star activity).6 – Lambda DNA incubated 16 hours with 70 u of EcoRI (star activity).

How to distinguish between star activity and incomplete digestion?

• Star activity results in additional DNA bands below the expected bands and no additional bands above the largest expected fragment. These additional bands become more intense, while the expected bands become less intense, when either the incubation time or the amount of enzyme is increased.

• Incomplete DNA digestion results in additional bands above the expected DNA bands on the gel. Additional bands disap-pear when the incubation time or amount of enzyme is increased. No additional bands below the smallest expected frag-ment are observed.

NoteCertain restriction enzymes remain associated with the substrate DNA after cleavage and cause a change in the mobility of the digestion products during electrophoresis. The resulting atypical pattern is not related to star activity. In these cases, use a loading dye with SDS (6X DNA Loading Dye & SDS Solution, #R1151), heat the sample for 10 min at 65°C and chill on ice prior to loading on the gel (see p.433). This effect is characteristic of the following

FastDigest® enzymes:FastDigest® BspMI (BveI), FastDigest® HgaI (CseI), FastDigest® AcuI (Eco57I), FastDigest® FokI, FastDigest® MboII, FastDigest® TauI, FastDigest® TspRI (TscAI) and

Conventional restriction enzymes:AarI, AloI, BdaI, BseXI (BbvI), BveI (BspMI), CseI (HgaI), Eco57I (AcuI), Eco57MI, EcoRII, FaqI (BsmFI), GsuI (BpmI), Lsp1109I (BbvI), LweI (SfaNI), MboII, MnlI, SchI (MlyI), TauI, TscAI (TspRI), TsoI, TstI.

Incomplete cleavage Star activity Complete digestion

1 2 3 4 5 6

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Digestion of Methylated DNA

DNA methylation is the process of transferring a methyl group from a donor molecule to either a cytosine or an adenine by DNA methyltrans-ferases. Such methylation is the most common and abundant DNA modification process in living organisms. Three types of methylated bases are predominantly found in DNA:

5-methylcytosine (m5C), N4-methylcytosine (m4C), N6-methyladenine (m6A).

Other modified bases, such as 5-hydroxy-methylcytosine (hm5C) and 5-hydroxymethy-luracil (hm5U), have also been described. The organism-specific pattern of methylation depends on the methyltransferases’ specificity.In prokaryotes, DNA cleavage by a cognate restriction enzyme is prevented by the methyla-tion of DNA by a sequence-specific methyltrans-ferase which is an integral component of every restriction-modification (R-M) system (4, 5).The majority of E.coli strains used for propagation of plasmid DNA contain two site-specific DNA methyltransferases – Dam and Dcm (6, 4). The methylase encoded by the dam gene methylates the N6-position of an adenine residue within the GATC sequence (8, 9). The methylase encoded by the dcm gene methylates the C5-position of the internal cytosine residue within the CCWGG sequence (7, 10). In addition to Dam and Dcm methylases, labora-tory strains of E.coli K12 and B may contain EcoKI or EcoBI enzymes, respectively, encoded by a type I R-M system. These methyltransferases modify adenine residues within their respective recognition sequences: AAC(N)6GTGC for EcoKI and TGA(N)8TGCT for EcoBI (3, 4).

References1. Luria, S.E., Human, M.L., A nonhereditary, host-induced varia-

tion of bacterial viruses, J. Bacteriol., 64, 557-569, 1952.2. Arber, W., Dussoix, D., Host specifi city of DNA producted

by Escherichia Coli: I. Host controlled modifi cation of bacteriophage lambda, J. Mol. Biol., 5, 18-36, 1962.

3. Roberts, R.J., et al., A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes, Nucleic Acids Res., 31, 1805-1812, 2003.

4. McClelland, M., The effect of sequence specific DNA me-thylation on restriction endonuclease cleavage, Nucleic Acids Res., 9, 5859-5866, 1981.

5. McClelland, M., et al., Effect of site-specific modification on restriction endonucleases and DNA modification meth-yltransferases, Nucleic Acids Res., 22, 3640-3659, 1994.

6. Marinus, M.G., Morris, N.R., Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12, J. Bacte-riol., 114, 1143-1150, 1973.

7. May, M.S., Hattman, S., Analysis of bacteriophage deoxy-ribonucleic acid sequences methylated by host- and R-factor-controlled enzymes, J. Bacteriol., 123, 768-770, 1975.

8. Hattman, S., et al., Sequence specificity of the P1 modi-fication methylase (M.EcoP1) and the DNA methylase (M.Ecodam) controlled by the Escherichia coli dam gene, J. Mol. Biol.,126, 367-380, 1978.

9. Geier, G.E., Modrich, P., Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of DpnI endonuclease, J. Biol. Chem., 254, 1408-1413, 1979.

10. Buryanov, Ya.I., et al., Site specificity and chromatographic properties of E.coli K12 and EcoRII DNA-cytosine methy-lases, FEBS Letters, 88, 251-254, 1978.

11. Waalwijk, C., Flavell, R.A., MspI, an isochizomer of HpaII which cleaves both unmethylated and methylated HpaII sites, Nucleic Acids Res., 5, 3231-3236, 1978.

12. Bird, A.P., et al., Methylated and unmethylated DNA com-partments in the sea urchin genome, Cell, 17, 889-902, 1979.

13. McClelland, M., The frequency and distribution of methylat-able DNA sequences in leguminous plant protein coding genes, J. Mol. Evol., 19, 346-354, 1983.

14. Dreiseikelmann, B., et al., The effect of differential methy-lation by Escherichia coli of plasmid DNA and phage T7 and lambda DNA on the cleavage by restriction endonuclease MboI from Moraxella bovis, Biochim. Biophys. Acta, 562, 418-428, 1979.

15. Butkus, V. et al., Investigation of restriction-modification enzymes from M.varians RFL19 with a new type of speci-ficity toward modification of substrate, Nucleic Acids Res., 13, 5727-5746, 1985.

DNA from higher eukaryotic organisms pos-sesses modified 5-methylcytosine residues within CpG or CpNpG contexts (5, 11-13). These tissue-specific methylation patterns are heritable. They participate in regulation of gene expression and cellular differentiation. In cases where a restriction enzyme target site overlaps a methylation site, the following diges-tion results are possible:

• no effect• partial inhibition• complete block

The ability to cleave methylated DNA is an intrinsic and unpredictable property of each restriction enzyme. Therefore, isoschizomers and neoschizomers which recognize the same DNA sequences can differ in their sensitivity to DNA methylation (Table 1.12). For instance, MboI (recognition sequence GATC) does not cleave DNA methylated by Dam methylase (14), while the isoschizomer Bsp143I is insensitive to Dam methylation. EcoRII does not cleave DNA at the CCWGG site if it is methylated by Dcm, while its neoisoschizomer MvaI will cleave this methylated site (15). Thus, to achieve effective DNA diges-tion, it is necessary to take into account both the type of DNA methylation and the sensitivity of the restriction enzyme to that type of methylation.All restriction enzymes produced by Fermentas have been examined for their sensitivity to Dam, Dcm, CpG, EcoKI and EcoBI methylation of substrate DNA. Detailed methylation sensitivity information for our restriction enzymes is listed in the tables on pp.176-181, as well as in the pro-duct descriptions and Certificates of Analysis.

Conventional restriction enzyme couple

Recognition and cleavage sites

Sensitivity to methylation

Acc65I (Asp718I) G↓GTACC Overlapping Dcm or CpG methylation may influence DNA cleavageKpnI GGTAC↓C Not influenced by Dcm or CpG methylationApaI GGGCC↓C Overlapping Dcm or CpG methylation may influence DNA cleavageBsp120I (PspOMI) G↓GGCCC Blocked by overlapping Dcm or CpG methylationBsp143I (Sau3AI) ↓GATC Not influenced by Dam, blocked by CpG methylationMboI ↓GATC Blocked by Dam methylated DNADpnI GA↓TC Cleaves only Dam methylated DNABspOI (BmtI) GCTAG↓C Not influenced by CpG methylationNheI G↓CTAGC Overlapping CpG methylation may influence DNA cleavageCfr9I (XmaI) C↓CCGGG CpG methylation may influence DNA cleavageSmaI CCC↓GGG Blocked by CpG methylationCsp6I (CviQI) G↓TAC Not influenced by CpG methylationRsaI GT↓AC Overlapping CpG methylation may influence DNA cleavageEcl136II (EcoICRI) GAG↓CTC Overlapping CpG methylation may influence DNA cleavageSacI GAGCT↓C Not influenced by CpG methylationEcoRII ↓CCWGG Blocked by Dcm methylationMvaI (BstNI) CC↓WGG Not influenced by Dcm methylationHpaII C↓CGG Blocked by CpG methylationMspI (HpaII) C↓CGG Not influenced by CpG methylation

Table 1.12. Fermentas isoschizomers and neoschizomers with differing sensitivities to target methylation.


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Note** – DpnI (Gm6A↓TC) – cleaves only Dam methylated DNA.

* – recognition sequence is indicated in bold.

m6A = N6-methyladenine.

– overlapping methylase sequences.

– cleavage not blocked.

– cleavage blocked.

– cleavage rate is slowed significantly by methylation.

?– the sensitivity to methylation has not been determined.


Table 1.14. Partially overlapping Dam methyltrans-ferase and restriction enzyme recognition sites.

?

?

?

Conventional FastDigest® Sequence* Effect

AloI5’...GAAC(N)4Gm6A TCC...3’

3’...CTTG(N)4C Tm6AGG...5’

BdaI5’...TGm6A TC(N)4TCA...3’

3’...AC Tm6AG(N)4AGT...5’

BseJIBsaBI

5’...Gm6A TC(N)3ATC...3’

3’...C Tm6AG(N)3TAG...5’

Bsh1285IBsiEI

5’...CGm6A TCG...3’

3’...GC Tm6AGC...5’

Bsp68INruI

5’...Gm6A TCGCGm6A TC...3’

3’...C Tm6AGCGC Tm6AG...5’

Bsu15I ClaI

5’...ATCGm6A TC...3’

3’...TAGC Tm6AG...5’

Hin4I

5’...Gm6A TC(N)4VTC...3’

3’...C Tm6AG(N)4BAG...5’

5’...GAY(N)4Gm6A TC...3’

3’...CTR(N)4C Tm6AG...5’

HphI5’...GGTGm6A TC...3’

3’...CCAC Tm6AG...5’

Kpn2I Kpn2I

5’...TCCGGm6A TC...3’

3’...AGGCC Tm6AG...5’

5’...Gm6A TCCGGm6A TC...3’

3’...C Tm6AGGCC Tm6AG...5’

MboII MboII

5’...GAAGm6A TC...3’

3’...CTTC Tm6AG...5’

PagIBspHI

5’...TCATGm6A TC...3’

3’...AGTAC Tm6AG...5’

5’...Gm6A TCATGm6A TC...3’

3’...C Tm6AGTAC Tm6AG...5’

PfoI PfoI

5’...TCCNGGm6A TC...3’

3’...AGGNCC Tm6AG...5’

SmoI5’...CTYRAGm6A TC...3’

3’...GARYTC Tm6AG...5’

TaqI TaqI

5’...TCGm6A TC...3’

3’...AGC Tm6AG...5’

TstI5’...CAC(N)4Gm6A TCC...3’

3’...GTC(N)4C Tm6AGG...5’

XbaI XbaI

5’...TCTAGm6A TC...5’

3’...AGATC Tm6AG...5’

Effect of Dam Methylation on DNA Cleavage

To cleave with a restriction enzyme which is sensitive to the Dam methylation, DNA should be purified from dam– E.coli strains. E.coli GM2163 dam–, dcm– (#M0099) is available upon request with any product purchase. Control digestions should be performed with Lambda DNA (dam–, dcm–), #SD0021.

Table 1.13. Completely overlapping Dam methyl-transferase and restriction enzyme recognition sites.


BamHI BamHI

GGm6ATCC

BclIBclI

TGm6ATCA

BglIIBglII

AGm6ATCT

Bsp143ISau3AI

Gm6ATC

BspPI GGm6ATC

DpnI**DpnI

Gm6ATC

MboIMboI

Gm6ATC

PsuIPsuI

RGm6ATCY

PvuIPvuI

CGm6ATCG

SfaAIAsiSI

GCGm6ATCGC

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Effect of Dcm Methylation on DNA Cleavage

To cleave with a restriction enzyme which is sensitive to Dcm methylation, DNA should be purified from dcm– E.coli strains. E.coli GM2163 dam–, dcm– (#M0099) is available upon request with any product purchase. Control digestions should be performed with Lambda DNA (dam–, dcm–), #SD0021.

Table 1.15. Completely overlapping Dcm methyl-transferase and restriction enzyme recognition sites.


EcoRII Cm5CWGG

MvaI MvaI

Cm5CWGG

PasI CCm5CWGGG

SexAI ACm5CWGGT

SgeI** Cm5CWGG

Table 1.16. Partially overlapping Dcm methyltrans-ferase and restriction enzyme recognition sites.

Note* – recognition sequence is indicated in bold.

** – SgeI cleaves only methylated DNA.

– overlapping methylase sequences. m5C = 5-methylcytosine.



– cleavage rate is slowed significantly by methylation.



Acc65IAcc65I

5’...GGTACm5CW GG...3’

3’...CCATG GWm5CC...5’

5’...Cm5CW GGTACm5CW GG...3’

3’...G GWm5CCATG GWm5CC...5’

AloI5’...GAAC(N)6TCm5CW GG...3’

3’...CTTG(N)6AG GWm5CC...5’

ApaI ApaI

5’...GGGCCm5CW GG...3’

3’...CCCGG GWm5CC...5’

BamHIBamHI

5’...Cm5CW GGATCm5CW GG...3’

3’...G GWm5CCTAG GWm5CC...5’

BfuI5’...GTATCm5CW GG...3’

3’...CATAG GWm5CC...5’

BglI BglI

5’...GCm5CW GGNNGGC...3’

3’...CG GWm5CCNNCCG...5’

Bme1390I ScrFI

5’...Cm5CW GG...3’

3’...G GWm5CC...5’

BseDI BsaJI

5’...Cm5CW GGG...3’

3’...G GWm5CCC...5’

BseGI BseGI

5’...Cm5CW GGATG...3’

3’...G GWm5CCTAC...5’

BseLI BslI

5’...Cm5CW GG(N)4GG...3’

3’...G GWm5CC(N)4CC...5’

5’...Cm5CW GGNCm5CW GG...3’

3’...G GWm5CCNG GWm5CC...5’

BseSI Bme1580I

5’...GKGCCm5CW GG...3’

3’...CMCGG GWm5CC...5’

BshNI BanI

5’...GGYRCm5CW GG...3’

3’...CCRYG GWm5CC...5’

5’...Cm5CW GGYRCm5CW GG...3’

3’...G GWm5CCRYG GWm5CC...5’

Bsp120I Bsp120I

5’...GGGCCm5CW GG...3’

3’...CCCGG GWm5CC...5’

BspLI NlaIV

5’...GGNNCm5CW GG...3’

3’...CCNNG GWm5CC...5’

5’...Cm5CW GGNNCm5CW GG...3’

3’...G GWm5CCNNG GWm5CC...5’

BspPI5’...Cm5CW GGATC...3’

3’...G GWm5CCTAG...5’

BstXI BstXI

5’...Cm5CA GG(N)4TGG...3’

3’...G GTm5CC(N)4ACC...5’

5’...Cm5CA GGNNCm5CT GG...3’

3’...G GTm5CCNNG GAm5CC...5’

BsuRI HaeIII

5’...GGCm5CW GG...3’

3’...CCG GWm5CC...5’

5’...Cm5CW GGCm5CW GG...3’

3’...G GWm5CCG GWm5CC...5’

CaiI AlwNI

5’...CAGNNCm5CT GG...3’

3’...GTCNNG GTm5CC...5’

CfrI5’...YGGCm5CW GG...3’

3’...RCCG GWm5CC...5’

Cfr13I Sau96I

5’...GGNCm5CW GG...3’

3’...CCNG GWm5CC...5’

Eco24I5’...Cm5CW GGGCCm5CW GG...3’

3’...G GWm5CCCGG GWm5CC...5’


Eco31I Eco31I

5’...Cm5CW GGTCTC...3’

3’...G GWm5CCAGAG...5’

Eco47I AvaII

5’...GGWCm5CW GG...3’

3’...CCWG GWm5CC...5’

Eco91I Eco91I

5’...Cm5CW GGTNACm5CW GG...3’

3’...G GWm5CCANTG GWm5CC...5’

Eco57MI5’...Cm5CT GGAG...3’

3’...G GAm5CCTC...5’

Eco147I StuI

5’...AGGCm5CT GG...3’

3’...TCCG GAm5CC...5’

EcoO109IEcoO109I

5’...RGGNCm5CT GG...3’

3’...YCCNG GAm5CC...5’

EheI EheI

5’...Cm5CW GGCGCm5CW GG...3’

3’...G GWm5CCGCG GWm5CC...5’

FaqI BsmFI

5’...Cm5CW GGGAC...3’

3’...G GWm5CCCTG...5’

FokI5’...Cm5CW GGATG...3’

3’...G GWm5CCTAC...5’

GsuI BpmI

5’...CTCm5CA GG...3’

3’...GAG GTm5CC...5’

Hin1I BsaHI

5’...GRCGCm5CW GG...3’

3’...CYGCG GWm5CC...5’

HphI5’...Cm5CW GGTGA...3’

3’...G GWm5CCACT...5’

KpnI KpnI

5’...GGTACm5CW GG...3’

3’...CCATG GWm5CC...5’

5’...Cm5CW GGTACm5CW GG...3’

3’...G GWm5CCATG GWm5CC...5’

MlsI MscI

5’...TGGCm5CA GG...3’

3’...ACCG GTm5CC...5’

PfoI PfoI

5’...TCm5CA GGA...3’

3’...AG GTm5CCT...5’

Psp5II PpuMI

5’...RGGWCm5CT GG...3’

3’...YCCWG GAm5CC...5’

SduI Bsp1286I

5’...Cm5CW GGGCCm5CW GG...3’

3’...G GWm5CCCGG GWm5CC...5’

SfiI SfiI

5’...GGCm5CW GGNNGGCC...3’

3’...CCG GWm5CCNNCCGG...5’

5’...Cm5CW GGCC(N)5GGCm5CW GG...3’

3’...G GWm5CCGG(N)5CCG GWm5CC...5’

SgsI AscI

5’...Cm5CW GGCGCGCm5CW GG...3’

3’...G GWm5CCGCGCG GWm5CC...5’

SspDI5’...Cm5CW GGCGCC...3’

3’...G GWm5CCGCGG...5’

TsoI5’...TARCm5CA GG...3’

3’...ATYG GTm5CC...5’

TstI5’...CAC(N)6TCm5CW GG...3’

3’...GTG(N)6AG GWm5CC...5’

Van91I PflMI

5’...Cm5CA GG(N)3TGG...3’

3’...G GTm5CC(N)3ACC...5’

XagI EcoNI

5’...Cm5CT GG(N)3AGG...3’

3’...G GAm5CC(N)3TCC...5’

5’...Cm5CT GGNCm5CA GG...3’

3’...G GAm5CCNG GTm5CC...5’

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Table 1.17. CpG is located inside the recognition site.

Effect of CpG Methylation on DNA Cleavage

Methylated DNA substrates were prepared using SssI methyltransferase.

ConventionalFastDigest® Sequence Effect

HpaII HpaII

CCGG

Kpn2I Kpn2I

TCCGGA

MauBIMauBI

CGCGCGCG

MbiI BsrBI

GAGCGG

MluI MluI

ACGCGT

MreI MreI

CGCCGGCG

MspI MspI

CCGG

NotI NotI

GCGGCCGC

NsbI FspI

TGCGCA

PauIBssHII

GCGCGC

PdiINaeI

GCCGGC

Pfl23IIBsiWI

CGTACG

Ppu21IBsaAI

YACGTR

Psp1406I AclI

AACGTT

PvuI PvuI

CGATCG

SalI SalI

GTCGAC

SfaAI AsiSI

GCGATCGC

SgrDI CGTCGACG

SgsI AscI

GGCGCGCC

SmaI SmaI

CCCGGG

SmuI CCCGC

SsiI AciI

CCGC

SspDI GGCGCC

TaiI TaiI

ACGT

TaqI TaqI

TCGA

TauI GCSGC

XhoI XhoI

CTCGAG

ConventionalFastDigest® Sequence Effect

AatIIAatII

GACGTC

AjiI CACGTC

BauI CACGAG

BcnINciI

CCSGG

Bsh1236IBsh1236I

CGCG

Bsh1285I BsiEI

CGRYCG

BshTI AgeI

ACCGGT

Bsp68INruI

TCGCGA

Bsp119I Bsp119I

TTCGAA

Bsu15I ClaI

ATCGAT

Cfr9I CCCGGG

Cfr10I BsrFI

RCCGGY

Cfr42I CCGCGG

CpoIRsrII

CGGWCCG

CseIHgaI

GACGC

Eco47III AfeI

AGCGCT

Eco52I EagI

CGGCCG

Eco72I PmlI

CACGTG

Eco88I AvaI

CYCGRG

Eco105I SnaBI

TACGTA

EheI EheI

GGCGCC

Esp3IBsmBI

CGTCTC

FspAI FspAI

RTGCGCAY

HaeII RGCGCY

HhaI HhaI

GCGC

Hin1I BsaHI

GRCGYC

Hin6I HinP1I

GCGC

Note



– cleavage rate is reduced significantly by methylation.


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Table 1.18. CpG partially overlaps the recognition site.


AarI5’...CACCTGm5C G...3’

3’...GTGGAC Gm5C...5’

AasIDrdI

5’...m5C GAC(N)6GTC...3’

3’... Gm5CTG(N)6CAG...5’

5’...m5C GAC(N)6GTm5C G...3’

3’... Gm5CTG(N)6CA Gm5C...5’

5’...GAm5C G(N)5GTC...3’

3’...CT Gm5C(N)5CAG...5’

5’...GAm5C G(N)4m5C GTC...3’

3’...CT Gm5C(N)4 Gm5CAG...5’

Acc65I Acc65I

5’...GGTACm5C G...3’

3’...CCATG Gm5C...5’

5’...m5C GGTACm5C G...3’

3’... Gm5CCATG Gm5C...5’

AdeI DraIII

5’...CAm5C GNNGTG...3’

3’...GT Gm5CNNCAC...5’

5’...CAm5C GNm5C GTG...3’

3’...GT Gm5CN Gm5CAC...5’

AjuIAjuI

5’...m5C GAA(N)7TTGG...3’

3’... Gm5CTT(N)7AACC...5’

AlfI5’...GCA(N)6TGm5C G...3’

3’...CGT(N)6AC Gm5C...5’

AloI

5’...m5C GAAC(N)6TCC...3’

3’... Gm5CTTG(N)6AGG...5’

5’...GAAC(N)6TCm5C G...3’

3’...CTTG(N)6AG Gm5C...5’

5’...GAAm5C G(N)5TCC...3’

3’...CTT Gm5C(N)5AGG...5’

Alw21I Alw21I

5’...m5C GWGCWm5C G...3’

3’... Gm5CWCGW Gm5C...5’

Alw26I Alw26I

5’...GTCTm5C G...3’

3’...CAGA Gm5C...5’

5’...m5C GTCTC...3’

3’... Gm5CAGAG...5’

Alw44I ApaLI

5’...GTGCAm5C G...3’

3’...CACGT Gm5C...5’

ApaI ApaI

5’...GGGCCm5C G...3’

3’...CCCGG Gm5C...5’

5’...m5C GGGCCm5C G...3’

3’... Gm5CCCGG Gm5C...5’

BamHI BamHI

5’...m5C GGATCm5C G...3’

3’... Gm5CCTAG Gm5C...5’

BfuI

5’...m5C GTATCC...3’

3’... Gm5CATAGG...5’

5’...GTATCm5C G...3’

3’...CATAG Gm5C...5’

BglI BglI

5’...GCm5C G(N)3m5C GGC...3’

3’...CG Gm5C(N)3 Gm5CCG...5’

5’...GCC(N)5GGm5C G...3’

3’...CGG(N)5CC Gm5C...5’

5’...m5C GCC(N)5GGm5C G...3’

3’... Gm5CGG(N)5CC Gm5C...5’

Bme1390I ScrFI

5’...Cm5C GGG...3’

3’...G Gm5CCC...5’

?

?


BoxI PshAI

5’...GAm5C G(N)3GTC...3’

3’...CT Gm5C(N)3CAG...5’

5’...GAm5C GNNm5C GTC...3’

3’...CT Gm5CNN Gm5CAG...5’

5’...GAC(N)4GTm5C G...3’

3’...CTG(N)4CA Gm5C...5’

BpiI BbsI

5’...GAAGAm5C G...3’

3’...CTTCT Gm5C...5’

5’...m5C GAAGAC...3’

3’... Gm5CTTCTG...5’

BplI BplI

5’...m5C GAG(N)5CTm5C G...3’

3’... Gm5CTC(N)5GA Gm5C...5’

Bpu10I Bpu10I

5’...CCTNAGm5C G...3’

3’...GGANTC Gm5C...5’

Bpu1102I BlpI

5’...m5C GCTNAGm5C G...3’

3’... Gm5CGANTC Gm5C...5’

BseDI BsaJI

5’...Cm5C Gm5C GG...3’

3’...G Gm5C Gm5CC...5’

BseGI BseGI

5’...m5C GGATG...3’

3’... Gm5CCTAC...5’

BseJIBsaBI

5’...GAT(N)4ATm5C G...3’

3’...CTA(N)4TA Gm5C...5’

5’...m5C GAT(N)4ATm5C G...3’

3’... Gm5CTA(N)4TA Gm5C...5’

BseLI BslI

5’...Cm5C G(N)6GG...3’

3’...G Gm5C(N)6CC...5’

5’...Cm5C G(N)5m5C GG...3’

3’...G Gm5C(N)5 Gm5CC...5’

BseMIBsrDI

5’...m5C GCAATG...3’

3’... Gm5CGTTAC...5’

BseSIBme1580I

5’...m5C GKGCMm5C G...3’

3’... Gm5CMCGK Gm5C...5’

BseXIBseXI

5’...m5C GCAGm5C G...3’

3’... Gm5CGTC Gm5C...5’

BshNI BanI

5’...GGYRCm5C G...3’

3’...CCRYG Gm5C...5’

5’...m5C GGYRCm5C G...3’

3’... Gm5CCRYG Gm5C...5’

5’...GGm5C GCC...3’

3’...CC Gm5CGG...5’

Bsp120I Bsp120I

5’...GGGCCm5C G...3’

3’...CCCGG Gm5C...5’

Bsp143I Sau3AI

5’...GATm5C G...3’

3’...CTA Gm5C...5’

BspLI NlaIV

5’...GGNNCm5C G...3’

3’...CCNNG Gm5C...5’

5’...m5C GGNNCm5C G...3’

3’... Gm5CCNNG Gm5C...5’

BspOI BmtI

5’...GCTAGm5C G...3’

3’...CGATC Gm5C...5’

5’...m5C GCTAGm5C G...3’

3’... Gm5CGATC Gm5C...5’

BspPI

5’...GGATm5C G...3’

3’...CCTA Gm5C...5’

5’...m5C GGATC...3’

3’... Gm5CCTAG...5’

?



Bst1107I BstZ17I

5’...GTATAm5C G...3’

3’...CATAT Gm5C...5’

5’...m5C GTATAm5C G...3’

3’... Gm5CATAT Gm5C...5’

BsuRI HaeIII

5’...m5C GGCm5C G...3’

3’... Gm5CCG Gm5C...5’

BveIBspMI

5’...ACCTGm5C G...3’

3’...TGGAC Gm5C...5’

CfrI5’...YGGCm5C G...3’

3’...RCCG Gm5C...5’

Cfr13I Sau96I

5’...GGNCm5C G...3’

3’...CCNG Gm5C...5’

Csp6I Csp6I

5’...m5C GTAm5C G...3’

3’... Gm5CAT Gm5C...5’

DpnI DpnI

5’...Gm6A Tm5C G...3’

3’...C Tm6A Gm5C...5’

Eam1104IEarI

5’...CTCTTm5C G...3’

3’...GAGAA Gm5C...5’

Eam1105I Eam1105I

5’...GAm5C G(N)3m5C GTC...3’

3’...CT Gm5C(N)3 Gm5CAG...5’

5’...GAC(N)5GTm5C G...3’

3’...CTG(N)5CA Gm5C...5’

5’...m5C GAC(N)5GTm5C G...3’

3’... Gm5CTG(N)5CA Gm5C...5’

Ecl136II Ecl136II

5’...GAGCTm5C G...3’

3’...CTCGA Gm5C...5’

5’...m5C GAGCTm5C G...3’

3’... Gm5CTCGA Gm5C...5’

Eco24I5’...m5C GRGCYm5C G...3’

3’... Gm5CYCGR Gm5C...5’

Eco31I Eco31I

5’...GGTCTm5C G...3’

3’...CCAGA Gm5C...5’

5’...m5C GGTCTC...3’

3’... Gm5CCAGAG...5’

Eco32I EcoRV

5’...m5C GATATm5C G...3’

3’... Gm5CTATA Gm5C...5’

Eco47I AvaII

5’...GGWCm5C G...3’

3’...CCWG Gm5C...5’

Eco91I Eco91I

5’...m5C GGTNACm5C G...3’

3’... Gm5CCANTG Gm5C...5’

EcoRI EcoRI

5’...m5C GAATTC...3’

3’... Gm5CTTAAG...5’

5’...m5C GAATTm5C G...3’

3’... Gm5CTTAA Gm5C...5’

FaqIBsmFI

5’...GGGAm5C G...3’

3’...CCCT Gm5C...5’

5’...m5C GGGAC...3’

3’... Gm5CCCTG...5’

FokI5’...m5C GGATG...3’

3’... Gm5CCTAC...5’’

Hin4I

5’...m5C GAY(N)5VTC...3’

3’... Gm5CTR(N)5BAG...5’

5’...GAY(N)5VTm5C G...3’

3’...CTR(N)5BA Gm5C...5’

5’...m5C GAY(N)5VTm5C G...3’

3’... Gm5CTR(N)5BA Gm5C...5’

?

?

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Table 1.18. CpG partially overlaps the recognition site.

?

?


HincII HincII

5’...GTYRAm5C G...3’

3’...CARYT Gm5C...5’

5’...m5C GTYRAm5C G...3’

3’... Gm5CARYT Gm5C...5’

5’...GTm5C GAC...3’

3’...CA Gm5CTG...5’

HinfI HinfI

5’...GANTm5C G...3’

3’...CTNA Gm5C...5’

5’...m5C GANTm5C G...3’

3’... Gm5CTNA Gm5C...5’

HphI5’...m5C GGTGA...3’

3’... Gm5CCACT...5’

Hpy8I Hpy8I

5’...GTNNAm5C G...3’

3’...CANNT Gm5C...5’

5’...m5C GTNNAm5C G...3’

3’... Gm5CANNT Gm5C...5’

HpyF10VI HpyF10VI

5’...GC(N)7Gm5C G...3’

3’...CG(N)7C Gm5C...5’

5’...m5C GC(N)7Gm5C G...3’

3’... Gm5CG(N)7C Gm5C...5’

5’...Gm5C G(N)6GC...3’

3’...C Gm5C(N)6CG...5’

5’...Gm5C G(N)5m5C GC...3’

3’...C Gm5C(N)5 Gm5CG...5’

KpnI KpnI

5’...m5C GGTACm5C G...3’

3’... Gm5CCATG Gm5C...5’

KspAI HpaI

5’...GTTAAm5C G...3’

3’...CAATT Gm5C...5’

5’...m5C GTTAAm5C G...3’

3’... Gm5CAATT Gm5C...5’

LguI SapI

5’...m5C GCTCTTC...3’

3’... Gm5CGAGAAG...5’

5’...GCTCTTm5C G...3’

3’...CGAGAA Gm5C...5’

Lsp1109I BbvI

5’...m5C GCAGm5C G...3’

3’... Gm5CGTC Gm5C...5’

LweI SfaNI

5’...GCATm5C G...3’

3’...CGTA Gm5C...5’

MbiI BsrBI

5’...m5C GAGCGG...3’

3’... Gm5CTCGCC...5’

MboI MboI

5’...m5C GATm5C G...3’

3’... Gm5CTA Gm5C...5’

MboII MboII

5’...m5C GAAGA...3’

3’... Gm5CTTCT...5’

MnlI MnlI

5’...CCTm5C G...3’

3’...GGA Gm5C...5’

MssI MssI

5’...GTTTAAAm5C G...3’

3’...CAAATTT Gm5C...5’

5’...m5C GTTTAAAm5C G...3’

3’... Gm5CAAATTT Gm5C...5’

Mva1269I Mva1269I

5’...GAATGm5C G...3’

3’...CTTAC Gm5C...5’

5’...m5C GAATGC...3’

3’... Gm5CTTACG...5’

NheI NheI

5’...GCTAGm5C G...3’

3’...CGATC Gm5C...5’

5’...m5C GCTAGm5C G...3’

3’... Gm5CGATC Gm5C...5’

?


SfiI SfiI

5’...m5C GGCC(N)5GGCm5C G...3’

3’... Gm5CCGG(N)5CCG Gm5C...5’

5’...GGCm5C G(N)4GGCC...3’

3’...CCG Gm5C(N)4CCGG...5’

5’...GGCm5C G(N)3m5C GGCC...3’

3’...CCG Gm5C(N)3 Gm5CCGG...5’

SgeI**

5’...m5C GNG...3’

3’... Gm5CNC...5’

5’...m5C Gm5C G...3’

3’... Gm5C Gm5C...5’

SmoI5’...CTm5C GAG...3’

3’...GA Gm5CTC...5’

TaaI TaaI

5’...Am5C GGT...3’

3’...T Gm5CCA...5’

TstI

5’...CAm5C G(N)5TCC...3’

3’...GT Gm5C(N)5AGG...5’

5’...CAC(N)5TCm5C G...3’

3’...GTG(N)5AG Gm5C...5’

XapI XapI

5’...m5C GAATTm5C G...3’

3’... Gm5CTTAA Gm5C...5’

XceI NspI

5’...RCATGm5C G...3’

3’...YGTAC Gm5C...5’

5’...m5C GCATGm5C G...3’

3’... Gm5CGTAC Gm5C...5’

XmiI AccI

5’...GTm5C GAC...3’

3’...CA Gm5CTG...5’

5’...GTMKAm5C G...3’

3’...CAKMT Gm5C...5’


** – SgeI cleaves only methylated DNA.

– overlapping methylase sequences. m5C = 5-methylcytosine.



–cleavage rate is reduced significantly by methylation.

?– the sensitivity to methylation has not been determined.



NmuCI NmuCI

5’...GTCAm5C G...3’

3’...CAGT Gm5C...5’

5’...m5C GTCAm5C G...3’

3’... Gm5CAGT Gm5C...5’

OliI AleI

5’...CAm5C GNNNGTG...3’

3’...GT Gm5CNNNCAC...5’

5’...CAm5C GNNm5C GTG...3’

3’...GT Gm5CNN Gm5CAC...5’

PaeI SphI

5’...m5C GCATGm5C G...3’

3’... Gm5CGTAC Gm5C...5’

PdmI PdmI

5’...GAA(N)4TTm5C G...3’

3’...CTT(N)4AA Gm5C...5’

5’...m5C GAA(N)4TTm5C G...3’

3’... Gm5CTT(N)4AA Gm5C...5’

PfeI TfiI

5’...GAWTm5C G...3’

3’...CTWA Gm5C...5’

PfoI PfoI

5’...TCm5C GGGA...3’

3’...AG Gm5CCCT...5’

PpiI

5’...m5C GAAC(N)5CTC...3’

3’... Gm5CTTG(N)5GAG...5’

5’...GAAC(N)5CTm5C G...3’

3’...CTTG(N)5GA Gm5C...5’

5’...GAAm5C G(N)4CTC...3’

3’...CTT Gm5C(N)4GAG...5’

PspFI5’...CCCAGm5C G...3’

3’...GGGTC Gm5C...5’

PsuI PsuI

5’...m5C GGATCm5C G...3’

3’... Gm5CCTAG Gm5C...5’

PsyI PsyI

5’...GAm5C GNNGTm5C G...3’

3’...CT Gm5CNNCA Gm5C...5’

RsaI RsaI

5’...GTAm5C G...3’

3’...CAT Gm5C...5’

5’...m5C GTAm5C G...3’

3’... Gm5CAT Gm5C...5’

RseI MslI

5’...CAm5C GNNNRTG...3’

3’...GT Gm5CNNNYAC...5’

SacI SacI

5’...m5C GAGCTm5C G...3’

3’... Gm5CTCGA Gm5C...5’

SanDI

5’...GGGWCCm5C G...3’

3’...CCCWGG Gm5C...5’

5’...m5C GGGWCCm5C G...3’

3’... Gm5CCWGG Gm5C...5’

SatI Fnu4HI

5’...Gm5C GGC...3’

3’...C Gm5CCG...5’

5’...GCNGm5C G...3’

3’...CGNC Gm5C...5’

SchI MlyI

5’...GAGTm5C G...3’

3’...CTCA Gm5C...5’

5’...m5C GAGTC...3’

3’... Gm5CTCAG...5’

SduIBsp1286I

5’...m5C GDGCHm5C G...3’

3’... Gm5CHCGD Gm5C...5’

SfaNI

5’...m5C GCATC...3’

3’... Gm5CGTAG...5’

5’...GCATm5C G...3’

3’...CGTA Gm5C...5’

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Effect of EcoKI and EcoBI Methylation on DNA Cleavage

Methylated DNA substrates were purified from E.coli K12 or E.coli B strains.

Table 1.19. EcoBI overlapping methylation.


AatIIAatII

5’...TGm6ACGTC(N)4 TGCT...3’

3’...AC TGCAG(N)4m6ACGA...5’

AluIAluI

5’...TGm6AGCT(N)5 TGCT...3’

3’...AC TCGA(N)5m6ACGA...5’

BclI BclI

5’...TGm6A TCA(N)5 TGCT...3’

3’...AC Tm6AGT(N)5m6ACGA...5’

BglII BglII

5’...TGm6AGATCT(N)3 TGCT...3’

3’...AC TCTAGA(N)3m6ACGA...5’

Bpu10IBpuI

5’...CCTGm6AGC(N)6 TGCT...3’

3’...GGAC TCG(N)6m6ACGA...5’

Bsp143ISau3AI

5’...TGm6ATC(N)6 TGCT...3’

3’...AC TAG(N)6m6ACGA...5’

EcoRI EcoRI

5’...TGm6AATTC(N)4 TGCT...3’

3’...AC TTAAG(N)4m6ACGA...5’

HincII HincII

5’...GTTGm6AC(N)7 TGCT...3’

3’...CAAC TG(N)7m6ACGA...5’

HindIII HindIII

5’...TGm6AAGCTT(N)3 TGCT...3’

3’...AC TTCGAA(N)3m6ACGA...5’

HinfI HinfI

5’...TGm6ANTC(N)5 TGCT...3’

3’...AC TNAG(N)5m6ACGA...5’

HphI 5’...GGTGm6A(N)8 TGCT...3’

3’...CCAC T(N)8m6ACGA...5’

MboI MboI

5’...TGm6ATC(N)6 TGCT...3’

3’...AC TAG(N)6m6ACGA...5’

MboII MboII

5’...TGm6AAGA(N)5 TGCT...3’

3’...AC TTCT(N)5m6ACGA...5’

MluI MluI

5’...TGm6ACGCGT(N)3 TGCT...3’

3’...AC TGCGCA(N)3m6ACGA...5’

MnlI MnlI

5’...TGm6AGG(N)6 TGCT...3’

3’...AC TCC(N)6m6ACGA...5’

NdeI NdeI

5’...TGm6A(N)4CATA TGCT...3’

3’...AC T(N)4GTATm6ACGA...5’

OliI AleI

5’...TGm6A(N)4CACG TGCT...3’

3’...AC T(N)4GTGCm6ACGA...5’

PaeI SphI

5’...TGm6A(N)5GCA TGCT...3’

3’...AC T(N)5CGTm6ACGA...5’

PagIBspHI

5’...TCATGm6A(N)8 TGCT...3’

3’...AGTAC T(N)8m6ACGA...5’

SacI SacI

5’...TGm6AGCTC(N)4 TGCT...3’

3’...AC TCGAG(N)4m6ACGA...5’

ScaI ScaI

5’...TGm6AGTACT(N)3 TGCT...3’

3’...AC TCATGA(N)3m6ACGA...5’

SspI SspI

5’...TGm6AATATT(N)3 TGCT...3’

3’...AC TTATAA(N)3m6ACGA...5’

TasI Tsp509I

5’...TGm6AATT(N)5 TGCT...3’

3’...AC TTAA(N)5m6ACGA...5’

Recognition sequences of the following endonu-cleases may also overlap with DNA sequences methylated by EcoBI and EcoKI.

The following conventional restriction enzymes and the respective FastDigest® enzymes have not been tested for sensitivity to EcoBI methylation: AarI, AdeI, AjiI, Alw21I, BauI, BcuI, BfiI, BglII, BoxI, BpiI, Bpu1102I, BseGI, BseJI, BseMI, BseNI, BseXI, BshTI, Bsp143II, BspPI, Bsu15I, BveI, CaiI, Cfr10I, CseI, DpnI, Eam1104I, Ecl136II, Eco24I, Eco31I, Eco32I, Eco47III, Eco57I, Eco57MI, Eco72I, Eco81I, Eco91I, Eco147I, EcoO109I, Esp3I, FspAI, FokI*, HaeII*, HindIII, Hin1I, Hin1II, Hin4I, Hpy8I, HpyF3I, LguI, Lsp1109I, LweI, MbiI, Mph1103I, MunI, Mva1269I, NdeI, NmuCI, PdmI, PfeI, Ppu21I, PscI, Psp5II, Psp1406I, PsuI, PsyI, PvuII, RseI, SchI, SduI, SexAI*, SfaNI*, SmiI, SmoI, SspI, TaaI, TaiI, TatI, TscAI, TstI, VspI, XagI, XapI and XceI.

The following conventional restriction enzymes and the respective FastDigest® enzymes have not been tested for sensitivity to EcoKI methylation: AanI, AdeI, AjiI, BauI, BcuI, BfiI, BseNI, BshNI, BshTI, Bsp119I, BveI, Cfr10I, Eco72I, Eco91I, FspAI, Hpy8I, MseI*, PacI, PdmI, Ppu21I, PscI, Psp1406I, SexAI*, SduI, TaaI, TaiI, TscAI, TsoI and XceI. * enzymes are available in FastDigest® format only.




–cleavage rate is reduced significantly by methylation.


Table 1.20. EcoKI overlapping methylation.


Alw21I Alw21I

5’...Am6AC(N)6G TGCWC...3’

3’...T TG(N)6Cm6ACGWG...5’

Alw44I ApaLI

5’...Am6AC(N)6G TGCAC...3’

3’...T TG(N)6Cm6ACGTG...5’

BseSI Bme1580I

5’...Am6AC(N)6G TGCMC...3’

3’...T TG(N)6Cm6ACGKG...5’

DraI DraI

5’...TTTAAm6AC(N)6G TGC...3’

3’...AAATT TG(N)6Cm6ACG...5’

HincII HincII

5’...GTYAm6AC(N)6G TGC...3’

3’...CART TG(N)6Cm6ACG...5’

KspAI HpaI

5’...GTTAm6AC(N)6G TGC...3’

3’...CAAT TG(N)6Cm6ACG...5’

MluI MluI

5’...Am6ACGCGTNNG TGC...3’

3’...T TGCGCANNCm6ACG...5’

MssI MssI

5’...GTTTAAm6AC(N)6G TGC...3’

3’...CAAATT TG(N)6Cm6ACG...5’

NsbI FspI

5’...Am6AC(N)6G TGCGCA...3’

3’...T TG(N)6Cm6ACGCGT...5’

OliI AleI

5’...Am6ACAC(N)4G TGC...3’

3’...T TGTG(N)4Cm6ACG...5’

RseI MslI

5’...GCm6ACNNNNRTG TT...3’

3’...CG TGNNNNYACm6AT...5’

Tru1I Tru1I

5’...TTAm6AC(N)6G TGC...3’

3’...AAT TG(N)6Cm6ACG...5’

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Newly Generated Cleavage Sites

Recognition Sites Resulting from Ligation of Blunt DNA Ends

Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends.


First restriction enzyme

Second restriction enzymeRestriction enzymes that cleave the newly generated recognition sequence

AanI (PsiI)/FastDi-gest® PsiI (AanI) (TTA↓TAA)

Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC), FaiI* (YA↓TA), FaiI* (YA↓TG) FaiIDraI/FastDigest® DraI (TTT↓AAA), Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT)

FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I

Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT) SetIEco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) AluI/FastDigest® AluI, CviJI, SetIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) CviJI

SspI/FastDigest® SspI (AAT↓ATT)FastDigest® MseI (SaqAI), TasI (Tsp509I)/FastDigest® Tsp509I (TasI), Tru1I (MseI)/FastDigest® Tru1I

AjiI (BmgBI)* (CAC↓GTC)

AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)

SetI

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetIEco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA)

MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI

Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG)

Eco72I (PmlI)/FastDigest® PmlI (Eco72I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI

FaiI* (YA↓TG) TscAI (TspRI)/FastDigest® TspRI (TscAI)MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIPdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC) BceAIZraI (GAC↓GTC) AjiI (BmgBI), MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI

AjiI (BmgBI)* (GAC↓GTG)


SetI

DpnI/FastDigest® DpnI (GA↓TC) HinfI/FastDigest® HinfIEcl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetIEco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG)

MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI

Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA)

CseI (HgaI)/FastDigest® HgaI (CseI)

EheI (SfoI)/FastDigest® EheI (GGC↓GCC)CseI (HgaI)/FastDigest® HgaI (CseI), Hin1I (BsaHI)/FastDi-gest® BsaHI (Hin1I)

MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIPdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC) BceAI

ZraI (GAC↓GTC)AatII/FastDigest® AatII, Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I), MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI, ZraI

AluI/FastDigest® AluI (AG↓CT)

AjiI (BmgBI)* (CAC↓GTC), AjiI (BmgBI)* (GAC↓GTG), Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG), ZraI (GAC↓GTC)

SetI

Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIBsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)

CviJI

CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)

AluI/FastDigest® AluI, CviJI, SetI

Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI

EheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI

Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG)

Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI


Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) CviJIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJIBsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG)

Bsh1236I (BstUI)/FastDigest® Bsh1236I


Note* Restriction enzymes that have degenerate recognition

sequences (i.e., recognize more than one sequence). Be aware that these restriction endonucleases will cleave sequences in addition to the one listed.

• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas conventional enzymes are shown in orange.

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Bsp68I (NruI)/Fast-Digest® NruI (RruI) (TCG↓CGA)

Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG)

Bsh1236I (BstUI)/FastDigest® Bsh1236I

Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIDraI/FastDigest® DraI (TTT↓AAA), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/Fast-Digest® HpaI (KspAI) (GTT↓AAC), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT), SspI/FastDigest® SspI (AAT↓ATT)

TaqI/FastDigest® TaqI

Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, TaqI/Fast-Digest® TaqI

Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) CviJIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJIHincII (HindII)/FastDigest® HincII* (GTY↓GAC) Hpy188I

Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC)

AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), FaiI* (YA↓TA), FaiI* (YA↓TG) FaiIAluI/FastDigest® AluI (AG↓CT), Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG), Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), HpyCH4V (TG↓CA), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA), MspA1I* (CMG↓CGG), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)

Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI

Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT)Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI, SetI

Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) AluI/FastDigest® AluI, CviJI, SetIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) CviJIHincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC) Hpy8I (MjaIV)/FastDigest® Hpy8I

HincII (HindII)/FastDigest® HincII* (GTY↓GAC)Hpy8I (MjaIV)/FastDigest® Hpy8I, XmiI (AccI)/FastDigest® AccI (XmiI)

RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT) MaeIIISspI/FastDigest® SspI (AAT↓ATT) TasI (Tsp509I)/FastDigest® Tsp509I (TasI)

BsuRI (HaeIII)/FastDi-gest® HaeIII (BsuRI) (GG↓CC)

AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)

CviJI

Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaICviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)

BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI

Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI

EheI (SfoI)/FastDigest® EheI (GGC↓GCC)BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI

FspAI/FastDigest® FspAI* (RTGC↓GCAC)BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

HincII (HindII)/FastDigest® HincII* (GTY↓GAC) FaqI (BsmFI)/FastDigest® BsmFI (FaqI)

CviJI* (AG↓CY)


SetI

AluI/FastDigest® AluI (AG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/Fast-Digest® BsrBI (MbiI)* (CCG↓CTC), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)


Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIBsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)

CviJI



CviJI* (GG↓CY)

AluI/FastDigest® AluI (AG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)

CviJI

Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIBsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)






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DpnI/FastDigest® DpnI (GA↓TC)

AjiI (BmgBI)* (CAC↓GTC), ZraI (GAC↓GTC)HinfI/FastDigest® HinfI, PleI, SchI (MlyI)/FastDigest® MlyI (SchI)

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) HinfI/FastDigest® HinfIEco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT) SetIEco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) HinfI/FastDigest® HinfI, PfeI (TfiI)/FastDigest® TfiI (PfeI)Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) AluI/FastDigest® AluI, CviJI, SetIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) CviJI

FspAI/FastDigest® FspAI* (RTGC↓GCAC)Alw21I (BsiHKAI)/FastDigest® Alw21I, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

SspI/FastDigest® SspI (AAT↓ATT) TasI (Tsp509I)/FastDigest® Tsp509I (TasI)

DraI/FastDigest® DraI (TTT↓AAA)

AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC)


Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqIFspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDi-gest® FspI (NsbI) (TGC↓GCA)

HpyCH4V

MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT)DraI/FastDigest® DraI, FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC)


SetI




CviJI

DpnI/FastDigest® DpnI (GA↓TC)HinfI/FastDigest® HinfI, PleI, SchI (MlyI)/FastDigest® MlyI (SchI)



MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)

AluI/FastDigest® AluI, Alw21I (BsiHKAI)/FastDigest® Alw21I, CviJI, Ecl136II (EcoICRI)/FastDigest® Ecl136II, Eco24I (BanII), SacI/FastDigest® SacI, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI), SetI

Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA)

AjiI (BmgBI)* (CAC↓GTC), ZraI (GAC↓GTC) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiIAjiI (BmgBI)* (GAC↓GTG), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDi-gest® BsaAI (Ppu21I)* (YAC↓GTG)



SetI

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetIMbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIPdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC) BceAI

Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA)Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI

Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT)

AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), DpnI/FastDigest® DpnI (GA↓TC), FaiI* (YA↓TA), FaiI* (YA↓TG)

SetI


CviJI

Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC)Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI, SetI

BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)






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Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)

AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA) FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1IAluI/FastDigest® AluI (AG↓CT), Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG), BsuRI (HaeIII)/Fast-Digest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), HpyCH4V (TG↓CA), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA), MspA1I* (CMG↓CGG), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)

Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI

Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, TaqI/Fast-Digest® TaqI

DpnI/FastDigest® DpnI (GA↓TC) HinfI/FastDigest® HinfI, PfeI (TfiI)/FastDigest® TfiI (PfeI)FspAI/FastDigest® FspAI* (RTGC↓GCAC), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA) HpyCH4VFspAI/FastDigest® FspAI* (RTGC↓GCAT) HpyCH4V, Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I)

Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT)

AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC), DpnI/FastDigest® DpnI (GA↓TC), FaiI* (YA↓TA), FaiI* (YA↓TG)


AluI/FastDigest® AluI (AG↓CT), Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG), Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), CviJI* (RG↓CT), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), HpyCH4V (TG↓CA), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)

CviJI

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) CviJI, MnlI/FastDigest® MnlIEco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) LweI (SfaNI)/FastDigest® SfaNI (BmsI)

EheI (SfoI)/FastDigest® EheI (GGC↓GCC)FastDigest® HaeII (BfoI), HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)

FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDi-gest® FspI (NsbI) (TGC↓GCA)

HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)

MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG)CviJI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

PdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC)SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDi-gest® TauI

SrfI (GCCC↓GGGC) Cac8I

Eco72I (PmlI)/FastDi-gest® PmlI (Eco72I) (CAC↓GTG)

AjiI (BmgBI)* (CAC↓GTC), ZraI (GAC↓GTC) AjiI (BmgBI), MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI

AjiI (BmgBI)* (GAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG)Eco72I (PmlI)/FastDigest® PmlI (Eco72I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI


SetI

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetIEco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA)


FaiI* (YA↓TG) TscAI (TspRI)/FastDigest® TspRI (TscAI)MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIPdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC) BceAI

EheI (SfoI)/FastDigest® EheI (GGC↓GCC)

AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC), DpnI/FastDigest® DpnI (GA↓TC), FaiI* (YA↓TA), FaiI* (YA↓TG)

CviJI

AjiI (BmgBI)* (CAC↓GTC), ZraI (GAC↓GTC) Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)AluI/FastDigest® AluI (AG↓CT), Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG), Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA), CviJI* (RG↓CT), HpyCH4V (TG↓CA), MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG)


BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)

BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, MnlI/FastDigest® MnlI

Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) LweI (SfaNI)/FastDigest® SfaNI (BmsI)

Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT)FastDigest® HaeII (BfoI), HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)



MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG)BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI



FaiI* (CA↓TR)

AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC)

FaiI

AjiI (BmgBI)* (GAC↓GTG), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), MspA1I* (CMG↓CTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG), PvuII/FastDigest® PvuII (CAG↓CTG)

TscAI (TspRI)/FastDigest® TspRI (TscAI)

Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT) SetIEco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) AluI/FastDigest® AluI, CviJI, SetIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) CviJISspI/FastDigest® SspI (AAT↓ATT) TasI (Tsp509I)/FastDigest® Tsp509I (TasI)

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FaiI* (TA↓TR)

AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC)

FaiI

Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT) SetIEco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) AluI/FastDigest® AluI, CviJI, SetIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) CviJISspI/FastDigest® SspI (AAT↓ATT) TasI (Tsp509I)/FastDigest® Tsp509I (TasI)

FspAI/FastDigest® FspAI* (ATGC↓GCAY)

DraI/FastDigest® DraI (TTT↓AAA), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/Fast-Digest® HpaI (KspAI) (GTT↓AAC), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT)

HpyCH4VI

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI

Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC)LweI (SfaNI)/FastDigest® SfaNI (BmsI), HpyCH4V, Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I)

Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), EheI (SfoI)/FastDigest® EheI (GGC↓GCC)HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)

MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA)HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), NsbI (FspI)/FastDigest® FspI (NsbI)


SrfI (GCCC↓GGGC) Cac8ISspI/FastDigest® SspI (AAT↓ATT) HpyCH4V, Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I)

FspAI/FastDigest® FspAI* (GTGC↓GCAY)

BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)

BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

DpnI/FastDigest® DpnI (GA↓TC)Alw21I (BsiHKAI)/FastDigest® Alw21I, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

DraI/FastDigest® DraI (TTT↓AAA), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDi-gest® HpaI (KspAI) (GTT↓AAC), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT), SspI/FastDigest® SspI (AAT↓ATT)

HpyCH4V

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlIEco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) LweI (SfaNI)/FastDigest® SfaNI (BmsI), HpyCH4V



NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA)HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), NsbI (FspI)/FastDigest® FspI (NsbI)


RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT)

Alw21I (BsiHKAI)/FastDigest® Alw21I, Alw44I (ApaLI)/FastDigest® ApaLI (AIw44I), BseSI (Bme1580I)/Fast-Digest® Bme1580I (BseSI), Hpy8I (MjaIV)/FastDigest® Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)


HincII (HindII)/FastDigest® HincII* (GTC↓RAC)

Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) Hpy188I

Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC)Hpy8I (MjaIV)/FastDigest® Hpy8I, XmiI (AccI)/FastDigest® AccI (XmiI)

DpnI/FastDigest® DpnI (GA↓TC) Alw26I (BsmAI)/FastDigest® Alw26IEcl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI

KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC)HincII (HindII)/FastDigest® HincII, Hpy8I (MjaIV)/FastDi-gest® Hpy8I

MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIRsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT) MaeIII, NmuCI (Tsp45I)/FastDigest® NmuCI

HincII (HindII)/FastDigest® HincII* (GTT↓RAC)

AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA), DraI/FastDigest® DraI (TTT↓AAA), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT)


Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqIBst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Hpy8I (MjaIV)/FastDigest® Hpy8IFspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDi-gest® FspI (NsbI) (TGC↓GCA)

HpyCH4V

KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC)HincII (HindII)/FastDigest® HincII, Hpy8I (MjaIV)/Fast-Digest® Hpy8I, KspAI (HpaI)/FastDigest® HpaI (KspAI), FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I

RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT) MaeIII

HpyCH4V (TG↓CA)



Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT) CviJIEheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI

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KspAI (HpaI)/FastDi-gest® HpaI (KspAI) (GTT↓AAC)

AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), DraI/FastDigest® DraI (TTT↓AAA), MssI (PmeI)/FastDi-gest® MssI (GTTT↓AAAC), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT)


Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqIBst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Hpy8I (MjaIV)/FastDigest® Hpy8IFspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDi-gest® FspI (NsbI) (TGC↓GCA)

HpyCH4V

HincII (HindII)/FastDigest® HincII* (GTY↓AAC)HincII (HindII)/FastDigest® HincII, Hpy8I (MjaIV)/Fast-Digest® Hpy8I, KspAI (HpaI)/FastDigest® HpaI (KspAI), FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I

HincII (HindII)/FastDigest® HincII* (GTY↓GAC)HincII (HindII)/FastDigest® HincII, Hpy8I (MjaIV)/FastDi-gest® Hpy8I

RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT) MaeIII

MbiI (BsrBI)/FastDi-gest® BsrBI (MbiI)* (CCG↓CTC)

AjiI (BmgBI)* (CAC↓GTC), AjiI (BmgBI)* (GAC↓GTG), Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), HincII (HindII)/FastDigest® HincII* (GTY↓GAC), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG), ZraI (GAC↓GTC)

HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

AluI/FastDigest® AluI (AG↓CT), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), CviJI* (RG↓CT), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), HpyCH4V (TG↓CA), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)

SsiI (AciI)/FastDigest® AciI (SsiI)

Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG), Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA)Bsh1236I (BstUI)/FastDigest® Bsh1236I, SsiI (AciI)/FastDigest® AciI (SsiI)


Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC)MbiI (BsrBI)/FastDigest® BsrBI (MbiI), SsiI (AciI)/FastDi-gest® AciI (SsiI)


Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT)CviJI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

EheI (SfoI)/FastDigest® EheI (GGC↓GCC)BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

MspA1I* (CMG↓CGG)BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Bsh1236I (BstUI)/FastDigest® Bsh1236I, BtgI, Cfr42I (SacII), MspA1I, SsiI (AciI)/FastDigest® AciI (SsiI)

MspA1I* (CMG↓CTG), PvuII/FastDigest® PvuII (CAG↓CTG) MspA1I, SsiI (AciI)/FastDigest® AciI (SsiI)

PdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC)BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

SmaI/FastDigest® SmaI (CCC↓GGG), SrfI (GCCC↓GGGC)

BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

MbiI (BsrBI)/FastDi-gest® BsrBI (MbiI)* (GAG↓CGG)


SetI




CviJI

DpnI/FastDigest® DpnI (GA↓TC)HinfI/FastDigest® HinfI, PleI, SchI (MlyI)/FastDigest® MlyI (SchI)

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC)




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MlsI (MscI)/FastDi-gest® MscI (MlsI) (TGG↓CCA)


CviJI

Bst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaIBsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT)






MspA1I* (CAG↓CKG)


SetI

AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)



CviJI


EheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJIFaiI* (YA↓TG) TscAI (TspRI)/FastDigest® TspRI (TscAI)MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG) MspA1I

PvuII/FastDigest® PvuII (CAG↓CTG)AluI/FastDigest® AluI, CviJI, MspA1I, PvuII/FastDigest® PvuII, SetI

MspA1I* (CCG↓CKG)

AjiI (BmgBI)* (CAC↓GTC), AjiI (BmgBI)* (GAC↓GTG), Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), HincII (HindII)/FastDigest® HincII* (GTY↓GAC), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG), ZraI (GAC↓GTC)


AluI/FastDigest® AluI (AG↓CT), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI) (GG↓CC), CviJI* (RG↓CC), CviJI* (RG↓CT), Eco147I (StuI)/FastDigest® StuI (Eco147I) (AGG↓CCT), HpyCH4V (TG↓CA), MlsI (MscI)/FastDigest® MscI (MlsI) (TGG↓CCA)


Bsh1236I (BstUI)/FastDigest® Bsh1236I (CG↓CG),Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA)Bsh1236I (BstUI)/FastDigest® Bsh1236I, SsiI (AciI)/FastDigest® AciI (SsiI)


Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)MbiI (BsrBI)/FastDigest® BsrBI (MbiI), SsiI (AciI)/FastDi-gest® AciI (SsiI)


Eco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT)CviJI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

EheI (SfoI)/FastDigest® EheI (GGC↓GCC)BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG)BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Bsh1236I (BstUI)/FastDigest® Bsh1236I, BtgI, Cfr42I (SacII), MspA1I, SsiI (AciI)/FastDigest® AciI (SsiI)


PvuII/FastDigest® PvuII (CAG↓CTG) MspA1I, SsiI (AciI)/FastDigest® AciI (SsiI)

SmaI/FastDigest® SmaI (CCC↓GGG), SrfI (GCCC↓GGGC)


MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC)


FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I,

Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqI

DraI/FastDigest® DraI (TTT↓AAA), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT)DraI/FastDigest® DraI, FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I


HpyCH4V

RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT) Hpy8I (MjaIV)/FastDigest® Hpy8I

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NsbI (FspI)/FastDi-gest® FspI (NsbI) (TGC↓GCA)

DraI/FastDigest® DraI (TTT↓AAA), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/Fast-Digest® HpaI (KspAI) (GTT↓AAC), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC), RsaI/FastDigest® RsaI (GT↓AC), ScaI/FastDigest® ScaI (AGT↓ACT), SmiI (SwaI)/FastDigest® SwaI (SmiI) (ATTT↓AAAT), SspI/FastDigest® SspI (AAT↓ATT)

HpyCH4V

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlIEco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) LweI (SfaNI)/FastDigest® SfaNI (BmsI) , HpyCH4V


FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT)HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), NsbI (FspI)/FastDigest® FspI (NsbI)




PdiI (NaeI)/FastDi-gest® NaeI (PdiI) (GCC↓GGC)

DpnI/FastDigest® DpnI (GA↓TC), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)

MnlI/FastDigest® MnlI

Eco32I (EcoRV)/FastDigest® EcoRV (Eco32I) (GAT↓ATC) BccIEco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), EheI (SfoI)/FastDigest® EheI (GGC↓GCC), FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA)

SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDi-gest® AciI (SsiI), TauI/FastDigest® TauI

MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG), SmaI/FastDigest® SmaI (CCC↓GGG), SrfI (GCCC↓GGGC)

BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (CAC↓GTR)

AjiI (BmgBI)* (CAC↓GTC), ZraI (GAC↓GTC) AjiI (BmgBI), MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI

AjiI (BmgBI)* (GAC↓GTG), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG)Eco72I (PmlI)/FastDigest® PmlI (Eco72I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI


SetI

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetI

Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA)MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI

FaiI* (YA↓TG) TscAI (TspRI)/FastDigest® TspRI (TscAI)MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIPdiI (NaeI)/FastDigest® NaeI (PdiI) (GCC↓GGC) BceAI

Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (TAC↓GTR)

AjiI (BmgBI)* (CAC↓GTC), ZraI (GAC↓GTC) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI

AjiI (BmgBI)* (GAC↓GTG), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG)MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI


SetI

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetI

Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA)Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI


PvuII/FastDigest® PvuII (CAG↓CTG)


SetI

AluI/FastDigest® AluI (AG↓CT), CviJI* (RG↓CT), Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC)



CviJI


EheI (SfoI)/FastDigest® EheI (GGC↓GCC) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJIFaiI* (YA↓TG) TscAI (TspRI)/FastDigest® TspRI (TscAI)MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG) MspA1I

MspA1I* (CMG↓CTG)AluI/FastDigest® AluI, CviJI, MspA1I, PvuII/FastDigest® PvuII, SetI

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RsaI/FastDigest® RsaI (GT↓AC)

AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA) FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1IBsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqIBst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC)

MaeIII

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) Alw26I (BsmAI)/FastDigest® Alw26I

FspAI/FastDigest® FspAI* (RTGC↓GCAC)

Alw21I (BsiHKAI)/FastDigest® Alw21I, Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I), BseSI (Bme1580I)/Fast-Digest® Bme1580I (BseSI), Hpy8I (MjaIV)/FastDigest® Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA) HpyCH4VHincII (HindII)/FastDigest® HincII* (GTY↓GAC) MaeIII, NmuCI (Tsp45I)/FastDigest® NmuCIMssI (PmeI)/FastDigest® MssI (GTTT↓AAAC) Hpy8I (MjaIV)/FastDigest® Hpy8IScaI/FastDigest® ScaI (AGT↓ACT) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI

ScaI/FastDigest® ScaI (AGT↓ACT)

AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA), FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1IBsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqIBst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC), HincII (HindII)/FastDigest® HincII* (GTY↓AAC), KspAI (HpaI)/FastDigest® HpaI (KspAI) (GTT↓AAC)

MaeIII

Ecl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) Alw26I (BsmAI)/FastDigest® Alw26I

FspAI/FastDigest® FspAI* (RTGC↓GCAC)

Alw21I (BsiHKAI)/FastDigest® Alw21I, Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I), BseSI (Bme1580I)/Fast-Digest® Bme1580I (BseSI), Hpy8I (MjaIV)/FastDigest® Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA) HpyCH4VHincII (HindII)/FastDigest® HincII* (GTY↓GAC) MaeIII, NmuCI (Tsp45I)/FastDigest® NmuCIMssI (PmeI)/FastDigest® MssI (GTTT↓AAAC) Hpy8I (MjaIV)/FastDigest® Hpy8IRsaI/FastDigest® RsaI (GT↓AC) Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI

SmaI/FastDigest® SmaI (CCC↓GGG)




SmuI (FauI), SsiI (AciI)/FastDigest® AciI (SsiI)

MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG)



SrfI (GCCC↓GGGC)

BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BmeT110I, BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BssKI, Cfr9I (XmaI), Eco88I (AvaI)/FastDigest® AvaI (Eco88I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, SmaI/FastDigest® SmaI

SmiI (SwaI)/FastDi-gest® SwaI (SmiI) (ATTT↓AAAT)



Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqI

DraI/FastDigest® DraI (TTT↓AAA), MssI (PmeI)/FastDigest® MssI (GTTT↓AAAC)DraI/FastDigest® DraI, FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I


HpyCH4V

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SrfI (GCCC↓GGGC)




Cac8I, SmuI (FauI), SsiI (AciI)/FastDigest® AciI (SsiI)

MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (GAG↓CGG), MspA1I* (CMG↓CGG)



SmaI/FastDigest® SmaI (CCC↓GGG)


SspI/FastDigest® SspI (AAT↓ATT)

AanI (PsiI)/FastDigest® PsiI (AanI) (TTA↓TAA)FastDigest® MseI (SaqAI), TasI (Tsp509I)/FastDigest® Tsp509I (TasI), Tru1I (MseI)/FastDigest® Tru1I

Bsp68I (NruI)/FastDigest® NruI (RruI) (TCG↓CGA) TaqI/FastDigest® TaqIBst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I) (GTA↓TAC), DpnI/FastDigest® DpnI (GA↓TC), FaiI* (YA↓TA), FaiI* (YA↓TG)

TasI (Tsp509I)/FastDigest® Tsp509I (TasI)

FspAI/FastDigest® FspAI* (RTGC↓GCAC), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA) HpyCH4VFspAI/FastDigest® FspAI* (RTGC↓GCAT) HpyCH4V, Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I)

ZraI (GAC↓GTC)

AjiI (BmgBI)* (CAC↓GTC)AatII/FastDigest® AatII, Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I), MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI, ZraI

AjiI (BmgBI)* (GAC↓GTG), Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I) (TAC↓GTA), Eco72I (PmlI)/FastDigest® PmlI (Eco72I) (CAC↓GTG), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTA), Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I)* (YAC↓GTG)



SetI

DpnI/FastDigest® DpnI (GA↓TC) HinfI/FastDigest® HinfIEcl136II (EcoICRI)/FastDigest® Ecl136II (GAG↓CTC), MbiI (BsrBI)/FastDigest® BsrBI (MbiI)* (CCG↓CTC) MnlI/FastDigest® MnlI, SetIEco47III (AfeI)/FastDigest® AfeI (Eco47III) (AGC↓GCT), FspAI/FastDigest® FspAI* (RTGC↓GCAC), FspAI/FastDigest® FspAI* (RTGC↓GCAT), NsbI (FspI)/FastDigest® FspI (NsbI) (TGC↓GCA)


EheI (SfoI)/FastDigest® EheI (GGC↓GCC)CseI (HgaI)/FastDigest® HgaI (CseI), Hin1I (BsaHI)/FastDi-gest® BsaHI (Hin1I)


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Recognition Sites Resulting from Ligation of Protruding Compatible DNA Ends

Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends.



Second restriction enzyme

Restriction enzyme that cleave the newly generated recognition sequence

AatII/FastDigest® AatII (GACGT↓C)

SetI* (ACGT), TaiI (MaeII)/FastDigest® TaiI (ACGT) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI

AbsI (CC↓TCGAGG)

Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG), SmoI (SmlI)* (C↓TCGAG), XhoI/FastDigest® XhoI (C↓TCGAG)

BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), MnlI/FastDigest® MnlI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI

PspXI* (VC↓TCGAGC), PspXI* (VC↓TCGAGT)BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), MnlI/FastDigest® MnlI, PspXI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI

PspXI* (VC↓TCGAGG)AbsI, BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), MnlI/FastDigest® MnlI, PspXI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI

SalI/FastDigest® SalI (G↓TCGAC) MnlI/FastDigest® MnlI, TaqI/FastDigest® TaqISgrDI (CG↓TCGACG) Hpy99I, MnlI/FastDigest® MnlI, TaqI/FastDigest® TaqI

Acc65I (Asp718I)/FastDi-gest® Acc65I (G↓GTACC)

BshNI (BanI)/FastDigest® BanI (BshNI)* (G↓GTACC)Acc65I (Asp718I)/FastDigest® Acc65I, BshNI (BanI)/FastDigest® BanI (BshNI), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Csp6I (CviQI)/FastDigest® Csp6I, KpnI/FastDigest® KpnI, RsaI/FastDigest® RsaI

Bsp1407I (BsrGI)/FastDigest® Bsp1407I (T↓GTACA), Pfl23II (BsiWI)/FastDi-gest® BsiWI (Pfl23II) (C↓GTACG), TatI/FastDigest® TatI* (W↓GTACA), TatI/FastDigest® TatI* (W↓GTACT)


AflIII* (A↓CATGT)

BtgI* (C↓CATGG), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG), FatI (↓CATG), NcoI/FastDigest® NcoI (C↓CATGG), PagI (BspHI)/FastDigest® BspHI (PagI) (T↓CATGA)

CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)

PscI (PciI) (A↓CATGT)AflIII, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), PscI (PciI), XceI (NspI)/FastDigest® NspI (XceI)

AflIII* (A↓CGCGT)

BtgI* (C↓CGCGG) Bsh1236I (BstUI)/FastDigest® Bsh1236IMauBI/FastDigest® MauBI (CG↓CGCGCG), SgsI (AscI)/FastDigest® AscI (SgsI) (GG↓CGCGCC), PauI (BssHII)/FastDigest® BssHII (PteI) (G↓CGCGC)

Bsh1236I (BstUI)/FastDigest® Bsh1236I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)

MluI/FastDigest® MluI (A↓CGCGT) AflIII, Bsh1236I (BstUI)/FastDigest® Bsh1236I, MluI/FastDigest® MluIAflIII* (A↓CGTGT) BtgI* (C↓CGTGG) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiIAlw21I (BsiHKAI)/FastDi-gest® Alw21I* (GAGCA↓C)

SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCA↓C)Alw21I (BsiHKAI)/FastDigest® Alw21I, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

Alw21I (BsiHKAI)/FastDi-gest® Alw21I* (GAGCT↓C)

Eco24I (BanII)* (GAGCT↓C), SacI/FastDigest® SacI (GAGCT↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCT↓C)


SetI* (AGCT) AluI/FastDigest® AluI, CviJI, SetI

Alw21I (BsiHKAI)/FastDi-gest® Alw21I* (GTGCA↓C)

BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GTGCA↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCA↓C)

Alw21I (BsiHKAI)/FastDigest® Alw21I, Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I), BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), Hpy8I (MjaIV)/FastDigest® Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I) (ATGCA↓T) HpyCH4VPstI/FastDigest® PstI (CTGCA↓G), SdaI (SbfI)/FastDigest® SbfI (SdaI) (CCTGCA↓GG)

BsgI, HpyCH4V

Alw21I (BsiHKAI)/FastDi-gest® Alw21I* (GTGCT↓C)

SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCT↓C)Alw21I (BsiHKAI)/FastDigest® Alw21I, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I) (G↓TGCAC)

BfmI (SfcI)/FastDigest® SfcI (BfmI)* (C↓TGCAG) BsgI, HpyCH4V

ApaI/FastDigest® ApaI (GGGCC↓C)

BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GGGCC↓C), Eco24I (BanII)* (GGGCC↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCC↓C)

ApaI/FastDigest® ApaI, BmgT120I, BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), Bsp120I (PspOMI)/FastDigest® Bsp120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

BamHI/FastDigest® BamHI (G↓GATCC)

BclI/FastDigest® BclI (T↓GATCA), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I) (↓GATC), MboI/FastDigest® MboI (↓GATC)

Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDi-gest® DpnI, MboI/FastDigest® MboI

BglII/FastDigest® BglII (A↓GATCT), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCT)

Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDi-gest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI

PsuI (BstYI)/FastDigest® PsuI* (R↓GATCC)BamHI/FastDigest® BamHI, Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), BspPI (AlwI), DpnI/Fast-Digest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI

BbeI (GGCGC↓C)FastDigest® HaeII (BfoI)* (RGCGC↓C)

BbeI, FastDigest® HaeII (BfoI), BshNI (BanI)/FastDigest® BanI (BshNI), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), EheI (SfoI)/FastDigest® EheI, HhaI/FastDigest® HhaI, Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I), Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), NarI, SspDI (KasI)

FastDigest® HaeII (BfoI)* (RGCGC↓T)FastDigest® HaeII (BfoI), HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)





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BclI/FastDigest® BclI (T↓GATCA)

BamHI/FastDigest® BamHI (G↓GATCC), BglII/FastDigest® BglII (A↓GATCT), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I) (↓GATC), MboI/FastDigest® MboI (↓GATC), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCC), PsuI (BstYI)/Fast-Digest® PsuI* (R↓GATCT)


BcuI (SpeI)/FastDigest® SpeI (BcuI) (A↓CTAGT)

Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CTAGG), NheI/FastDigest® NheI (G↓CTAGC), XbaI/FastDigest® XbaI (T↓CTAGA), XmaJI (AvrII)/FastDi-gest® AvrII (XmaJI) (C↓CTAGG)

FspBI (BfaI)/FastDigest® BfaI (FspBI)

BfmI (SfcI)/FastDigest® SfcI (BfmI)* (C↓TGCAG)

Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I) (G↓TGCAC) HpyCH4V

BglII/FastDigest® BglII (A↓GATCT)

BamHI/FastDigest® BamHI (G↓GATCC), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCC)

Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI



PsuI (BstYI)/FastDigest® PsuI* (R↓GATCT)BglII/FastDigest® BglII, Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI

BmeT110I* (CC↓CGRG)

Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGCC), Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I) (G↓CGC), NarI (GG↓CGCC), SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)

SmuI (FauI), SsiI (AciI)/FastDigest® AciI (SsiI)

HpaII/FastDigest® HpaII (C↓CGG), MspI (HpaII)/FastDigest® MspI (C↓CGG), SsiI (AciI)/FastDigest® AciI (SsiI)* (G↓CGG)


BmeT110I* (CT↓CGRG)Bsp119I (BstBI)/FastDigest® Bsp119I (TT↓CGAA), Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I) (AT↓CGAT), TaqI/FastDigest® TaqI (T↓CGA), XmiI (AccI)/FastDi-gest® AccI (XmiI)* (GT↓CGAC)


BsaWI* (A↓CCGGW)

BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), SgrAI* (CR↓CCGGTG)

BsaWI, BshTI (AgeI)/FastDigest® AgeI (BshTI), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), MreI (Sse232I)/Fast-Digest® MreI (CG↓CCGGCG), NgoMIV (G↓CCGGC), SgrAI* (CR↓CCGGCG)

Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

Cfr9I (XmaI) (C↓CCGGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)


Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) BsaWI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

BsaWI* (T↓CCGGW)

BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), SgrAI* (CR↓CCGGTG)

BsaWI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI





Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA)BsaWI, HpaII/FastDigest® HpaII, Hpy188III, Kpn2I (BspEI)/FastDigest® Kpn2I, MspI (HpaII)/FastDigest® MspI

BseSI (Bme1580I)/FastDi-gest® Bme1580I (BseSI)* (GGGCA↓C)

SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCA↓C)BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)

BseSI (Bme1580I)/FastDi-gest® Bme1580I (BseSI)* (GGGCC↓C)

ApaI/FastDigest® ApaI (GGGCC↓C), Eco24I (BanII)* (GGGCC↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCC↓C)


BseSI (Bme1580I)/FastDi-gest® Bme1580I (BseSI)* (GTGCA↓C)

Alw21I (BsiHKAI)/FastDigest® Alw21I* (GTGCA↓C), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)* (GTGCA↓C)



BsgI, HpyCH4V

BseSI (Bme1580I)/FastDi-gest® Bme1580I (BseSI)* (GTGCC↓C)

SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCC↓C)BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)

Bsh1285I (BsiEI)/FastDi-gest® BsiEI (Bsh1285I)* (CGAT↓CG)

PacI/FastDigest® PacI (TTAAT↓TAA) FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I

PvuI/FastDigest® PvuI (CGAT↓CG), SfaAI (AsiSI)/FastDigest® AsiSI (SfaAI) (GCGAT↓CGC)

Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDi-gest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI

BshNI (BanI)/FastDigest® BanI (BshNI)* (G↓GCGCC)

SspDI (KasI) (G↓GCGCC)


BshNI (BanI)/FastDigest® BanI (BshNI)* (G↓GTACC)

Acc65I (Asp718I)/FastDigest® Acc65I (G↓GTACC)Acc65I (Asp718I)/FastDigest® Acc65I, BshNI (BanI)/FastDigest® BanI (BshNI), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Csp6I (CviQI)/FastDigest® Csp6I, KpnI/FastDigest® KpnI, RsaI/FastDigest® RsaI

Bsp1407I (BsrGI)/FastDigest® Bsp1407I (T↓GTACA), Pfl23II (BsiWI)/FastDi-gest® BsiWI (Pfl23II) (C↓GTACG), TatI/FastDigest® TatI* (W↓GTACA), TatI/FastDigest® TatI* (W↓GTACT)


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BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT)

BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) BsaWI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), SgrAI* (CR↓CCGGTG)






Bsp119I (BstBI)/FastDigest® Bsp119I (TT↓CGAA)

BmeT110I* (CY↓CGAG), Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I) (AT↓CGAT), TaqI/FastDigest® TaqI (T↓CGA), XmiI (AccI)/FastDigest® AccI (XmiI)* (GT↓CGAC)


Bsp120I (PspOMI)/FastDi-gest® Bsp120I (G↓GGCCC)

CfrI (EaeI)* (Y↓GGCCA), CfrI (EaeI)* (Y↓GGCCG), Eco52I (EagI)/FastDigest® EagI (Eco52I) (C↓GGCCG)

BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), CviJI

NotI/FastDigest® NotI (GC↓GGCCGC)BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/Fast-Digest® Sau96I (Cfr13I), CviJI, SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDigest® AciI (SsiI), TauI/FastDigest® TauI

Bsp1407I (BsrGI)/FastDi-gest® Bsp1407I (T↓GTACA)

Acc65I (Asp718I)/FastDigest® Acc65I (G↓GTACC), BshNI (BanI)/FastDi-gest® BanI (BshNI)* (G↓GTACC), Pfl23II (BsiWI)/FastDigest® BsiWI (Pfl23II) (C↓GTACG)


TatI/FastDigest® TatI* (W↓GTACA)Bsp1407I (BsrGI)/FastDigest® Bsp1407I, Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI, TatI/FastDigest® TatI

TatI/FastDigest® TatI* (W↓GTACT)Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI, TatI/FastDigest® TatI

Bsp143I (Sau3AI)/FastDi-gest® Sau3AI (Bsp143I) (↓GATC)

BamHI/FastDigest® BamHI (G↓GATCC), BclI/FastDigest® BclI (T↓GATCA), BglII/FastDigest® BglII (A↓GATCT), MboI/FastDigest® MboI (↓GATC), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCC), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCT)


BspTI (AflII)/FastDigest® AflII (BspTI) (C↓TTAAG)

SmoI (SmlI)* (C↓TTAAG)BspTI (AflII)/FastDigest® AflII (BspTI), FastDigest® MseI (SaqAI), SmoI (SmlI), Tru1I (MseI)/FastDigest® Tru1I

Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I) (AT↓CGAT)

BmeT110I* (CY↓CGAG), Bsp119I (BstBI)/FastDigest® Bsp119I (TT↓CGAA), TaqI/FastDigest® TaqI (T↓CGA), XmiI (AccI)/FastDigest® AccI (XmiI)* (GT↓CGAC)


BtgI* (C↓CACGG) AflIII* (A↓CACGT) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI

BtgI* (C↓CATGG)

AflIII* (A↓CATGT), FatI (↓CATG), PagI (BspHI)/FastDigest® BspHI (PagI) (T↓CATGA), PscI (PciI) (A↓CATGT)


Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG), NcoI/FastDigest® NcoI (C↓CATGG)

BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BtgI, CviAII, Eco130I (StyI)/FastDi-gest® StyI (Eco130I), FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), NcoI/FastDigest® NcoI

BtgI* (C↓CGCGG)AflIII* (A↓CGCGT), MluI/FastDigest® MluI (A↓CGCGT) Bsh1236I (BstUI)/FastDigest® Bsh1236I, SsiI (AciI)/FastDigest® AciI (SsiI)MauBI/FastDigest® MauBI (CG↓CGCGCG), SgsI (AscI)/FastDigest® AscI (SgsI) (GG↓CGCGCC), PauI (BssHII)/FastDigest® BssHII (PteI) (G↓CGCGC)

Bsh1236I (BstUI)/FastDigest® Bsh1236I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), SsiI (AciI)/FastDigest® AciI (SsiI)

Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (A↓CCGGY)

BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) BsaWI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), SgrAI* (CR↓CCGGTG)




MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG), NgoMIV (G↓CCGGC), SgrAI* (CR↓CCGGCG)


Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (G↓CCGGY)

BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), SgrAI* (CR↓CCGGTG)




MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG), NgoMIV (G↓CCGGC), SgrAI* (CR↓CCGGCG)

Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)

Cfr42I (SacII) (CCGC↓GG) Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)* (CGGC↓CG) SsiI (AciI)/FastDigest® AciI (SsiI)

Cfr9I (XmaI) (C↓CCGGG)

BsaWI* (W↓CCGGA), BsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), Kpn2I (BspEI)/FastDi-gest® Kpn2I (T↓CCGGA), MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG), NgoMIV (G↓CCGGC), SgrAI* (CR↓CCGGCG), SgrAI* (CR↓CCGGTG)


Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)


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CfrI (EaeI)* (C↓GGCCR)

Bsp120I (PspOMI)/FastDigest® Bsp120I (G↓GGCCC)BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), CviJI

Eco52I (EagI)/FastDigest® EagI (Eco52I) (C↓GGCCG)Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I)

NotI/FastDigest® NotI (GC↓GGCCGC)

Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDigest® AciI (SsiI), TauI/FastDigest® TauI

CfrI (EaeI)* (T↓GGCCR)


Eco52I (EagI)/FastDigest® EagI (Eco52I) (C↓GGCCG) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI

NotI/FastDigest® NotI (GC↓GGCCGC)BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDigest® AciI (SsiI), TauI/FastDi-gest® TauI

CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GACCG)

Eco47I (AvaII)/FastDigest® AvaII (Eco47I)* (G↓GACC)BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)

FastDigest® SanDI (KflI)* (GG↓GACCC), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GACCC)

BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)

Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GACCT)BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), SetI

CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GTCCG)

Eco47I (AvaII)/FastDigest® AvaII (Eco47I)* (G↓GTCC), Psp5II (PpuMI)/FastDi-gest® PpuMI (Psp5II)* (RG↓GTCCT)

BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)

FastDigest® SanDI (KflI)* (GG↓GTCCC), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GTCCC)


Csp6I (CviQI)/FastDigest® Csp6I (G↓TAC)

NdeI/FastDigest® NdeI (CA↓TATG) FaiI

CviAII (C↓ATG) XmiI (AccI)/FastDigest® AccI (XmiI)* (GT↓ATAC) FaiI

Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG)



BtgI* (C↓CATGG), NcoI/FastDigest® NcoI (C↓CATGG)BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BtgI, CviAII, Eco130I (StyI)/FastDi-gest® StyI (Eco130I), FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), NcoI/FastDigest® NcoI

Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CTAGG)

BcuI (SpeI)/FastDigest® SpeI (BcuI) (A↓CTAGT), NheI/FastDigest® NheI (G↓CTAGC), XbaI/FastDigest® XbaI (T↓CTAGA)


XmaJI (AvrII)/FastDigest® AvrII (XmaJI) (C↓CTAGG)BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I), FspBI (BfaI)/FastDigest® BfaI (FspBI), XmaJI (AvrII)/FastDigest® AvrII (XmaJI)

Eco24I (BanII)* (GAGCC↓C) SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCC↓C) CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

Eco24I (BanII)* (GAGCT↓C)Alw21I (BsiHKAI)/FastDigest® Alw21I* (GAGCT↓C), SacI/FastDigest® SacI (GAGCT↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCT↓C)



Eco24I (BanII)* (GGGCC↓C)ApaI/FastDigest® ApaI (GGGCC↓C), BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GGGCC↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCC↓C)


Eco24I (BanII)* (GGGCT↓C) SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCT↓C) CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

Eco47I (AvaII)/FastDigest® AvaII (Eco47I)* (G↓GACC)

CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GACCG)BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I)

FastDigest® SanDI (KflI)* (GG↓GACCC), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GACCC)


Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GACCT)BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), SetI

Eco47I (AvaII)/FastDigest® AvaII (Eco47I)* (G↓GTCC)

CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GTCCG), Psp5II (PpuMI)/FastDi-gest® PpuMI (Psp5II)* (RG↓GTCCT)


FastDigest® SanDI (KflI)* (GG↓GTCCC), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GTCCC)


Eco52I (EagI)/FastDigest® EagI (Eco52I) (C↓GGCCG)


CfrI (EaeI)* (Y↓GGCCA) BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI

CfrI (EaeI)* (Y↓GGCCG)Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I)


Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDigest® AciI (SsiI), TauI/FastDigest® TauI

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Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓CCGGG)

BsaWI* (W↓CCGGA), BsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), Kpn2I (BspEI)/FastDi-gest® Kpn2I (T↓CCGGA), MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG), NgoMIV (G↓CCGGC), SgrAI* (CR↓CCGGCG), SgrAI* (CR↓CCGGTG)


Cfr9I (XmaI) (C↓CCGGG)


Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG)

AbsI (CC↓TCGAGG), PspXI* (VC↓TCGAGC), PspXI* (VC↓TCGAGG), PspXI* (VC↓TCGAGT), SmoI (SmlI)* (C↓TCGAG), XhoI/FastDigest® XhoI (C↓TCGAG)

BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI

SalI/FastDigest® SalI (G↓TCGAC) TaqI/FastDigest® TaqISgrDI (CG↓TCGACG) Hpy99I, TaqI/FastDigest® TaqI

EcoRI/FastDigest® EcoRI (G↓AATTC)

MunI (MfeI)/FastDigest® MfeI (MunI) (C↓AATTG), TasI (Tsp509I)/FastDigest® Tsp509I (TasI) (↓AATT)


XapI (ApoI)/FastDigest® XapI* (R↓AATTC)EcoRI/FastDigest® EcoRI, TasI (Tsp509I)/FastDigest® Tsp509I (TasI), XapI (ApoI)/FastDigest® XapI

XapI (ApoI)/FastDigest® XapI* (R↓AATTT) TasI (Tsp509I)/FastDigest® Tsp509I (TasI), XapI (ApoI)/FastDigest® XapI

EcoRII* (↓CCAGG) FastDigest® SexAI (CsiI)* (A↓CCAGGT)Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, EcoRII, MvaI (BstNI)/FastDigest® MvaI, SetI

EcoRII* (↓CCTGG) FastDigest® SexAI (CsiI)* (A↓CCTGGT)Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, EcoRII, MvaI (BstNI)/FastDigest® MvaI

FatI (↓CATG)AflIII* (A↓CATGT), BtgI* (C↓CATGG), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG), NcoI/FastDigest® NcoI (C↓CATGG), PagI (BspHI)/FastDigest® BspHI (PagI) (T↓CATGA), PscI (PciI) (A↓CATGT)


FspBI (BfaI)/FastDigest® BfaI (FspBI) (C↓TAG)

NdeI/FastDigest® NdeI (CA↓TATG) FaiI

FastDigest® HaeII (BfoI)* (AGCGC↓Y)

BbeI (GGCGC↓C)FastDigest® HaeII (BfoI), HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)

FastDigest® HaeII (BfoI)* (GGCGC↓Y)

BbeI (GGCGC↓C)


Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GA↓CGYC)

Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I) (G↓CGC), SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)


MaeII (A↓CGT), Psp1406I (AclI)/FastDigest® AclI (Psp1406I) (AA↓CGTT) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiINarI (GG↓CGCC) CseI (HgaI)/FastDigest® HgaI (CseI), Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)

Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GG↓CGYC)

Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I) (G↓CGC), SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)


NarI (GG↓CGCC)


Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II) (CATG↓)

PaeI (SphI)/FastDigest® SphI (PaeI) (GCATG↓C), XceI (NspI)/FastDigest® NspI (XceI)* (RCATG↓C), XceI (NspI)/FastDigest® NspI (XceI)* (RCATG↓T)


Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I) (G↓CGC)

Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGCC), NarI (GG↓CGCC), SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)


HpaII/FastDigest® HpaII (C↓CGG)

BmeT110I* (CY↓CGGG)BcnI (NciI)/FastDigest® NciI (BcnI), Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI



MspI (HpaII)/FastDigest® MspI (C↓CGG), SsiI (AciI)/FastDigest® AciI (SsiI)* (G↓CGG)


Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA)

BsaWI* (W↓CCGGA)BsaWI, HpaII/FastDigest® HpaII, Hpy188III, Kpn2I (BspEI)/FastDigest® Kpn2I, MspI (HpaII)/FastDigest® MspI

BsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), SgrAI* (CR↓CCGGTG)

BsaWI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI





MaeII (A↓CGT)Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGTC), Psp1406I (AclI)/FastDi-gest® AclI (Psp1406I) (AA↓CGTT)


MauBI/FastDigest® MauBI (CG↓CGCGCG)

AflIII* (A↓CGCGT), BtgI* (C↓CGCGG), MluI/FastDigest® MluI (A↓CGCGT)Bsh1236I (BstUI)/FastDigest® Bsh1236I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)

PauI (BssHII)/FastDigest® BssHII (PteI) (G↓CGCGC), SgsI (AscI)/FastDigest® AscI (SgsI) (GG↓CGCGCC)

Bsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), PauI (BssHII)/FastDigest® BssHII (PteI)

197


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MboI/FastDigest® MboI (↓GATC)

BamHI/FastDigest® BamHI (G↓GATCC), BclI/FastDigest® BclI (T↓GATCA), BglII/FastDigest® BglII (A↓GATCT), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I) (↓GATC), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCC), PsuI (BstYI)/FastDigest® PsuI* (R↓GATCT)


MluI/FastDigest® MluI (A↓CGCGT)

AflIII* (A↓CGCGT) AflIII, Bsh1236I (BstUI)/FastDigest® Bsh1236I, MluI/FastDigest® MluIBtgI* (C↓CGCGG) Bsh1236I (BstUI)/FastDigest® Bsh1236IMauBI/FastDigest® MauBI (CG↓CGCGCG), SgsI (AscI)/FastDigest® AscI (SgsI) (GG↓CGCGCC), PauI (BssHII)/FastDigest® BssHII (PteI) (G↓CGCGC)

Bsh1236I (BstUI)/FastDigest® Bsh1236I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I)

Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I) (ATGCA↓T)

Alw21I (BsiHKAI)/FastDigest® Alw21I* (GTGCA↓C), BseSI (Bme1580I)/Fast-Digest® Bme1580I (BseSI)* (GTGCA↓C), PstI/FastDigest® PstI (CTGCA↓G), SdaI (SbfI)/FastDigest® SbfI (SdaI) (CCTGCA↓GG), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)* (GTGCA↓C)

HpyCH4V

MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG)

BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT)


Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), NgoMIV (G↓CCGGC)Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)



SgrAI* (CR↓CCGGCG)Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MreI (Sse232I)/FastDigest® MreI, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI), SgrAI

SgrAI* (CR↓CCGGTG)Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, SgrAI

FastDigest® MseI (SaqAI) (T↓TAA)

NdeI/FastDigest® NdeI (CA↓TATG) FaiITru1I (MseI)/FastDigest® Tru1I (T↓TAA), VspI (AseI)/FastDigest® AseI (VspI) (AT↓TAAT)


MspI (HpaII)/FastDigest® MspI (C↓CGG)




HpaII/FastDigest® HpaII (C↓CGG), SsiI (AciI)/FastDigest® AciI (SsiI)* (G↓CGG)


MunI (MfeI)/FastDigest® MfeI (MunI) (C↓AATTG)

EcoRI/FastDigest® EcoRI (G↓AATTC), TasI (Tsp509I)/FastDigest® Tsp509I (TasI) (↓AATT), XapI (ApoI)/FastDigest® XapI* (R↓AATTC), XapI (ApoI)/FastDi-gest® XapI* (R↓AATTT)


NarI (GG↓CGCC)

Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGCC)


Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGTC) Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I) (G↓CGC), SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)


NcoI/FastDigest® NcoI (C↓CATGG)



BtgI* (C↓CATGG), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG)BseDI (BsaJI)/FastDigest® BsaJI (BseDI), BtgI, CviAII, Eco130I (StyI)/FastDi-gest® StyI (Eco130I), FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), NcoI/FastDigest® NcoI

NdeI/FastDigest® NdeI (CA↓TATG)

Csp6I (CviQI)/FastDigest® Csp6I (G↓TAC), FspBI (BfaI)/FastDigest® BfaI (FspBI) (C↓TAG), FastDigest® MseI (SaqAI) (T↓TAA), Tru1I (MseI)/FastDigest® Tru1I (T↓TAA), VspI (AseI)/FastDigest® AseI (VspI) (AT↓TAAT)

FaiI

NgoMIV (G↓CCGGC)

BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT), SgrAI* (CR↓CCGGTG)


Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), MreI (Sse232I)/Fast-Digest® MreI (CG↓CCGGCG), SgrAI* (CR↓CCGGCG)

Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)



NheI/FastDigest® NheI (G↓CTAGC)

BcuI (SpeI)/FastDigest® SpeI (BcuI) (A↓CTAGT), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CTAGG), XbaI/FastDigest® XbaI (T↓CTAGA), XmaJI (AvrII)/FastDigest® AvrII (XmaJI) (C↓CTAGG)



Bsp120I (PspOMI)/FastDigest® Bsp120I (G↓GGCCC)BmgT120I, BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), CviJI, SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDigest® TauI

CfrI (EaeI)* (Y↓GGCCA)BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDigest® TauI

CfrI (EaeI)* (Y↓GGCCG), Eco52I (EagI)/FastDigest® EagI (Eco52I) (C↓GGCCG)Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDigest® TauI

198

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PacI/FastDigest® PacI (TTAAT↓TAA)

Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)* (CGAT↓CG), PvuI/FastDi-gest® PvuI (CGAT↓CG), SfaAI (AsiSI)/FastDigest® AsiSI (SfaAI) (GCGAT↓CGC)


PaeI (SphI)/FastDigest® SphI (PaeI) (GCATG↓C)

Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II) (CATG) CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)

XceI (NspI)/FastDigest® NspI (XceI)* (RCATG↓C)Cac8I, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), PaeI (SphI)/FastDigest® SphI (PaeI), XceI (NspI)/FastDigest® NspI (XceI)

XceI (NspI)/FastDigest® NspI (XceI)* (RCATG↓T)CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), XceI (NspI)/FastDi-gest® NspI (XceI)

PagI (BspHI)/FastDigest® BspHI (PagI) (T↓CATGA)

AflIII* (A↓CATGT), BtgI* (C↓CATGG), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG), FatI (↓CATG), NcoI/FastDigest® NcoI (C↓CATGG), PscI (PciI) (A↓CATGT)


PasI* (CC↓CAGGG) TseI* (G↓CAGC) BseYI

PauI (BssHII)/FastDigest® BssHII (PteI) (G↓CGCGC)


MauBI/FastDigest® MauBI (CG↓CGCGCG), SgsI (AscI)/FastDigest® AscI (SgsI) (GG↓CGCGCC)


Pfl23II (BsiWI)/FastDigest® BsiWI (Pfl23II) (C↓GTACG)

Acc65I (Asp718I)/FastDigest® Acc65I (G↓GTACC), BshNI (BanI)/FastDigest® BanI (BshNI)* (G↓GTACC), Bsp1407I (BsrGI)/FastDigest® Bsp1407I (T↓GTACA), TatI/FastDigest® TatI* (W↓GTACA), TatI/FastDigest® TatI* (W↓GTACT)


PscI (PciI) (A↓CATGT)

AflIII* (A↓CATGT)AflIII, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), PscI (PciI), XceI (NspI)/FastDigest® NspI (XceI)

BtgI* (C↓CATGG), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CATGG), FatI (↓CATG), NcoI/FastDigest® NcoI (C↓CATGG), PagI (BspHI)/FastDigest® BspHI (PagI) (T↓CATGA)


Psp1406I (AclI)/FastDigest® AclI (Psp1406I) (AA↓CGTT)

Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGTC), MaeII (A↓CGT) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI

Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (AG↓GACCY)

CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GACCG), Eco47I (AvaII)/FastDi-gest® AvaII (Eco47I)* (G↓GACC)


FastDigest® SanDI (KflI)* (GG↓GACCC)BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)

Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (AG↓GTCCY)

CpoI (RsrII)/FastDigest® RsrII (CpoI)* (CG↓GTCCG), Eco47I (AvaII)/FastDi-gest® AvaII (Eco47I)* (G↓GTCC)

BmgT120I, Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), SetI

FastDigest® SanDI (KflI)* (GG↓GTCCC)

BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II), SetI

Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (GG↓GACCY)


BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/Fast-Digest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), FaqI (BsmFI)/FastDigest® BsmFI (FaqI)

FastDigest® SanDI (KflI)* (GG↓GACCC)

BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, FaqI (BsmFI)/FastDigest® BsmFI (FaqI), FastDigest® SanDI (KflI), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)

Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (GG↓GTCCY)




BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, FastDigest® SanDI (KflI), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)

PspXI* (AC↓TCGAGB)

AbsI (CC↓TCGAGG)BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), PspXI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI




PspXI* (CC↓TCGAGB)

AbsI (CC↓TCGAGG)AbsI, BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), MnlI/FastDigest® MnlI, PspXI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI


BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), MnlI/FastDigest® MnlI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI

SalI/FastDigest® SalI (G↓TCGAC) MnlI/FastDigest® MnlI, TaqI/FastDigest® TaqISgrDI (CG↓TCGACG) Hpy99I, MnlI/FastDigest® MnlI, TaqI/FastDigest® TaqI

PspXI* (GC↓TCGAGB)

AbsI (CC↓TCGAGG)BmeT110I, Eco88I (AvaI)/FastDigest® AvaI (Eco88I), PspXI, SmoI (SmlI), TaqI/FastDigest® TaqI, XhoI/FastDigest® XhoI




199


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PstI/FastDigest® PstI (CTGCA↓G)

Alw21I (BsiHKAI)/FastDigest® Alw21I* (GTGCA↓C), BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GTGCA↓C), Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I) (ATGCA↓T), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCA↓C)

HpyCH4V

SdaI (SbfI)/FastDigest® SbfI (SdaI) (CCTGCA↓GG) BfmI (SfcI)/FastDigest® SfcI (BfmI), HpyCH4V, PstI/FastDigest® PstI

PsuI (BstYI)/FastDigest® PsuI* (A↓GATCY)

BamHI/FastDigest® BamHI (G↓GATCC)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI



BglII/FastDigest® BglII (A↓GATCT)BglII/FastDigest® BglII, Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI

PsuI (BstYI)/FastDigest® PsuI* (G↓GATCY)

BamHI/FastDigest® BamHI (G↓GATCC)BamHI/FastDigest® BamHI, Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspLI (NlaIV)/FastDigest® NlaIV (BspLI), BspPI (AlwI), DpnI/Fast-Digest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI


Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDi-gest® DpnI, MboI/FastDigest® MboI

BglII/FastDigest® BglII (A↓GATCT)Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDi-gest® DpnI, MboI/FastDigest® MboI, PsuI (BstYI)/FastDigest® PsuI

PvuI/FastDigest® PvuI (CGAT↓CG)

Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)* (CGAT↓CG), SfaAI (AsiSI)/FastDigest® AsiSI (SfaAI) (GCGAT↓CGC)



SacI/FastDigest® SacI (GAGCT↓C)

Alw21I (BsiHKAI)/FastDigest® Alw21I* (GAGCT↓C), Eco24I (BanII)* (GAGCT↓C), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCT↓C)



SalI/FastDigest® SalI (G↓TCGAC)

AbsI (CC↓TCGAGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG), PspXI* (VC↓TCGAGC), PspXI* (VC↓TCGAGG), PspXI* (VC↓TCGAGT), SmoI (SmlI)* (C↓TCGAG), XhoI/FastDigest® XhoI (C↓TCGAG)


SgrDI (CG↓TCGACG)HincII (HindII)/FastDigest® HincII, Hpy8I (MjaIV)/FastDigest® Hpy8I, Hpy99I, SalI/FastDigest® SalI, TaqI/FastDigest® TaqI, XmiI (AccI)/FastDigest® AccI (XmiI)

FastDigest® SanDI (KflI)* (GG↓GACCC)


BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/Fast-Digest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), FaqI (BsmFI)/FastDigest® BsmFI (FaqI)

Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GACCC)

BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, FaqI (BsmFI)/FastDigest® BsmFI (FaqI), FastDigest® SanDI (KflI), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)

Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GACCT)

BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, FaqI (BsmFI)/FastDigest® BsmFI (FaqI), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II), SetI




Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GTCCC)

BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, FastDigest® SanDI (KflI), Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)

Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)* (RG↓GTCCT)BmgT120I, BspLI (NlaIV)/FastDigest® NlaIV (BspLI), Cfr13I (Sau96I)/FastDi-gest® Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest® AvaII (Eco47I), EcoO109I (DraII)/FastDigest® EcoO109I, Psp5II (PpuMI)/FastDigest® PpuMI (Psp5II)

SdaI (SbfI)/FastDigest® SbfI (SdaI) (CCTGCA↓GG)

Alw21I (BsiHKAI)/FastDigest® Alw21I* (GTGCA↓C), BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GTGCA↓C), Mph1103I (NsiI)/FastDigest® NsiI (Mph1103I) (ATGCA↓T), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCA↓C)

HpyCH4V

PstI/FastDigest® PstI (CTGCA↓G) BfmI (SfcI)/FastDigest® SfcI (BfmI), HpyCH4V, PstI/FastDigest® PstISduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCA↓C)

Alw21I (BsiHKAI)/FastDigest® Alw21I* (GAGCA↓C)Alw21I (BsiHKAI)/FastDigest® Alw21I, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCC↓C)

Eco24I (BanII)* (GAGCC↓C) CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GAGCT↓C)

Alw21I (BsiHKAI)/FastDigest® Alw21I* (GAGCT↓C), Eco24I (BanII)* (GAGCT↓C), SacI/FastDigest® SacI (GAGCT↓C)


SetI* (AGCT) AluI/FastDigest® AluI, CviJI, SetISduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCA↓C)

BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GGGCA↓C)BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)

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SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCC↓C)

ApaI/FastDigest® ApaI (GGGCC↓C), BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GGGCC↓C), Eco24I (BanII)* (GGGCC↓C)


SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GGGCT↓C)

Eco24I (BanII)* (GGGCT↓C) CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCA↓C)

Alw21I (BsiHKAI)/FastDigest® Alw21I* (GTGCA↓C), BseSI (Bme1580I)/Fast-Digest® Bme1580I (BseSI)* (GTGCA↓C)



BsgI, HpyCH4V

SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCC↓C)

BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI)* (GTGCC↓C)BseSI (Bme1580I)/FastDigest® Bme1580I (BseSI), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)

SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)* (GTGCT↓C)

Alw21I (BsiHKAI)/FastDigest® Alw21I* (GTGCT↓C)Alw21I (BsiHKAI)/FastDigest® Alw21I, SduI (Bsp1286I)/FastDigest® Bsp1286I (SduI)

SetI* (ACGT) AatII/FastDigest® AatII (GACGT↓C), TaiI (MaeII)/FastDigest® TaiI (ACGT) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI

SetI* (AGCT)Alw21I (BsiHKAI)/FastDigest® Alw21I* (GAGCT↓C), Eco24I (BanII)* (GAGCT↓C), SacI/FastDigest® SacI (GAGCT↓C), SduI (Bsp1286I)/FastDi-gest® Bsp1286I (SduI)* (GAGCT↓C)


FastDigest® SexAI (CsiI)* (A↓CCAGGT)

EcoRII* (↓CCAGG)Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, EcoRII, MvaI (BstNI)/FastDigest® MvaI

FastDigest® SexAI (CsiI)* (A↓CCTGGT)

EcoRII* (↓CCTGG)Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I), BssKI, EcoRII, MvaI (BstNI)/FastDigest® MvaI, SetI

SfaAI (AsiSI)/FastDigest® AsiSI (SfaAI) (GCGAT↓CGC)

Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)* (CGAT↓CG), PvuI/FastDi-gest® PvuI (CGAT↓CG)



SgrAI* (CA↓CCGGYG)

BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) BsaWI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT)


Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), NgoMIV (G↓CCGGC)Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI



MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG)Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, SgrAI

SgrAI* (CG↓CCGGYG)

BsaWI* (W↓CCGGA), Kpn2I (BspEI)/FastDigest® Kpn2I (T↓CCGGA) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIBsaWI* (W↓CCGGT), BshTI (AgeI)/FastDigest® AgeI (BshTI) (A↓CCGGT), Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGT)


Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)* (R↓CCGGC), NgoMIV (G↓CCGGC)Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)



MreI (Sse232I)/FastDigest® MreI (CG↓CCGGCG)Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), HpaII/FastDigest® HpaII, MreI (Sse232I)/FastDigest® MreI, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI), SgrAI

SgrDI (CG↓TCGACG)

AbsI (CC↓TCGAGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG), PspXI* (VC↓TCGAGC), PspXI* (VC↓TCGAGG), PspXI* (VC↓TCGAGT), SmoI (SmlI)* (C↓TCGAG), XhoI/FastDigest® XhoI (C↓TCGAG)

Hpy99I, TaqI/FastDigest® TaqI

SalI/FastDigest® SalI (G↓TCGAC)HincII (HindII)/FastDigest® HincII, Hpy8I (MjaIV)/FastDigest® Hpy8I, Hpy99I, SalI/FastDigest® SalI, TaqI/FastDigest® TaqI, XmiI (AccI)/FastDigest® AccI (XmiI)

SgsI (AscI)/FastDigest® AscI (SgsI) (GG↓CGCGCC)


MauBI/FastDigest® MauBI (CG↓CGCGCG), PauI (BssHII)/FastDigest® BssHII (PteI) (G↓CGCGC)


SmoI (SmlI)* (C↓TCGAG)

AbsI (CC↓TCGAGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG), PspXI* (VC↓TCGAGC), PspXI* (VC↓TCGAGG), PspXI* (VC↓TCGAGT), XhoI/FastDigest® XhoI (C↓TCGAG)



SmoI (SmlI)* (C↓TTAAG) BspTI (AflII)/FastDigest® AflII (BspTI) (C↓TTAAG)BspTI (AflII)/FastDigest® AflII (BspTI), FastDigest® MseI (SaqAI), SmoI (SmlI), Tru1I (MseI)/FastDigest® Tru1I

SsiI (AciI)/FastDigest® AciI (SsiI)* (C↓CGC)


Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGCC), Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I) (G↓CGC), NarI (GG↓CGCC)


HpaII/FastDigest® HpaII (C↓CGG), MspI (HpaII)/FastDigest® MspI (C↓CGG) HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

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SsiI (AciI)/FastDigest® AciI (SsiI)* (G↓CGG)

Hin1I (BsaHI)/FastDigest® BsaHI (Hin1I)* (GR↓CGCC), Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I) (G↓CGC), NarI (GG↓CGCC)


SspDI (KasI) (G↓GCGCC) BshNI (BanI)/FastDigest® BanI (BshNI)* (G↓GCGCC)


TaiI (MaeII)/FastDigest® TaiI (ACGT)

AatII/FastDigest® AatII (GACGT↓C), SetI* (ACGT) MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI

TaqI/FastDigest® TaqI (T↓CGA)

BmeT110I* (CY↓CGAG), Bsp119I (BstBI)/FastDigest® Bsp119I (TT↓CGAA), Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I) (AT↓CGAT), XmiI (AccI)/FastDigest® AccI (XmiI)* (GT↓CGAC)


TasI (Tsp509I)/FastDigest® Tsp509I (TasI) (↓AATT)

EcoRI/FastDigest® EcoRI (G↓AATTC), MunI (MfeI)/FastDigest® MfeI (MunI) (C↓AATTG), XapI (ApoI)/FastDigest® XapI* (R↓AATTC), XapI (ApoI)/FastDi-gest® XapI* (R↓AATTT)


TatI/FastDigest® TatI* (A↓GTACW)



Bsp1407I (BsrGI)/FastDigest® Bsp1407I (T↓GTACA)Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI, TatI/FastDigest® TatI

TatI/FastDigest® TatI* (T↓GTACW)



Bsp1407I (BsrGI)/FastDigest® Bsp1407I (T↓GTACA)Bsp1407I (BsrGI)/FastDigest® Bsp1407I, Csp6I (CviQI)/FastDigest® Csp6I, RsaI/FastDigest® RsaI, TatI/FastDigest® TatI

Tru1I (MseI)/FastDigest® Tru1I (T↓TAA)

NdeI/FastDigest® NdeI (CA↓TATG) FaiIFastDigest® MseI (SaqAI) (T↓TAA), VspI (AseI)/FastDigest® AseI (VspI) (AT↓TAAT)


VspI (AseI)/FastDigest® AseI (VspI) (AT↓TAAT)

NdeI/FastDigest® NdeI (CA↓TATG) FaiIFastDigest® MseI (SaqAI) (T↓TAA), Tru1I (MseI)/FastDigest® Tru1I (T↓TAA) FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1I

XapI (ApoI)/FastDigest® XapI* (A↓AATTY)

EcoRI/FastDigest® EcoRI (G↓AATTC) TasI (Tsp509I)/FastDigest® Tsp509I (TasI), XapI (ApoI)/FastDigest® XapIMunI (MfeI)/FastDigest® MfeI (MunI) (C↓AATTG), TasI (Tsp509I)/FastDigest® Tsp509I (TasI) (↓AATT)


XapI (ApoI)/FastDigest® XapI* (G↓AATTY)

EcoRI/FastDigest® EcoRI (G↓AATTC)EcoRI/FastDigest® EcoRI, TasI (Tsp509I)/FastDigest® Tsp509I (TasI), XapI (ApoI)/FastDigest® XapI

MunI (MfeI)/FastDigest® MfeI (MunI) (C↓AATTG), TasI (Tsp509I)/FastDigest® Tsp509I (TasI) (↓AATT)


XbaI/FastDigest® XbaI (T↓CTAGA)

BcuI (SpeI)/FastDigest® SpeI (BcuI) (A↓CTAGT), Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CTAGG), NheI/FastDigest® NheI (G↓CTAGC), XmaJI (AvrII)/FastDigest® AvrII (XmaJI) (C↓CTAGG)


XceI (NspI)/FastDigest® NspI (XceI)* (ACATG↓Y)


PaeI (SphI)/FastDigest® SphI (PaeI) (GCATG↓C)CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), XceI (NspI)/FastDi-gest® NspI (XceI)

XceI (NspI)/FastDigest® NspI (XceI)* (GCATG↓Y)


PaeI (SphI)/FastDigest® SphI (PaeI) (GCATG↓C)Cac8I, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), PaeI (SphI)/FastDigest® SphI (PaeI), XceI (NspI)/FastDigest® NspI (XceI)

XhoI/FastDigest® XhoI (C↓TCGAG)

AbsI (CC↓TCGAGG), Eco88I (AvaI)/FastDigest® AvaI (Eco88I)* (C↓TCGAG), PspXI* (VC↓TCGAGC), PspXI* (VC↓TCGAGG), PspXI* (VC↓TCGAGT), SmoI (SmlI)* (C↓TCGAG)



XmaJI (AvrII)/FastDigest® AvrII (XmaJI) (C↓CTAGG)

BcuI (SpeI)/FastDigest® SpeI (BcuI) (A↓CTAGT), NheI/FastDigest® NheI (G↓CTAGC), XbaI/FastDigest® XbaI (T↓CTAGA)


Eco130I (StyI)/FastDigest® StyI (Eco130I)* (C↓CTAGG)BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I), FspBI (BfaI)/FastDigest® BfaI (FspBI), XmaJI (AvrII)/FastDigest® AvrII (XmaJI)

XmiI (AccI)/FastDigest® AccI (XmiI)* (GT↓ATAC)

CviAII (C↓ATG) FaiI

XmiI (AccI)/FastDigest® AccI (XmiI)* (GT↓CGAC)

BmeT110I* (CY↓CGAG), Bsp119I (BstBI)/FastDigest® Bsp119I (TT↓CGAA), Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I) (AT↓CGAT), TaqI/FastDigest® TaqI (T↓CGA)


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Recognition Sites Resulting from Fill-in of 5’-overhang and Self-ligation

Table 1.23. Newly generated recognition sites resulting from fill-in of 5’-overhang and self-ligation.


Restriction enzyme

Recognition sequence

Newly generated sequence after reaction

Restriction enzymes that cleave the newly generated recognition sequence

AbsI CC↓TCGAGG CCTCGATCGAGGBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, [MnlI/FastDigest® MnlI], PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]

Acc65I (Asp718I)/FastDigest® Acc65I G↓GTACC GGTACGTACC[Csp6I (CviQI)/FastDigest® Csp6I], Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), [RsaI/FastDigest® RsaI], SetI, TaiI (MaeII)/FastDigest® TaiI

AflIII* A↓CACGT ACACGCACGT [MaeII], [SetI], [TaiI (MaeII)/FastDigest® TaiI]

AflIII* A↓CATGT ACATGCATGT[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I), [XceI (NspI)/FastDigest® NspI (XceI)]

AflIII* A↓CGCGT ACGCGCGCGT[Bsh1236I (BstUI)/FastDigest® Bsh1236I], Cac8I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I), MauBI/FastDigest® MauBI, PauI (BssHII)/FastDigest® BssHII (PteI)

AflIII* A↓CGTGT ACGTGCGTGT [MaeII], [SetI], [TaiI (MaeII)/FastDigest® TaiI]Alw44I (ApaLI)/FastDigest® ApaLI (Alw44I)

G↓TGCAC GTGCATGCACCac8I, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), [HpyCH4V], PaeI (SphI)/FastDi-gest® SphI (PaeI), XceI (NspI)/FastDigest® NspI (XceI)

BamHI/FastDigest® BamHI G↓GATCC GGATCGATCC[Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)], [BspPI (AlwI)], Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I), [DpnI/FastDigest® DpnI], [MboI/FastDigest® MboI], TaqI/FastDigest® TaqI

BbvCI CC↓TCAGC CCTCATCAGC [MnlI/FastDigest® MnlI]

BclI/FastDigest® BclI T↓GATCA TGATCGATCA[Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I), [DpnI/FastDigest® DpnI], [MboI/FastDigest® MboI], TaqI/FastDigest® TaqI

BcnI (NciI)/FastDigest® NciI (BcnI) CC↓SGG CCSSGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI)

BcnI (NciI)/FastDigest® NciI (BcnI)* CC↓CGG CCCCGG[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BseDI (BsaJI)/FastDigest® BsaJI (BseDI), [BssKI], [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]

BcnI (NciI)/FastDigest® NciI (BcnI)* CC↓GGG CCGGGG[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BseDI (BsaJI)/FastDigest® BsaJI (BseDI), [BssKI], [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]

BcuI (SpeI)/FastDigest® SpeI (BcuI) A↓CTAGT ACTAGCTAGT AluI/FastDigest® AluI, CviJI, [FspBI (BfaI)/FastDigest® BfaI (FspBI)], SetIBfmI (SfcI)/FastDigest® SfcI (BfmI) C↓TRYAG CTRYATRYAG FaiIBfmI (SfcI)/FastDigest® SfcI (BfmI)* C↓TACAG CTACATACAG FaiIBfmI (SfcI)/FastDigest® SfcI (BfmI)* C↓TATAG CTATATATAG [FaiI]

BfmI (SfcI)/FastDigest® SfcI (BfmI)* C↓TGCAG CTGCATGCAGCac8I, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II), [HpyCH4V], PaeI (SphI)/FastDi-gest® SphI (PaeI), XceI (NspI)/FastDigest® NspI (XceI)

BfmI (SfcI)/FastDigest® SfcI (BfmI)* C↓TGTAG CTGTATGTAG FaiI

BglII/FastDigest® BglII A↓GATCT AGATCGATCT[Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I), [DpnI/FastDigest® DpnI], [MboI/FastDigest® MboI], TaqI/FastDigest® TaqI

Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)

CC↓NGG CCNNGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI)

Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)*

CC↓AGG CCAAGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I)


CC↓CGG CCCCGG[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BseDI (BsaJI)/FastDigest® BsaJI (BseDI), [BssKI], [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]


CC↓GGG CCGGGG[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BseDI (BsaJI)/FastDigest® BsaJI (BseDI), [BssKI], [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]


CC↓TGG CCTTGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I)

BmeT110I CY↓CGRG CYCGCGRG Bsh1236I (BstUI)/FastDigest® Bsh1236IBmgT120I GG↓NCC GGNNCC BspLI (NlaIV)/FastDigest® NlaIV (BspLI)BmgT120I* GG↓ACC GGAACC BspLI (NlaIV)/FastDigest® NlaIV (BspLI)

BmgT120I* GG↓CCC GGCCCC[BmgT120I], BspLI (NlaIV)/FastDigest® NlaIV (BspLI), [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I)], [CviJI]

BmgT120I* GG↓GCC GGGGCC[BmgT120I], BspLI (NlaIV)/FastDigest® NlaIV (BspLI), [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I)], [CviJI]

BmgT120I* GG↓TCC GGTTCC BspLI (NlaIV)/FastDigest® NlaIV (BspLI)Bpu10I/FastDigest® Bpu10I* CC↓TCAGC CCTCATCAGC [MnlI/FastDigest® MnlI]

BsaWI W↓CCGGW WCCGGCCGGWBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]



[ ] Enzymes that cleave the target both before and after the ligation.


Single letter codeR = G or A; K = G or T; B = C, G or T;Y = C or T; S = C or G; D = A, G or T;W = A or T; H = A, C or T; N = G, A, T or C.M = A or C; V = A, C or G;

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Restriction enzyme




BseYI C↓CCAGC CCCAGCCAGC [BseYI], Cac8I, CviJIBshNI (BanI)/FastDigest® BanI (BshNI)* G↓GCACC GGCACGCACC Cac8I

BshNI (BanI)/FastDigest® BanI (BshNI)* G↓GCGCC GGCGCGCGCCBsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I)], PauI (BssHII)/FastDigest® BssHII (PteI)

BshNI (BanI)/FastDigest® BanI (BshNI)* G↓GTACC GGTACGTACC[Csp6I (CviQI)/FastDigest® Csp6I], Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), [RsaI/FastDigest® RsaI], SetI, TaiI (MaeII)/FastDigest® TaiI

BshNI (BanI)/FastDigest® BanI (BshNI)* G↓GTGCC GGTGCGTGCC Cac8I

BshTI (AgeI)/FastDigest® AgeI (BshTI) A↓CCGGT ACCGGCCGGTBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), [Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]

Bsp119I (BstBI)/FastDigest® Bsp119I TT↓CGAA TTCGCGAA Bsh1236I (BstUI)/FastDigest® Bsh1236I, Hpy188III, Bsp68I (NruI)/FastDigest® NruI (RruI)

Bsp120I (PspOMI)/FastDigest® Bsp120I

G↓GGCCC GGGCCGGCCC[BmgT120I], [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), [Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I)], [CviJI], FseI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)

Bsp1407I (BsrGI)/FastDigest® Bsp1407I

T↓GTACA TGTACGTACA[Csp6I (CviQI)/FastDigest® Csp6I], Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), [RsaI/FastDigest® RsaI], SetI, TaiI (MaeII)/FastDigest® TaiI

Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)

↓GATC GATCGATC[Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I), [DpnI/FastDigest® DpnI], [MboI/FastDigest® MboI], TaqI/FastDigest® TaqI

BspTI (AflII)/FastDigest® AflII (BspTI) C↓TTAAG CTTAATTAAGPacI/FastDigest® PacI (TTAAT↓TAA), [FastDigest® MseI (SaqAI)], TasI (Tsp509I)/FastDigest® Tsp509I (TasI), [Tru1I (MseI)/FastDigest® Tru1I]

BssKI ↓CCNGG CCNGGCCNGG[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI

BssKI* ↓CCAGG CCAGGCCAGG[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]

BssKI* ↓CCCGG CCCGGCCCGG[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BmgT120I, [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI, [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]

BssKI* ↓CCGGG CCGGGCCGGG[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BmgT120I, [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI, [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]

BssKI* ↓CCTGG CCTGGCCTGG[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]

Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I) AT↓CGAT ATCGCGAT Bsh1236I (BstUI)/FastDigest® Bsh1236I, Hpy188III, Bsp68I (NruI)/FastDigest® NruI (RruI)

BtgI* C↓CATGG CCATGCATGG[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I)

BtgI* C↓CGCGG CCGCGCGCGG[Bsh1236I (BstUI)/FastDigest® Bsh1236I], Cac8I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I), MauBI/FastDigest® MauBI, [SsiI (AciI)/FastDigest® AciI (SsiI)], PauI (BssHII)/FastDigest® BssHII (PteI)

Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)

R↓CCGGY RCCGGCCGGYBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), [Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]

Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I)*

G↓GCCC GGCCGCCC[BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [CviJI], SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDigest® AciI (SsiI), TauI/FastDigest® TauI

Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I)*

G↓GGCC GGGCGGCC[BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [CviJI], SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDigest® TauI

Cfr9I (XmaI) C↓CCGGG CCCGGCCGGG

[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]

CfrI (EaeI) Y↓GGCCR YGGCCGGCCR[BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), [CfrI (EaeI)], [CviJI], FseI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)

CpoI (RsrII)/FastDigest® RsrII (CpoI)* CG↓GACCG CGGACGACCG Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)CpoI (RsrII)/FastDigest® RsrII (CpoI)* CG↓GTCCG CGGTCGTCCG Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)

FastDigest® SexAI (CsiI) A↓CCWGGT ACCWGGCCWGGT[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]

FastDigest® SexAI (CsiI)* A↓CCAGGT ACCAGGCCAGGT[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI], [SetI]

FastDigest® SexAI (CsiI)* A↓CCTGGT ACCTGGCCTGGT[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI], [SetI]

Csp6I (CviQI)/FastDigest® Csp6I G↓TAC GTATACBst1107I (BstZ17I)/FastDigest® BstZ17I (Bst1107I), FaiI, Hpy8I (MjaIV)/FastDigest® Hpy8I, XmiI (AccI)/FastDigest® AccI (XmiI)

CviAII C↓ATG CATATG [FaiI], NdeI/FastDigest® NdeIEco130I (StyI)/FastDigest® StyI (Eco130I)*

C↓CATGG CCATGCATGG[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I)

Eco130I (StyI)/FastDigest® StyI (Eco130I)*

C↓CTAGG CCTAGCTAGG AluI/FastDigest® AluI, CviJI, [FspBI (BfaI)/FastDigest® BfaI (FspBI)], SetI

Eco52I (EagI)/FastDigest® EagI (Eco52I)

C↓GGCCG CGGCCGGCCG

[Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)], [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), [CfrI (EaeI)], [CviJI], [Eco52I (EagI)/FastDigest® EagI (Eco52I)], FseI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI)

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Restriction enzyme




Eco81I (Bsu36I)/FastDigest® Bsu36I (Eco81I)*

CC↓TCAGG CCTCATCAGG [MnlI/FastDigest® MnlI]

Eco88I (AvaI)/FastDigest® AvaI (Eco88I)

C↓YCGRG CYCGRYCGRG Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)

Eco88I (AvaI)/FastDigest® AvaI (Eco88I)*

C↓CCGAG CCCGACCGAG Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)


C↓CCGGG CCCGGCCGGG

[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]


C↓TCGAG CTCGATCGAGBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]


C↓TCGGG CTCGGTCGGG Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)

Eco91I (BstEII)/FastDigest® Eco91I G↓GTNACC GGTNACGTNACC MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest® TaiIEco91I (BstEII)/FastDigest® Eco91I* G↓GTAACC GGTAACGTAACC MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest® TaiIEco91I (BstEII)/FastDigest® Eco91I* G↓GTCACC GGTCACGTCACC AjiI (BmgBI), MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiIEco91I (BstEII)/FastDigest® Eco91I* G↓GTGACC GGTGACGTGACC [HphI], MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiIEco91I (BstEII)/FastDigest® Eco91I* G↓GTTACC GGTTACGTTACC MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest® TaiIEcoO109I (DraII)/FastDigest® EcoO109I*

RG↓GCCCY RGGCCGCCCY[BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [CviJI], SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), SsiI (AciI)/FastDigest® AciI (SsiI), TauI/FastDigest® TauI

EcoO109I (DraII)/FastDigest® EcoO109I*

RG↓GGCCY RGGGCGGCCY[BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [CviJI], SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI), TauI/FastDigest® TauI

EcoRI/FastDigest® EcoRI G↓AATTC GAATTAATTCPdmI (XmnI)/FastDigest® PdmI, FastDigest® MseI (SaqAI), [TasI (Tsp509I)/FastDigest® Tsp509I (TasI)], Tru1I (MseI)/FastDigest® Tru1I, VspI (AseI)/FastDigest® AseI (VspI)

EcoRII ↓CCWGG CCWGGCCWGG[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]

EcoRII* ↓CCAGG CCAGGCCAGG[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]

EcoRII* ↓CCTGG CCTGGCCTGG[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]

FatI ↓CATG CATGCATG[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I)

FspBI (BfaI)/FastDigest® BfaI (FspBI) C↓TAG CTATAG BfmI (SfcI)/FastDigest® SfcI (BfmI), FaiIHin1I (BsaHI)/FastDigest® BsaHI (Hin1I) GR↓CGYC GRCGCGYC Bsh1236I (BstUI)/FastDigest® Bsh1236IHin6I (HinP1I)/FastDigest® HinP1I (Hin6I)

G↓CGC GCGCGCBsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I)], PauI (BssHII)/FastDigest® BssHII (PteI)

HindIII/FastDigest® HindIII A↓AGCTT AAGCTAGCTT[AluI/FastDigest® AluI], BspOI (BmtI)/FastDigest® BmtI (BspOI), Cac8I, [CviJI], FspBI (BfaI)/FastDigest® BfaI (FspBI), NheI/FastDigest® NheI, [SetI]

HpaII/FastDigest® HpaII C↓CGG CCGCGGBseDI (BsaJI)/FastDigest® BsaJI (BseDI), Bsh1236I (BstUI)/FastDigest® Bsh1236I, BtgI, Cfr42I (SacII), MspA1I, SsiI (AciI)/FastDigest® AciI (SsiI)

Kpn2I (BspEI)/FastDigest® Kpn2I T↓CCGGA TCCGGCCGGABsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]

MaeII A↓CGT ACGCGT AflIII, Bsh1236I (BstUI)/FastDigest® Bsh1236I, MluI/FastDigest® MluIMaeIII ↓GTNAC GTNACGTNAC MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest® TaiIMaeIII* ↓GTAAC GTAACGTAAC MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest® TaiIMaeIII* ↓GTCAC GTCACGTCAC AjiI (BmgBI), MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiIMaeIII* ↓GTGAC GTGACGTGAC MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiIMaeIII* ↓GTTAC GTTACGTTAC MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest® TaiI

MauBI/FastDigest® MauBI CG↓CGCGCG CGCGCGCGCGCG[Bsh1236I (BstUI)/FastDigest® Bsh1236I], [Cac8I], [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I)], [MauBI/FastDigest® MauBI], [PauI (BssHII)/FastDigest® BssHII (PteI)]

MboI/FastDigest® MboI ↓GATC GATCGATC[Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I), [DpnI/FastDigest® DpnI], [MboI/FastDigest® MboI], TaqI/FastDigest® TaqI

MluI/FastDigest® MluI A↓CGCGT ACGCGCGCGT[Bsh1236I (BstUI)/FastDigest® Bsh1236I], Cac8I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I), MauBI/FastDigest® MauBI, PauI (BssHII)/FastDigest® BssHII (PteI)

MreI (Sse232I)/FastDigest® MreI CG↓CCGGCG CGCCGGCCGGCG

Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), [Cac8I], [Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI], [NgoMIV], [PdiI (NaeI)/FastDigest® NaeI (PdiI)]

FastDigest® MseI (SaqAI) T↓TAA TTATAA AanI (PsiI)/FastDigest® PsiI (AanI), FaiI

MspI (HpaII)/FastDigest® MspI C↓CGG CCGCGGBseDI (BsaJI)/FastDigest® BsaJI (BseDI), Bsh1236I (BstUI)/FastDigest® Bsh1236I, BtgI, Cfr42I (SacII), MspA1I, SsiI (AciI)/FastDigest® AciI (SsiI)

MunI (MfeI)/FastDigest® MfeI (MunI) C↓AATTG CAATTAATTGFastDigest® MseI (SaqAI), [TasI (Tsp509I)/FastDigest® Tsp509I (TasI)], Tru1I (MseI)/FastDigest® Tru1I, VspI (AseI)/FastDigest® AseI (VspI)

MvaI (BstNI)/FastDigest® MvaI CC↓WGG CCWWGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I)MvaI (BstNI)/FastDigest® MvaI* CC↓AGG CCAAGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I)MvaI (BstNI)/FastDigest® MvaI* CC↓TGG CCTTGG BseDI (BsaJI)/FastDigest® BsaJI (BseDI), Eco130I (StyI)/FastDigest® StyI (Eco130I)

205


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Restriction enzyme




NarI GG↓CGCC GGCGCGCCBsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/FastDi-gest® HinP1I (Hin6I)], SgsI (AscI)/FastDigest® AscI (SgsI), PauI (BssHII)/FastDigest® BssHII (PteI)

NcoI/FastDigest® NcoI C↓CATGG CCATGCATGG[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I)

NdeI/FastDigest® NdeI CA↓TATG CATATATG [FaiI]

NgoMIV G↓CCGGC GCCGGCCGGC

Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), [Cac8I], [Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI], [NgoMIV], [PdiI (NaeI)/FastDigest® NaeI (PdiI)]

NheI/FastDigest® NheI G↓CTAGC GCTAGCTAGCAluI/FastDigest® AluI, [BspOI (BmtI)/FastDigest® BmtI (BspOI)], [Cac8I], CviJI, [FspBI (BfaI)/FastDigest® BfaI (FspBI)], [NheI/FastDigest® NheI], SetI

NmuCI (Tsp45I)/FastDigest® NmuCI ↓GTSAC GTSACGTSAC MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiINmuCI (Tsp45I)/FastDigest® NmuCI* ↓GTCAC GTCACGTCAC AjiI (BmgBI), MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiINmuCI (Tsp45I)/FastDigest® NmuCI* ↓GTGAC GTGACGTGAC MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest® NmuCI], SetI, TaiI (MaeII)/FastDigest® TaiI

NotI/FastDigest® NotI GC↓GGCCGC GCGGCCGGCCGC

[Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)], [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], Cac8I, Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I), [CfrI (EaeI)], [CviJI], [Eco52I (EagI)/FastDigest® EagI (Eco52I)], FseI, HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI, NgoMIV, PdiI (NaeI)/FastDigest® NaeI (PdiI), [SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)], [SsiI (AciI)/FastDigest® AciI (SsiI)], [TauI/FastDigest® TauI]

PagI (BspHI)/FastDigest® BspHI (PagI) T↓CATGA TCATGCATGA[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I)

PasI* CC↓CAGGG CCCAGCAGGG BseYI, EcoP15I

PauI (BssHII)/FastDigest® BssHII (PteI) G↓CGCGC GCGCGCGCGC[Bsh1236I (BstUI)/FastDigest® Bsh1236I], [Cac8I], [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I)], MauBI/FastDigest® MauBI, [PauI (BssHII)/FastDigest® BssHII (PteI)]

Pfl23II (BsiWI)/FastDigest® BsiWI (Pfl23II)

C↓GTACG CGTACGTACG[Csp6I (CviQI)/FastDigest® Csp6I], Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, [Pfl23II (BsiWI)/FastDigest® BsiWI (Pfl23II)], Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), [RsaI/FastDi-gest® RsaI], SetI, TaiI (MaeII)/FastDigest® TaiI

PfoI/FastDigest® PfoI T↓CCNGGA TCCNGGCCNGGA[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI

PfoI/FastDigest® PfoI* T↓CCAGGA TCCAGGCCAGGA[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]

PfoI/FastDigest® PfoI* T↓CCCGGA TCCCGGCCCGGA[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BmgT120I, [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI, [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]

PfoI/FastDigest® PfoI* T↓CCGGGA TCCGGGCCGGGA[BcnI (NciI)/FastDigest® NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], BmgT120I, [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest® Sau96I (Cfr13I), CviJI, [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]

PfoI/FastDigest® PfoI* T↓CCTGGA TCCTGGCCTGGA[Bme1390I (ScrFI)/FastDigest® ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest® MvaI]

PscI (PciI) A↓CATGT ACATGCATGT[CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest® NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDi-gest® NsiI (Mph1103I), [XceI (NspI)/FastDigest® NspI (XceI)]

Psp1406I (AclI)/FastDigest® AclI (Psp1406I)

AA↓CGTT AACGCGTT AflIII, Bsh1236I (BstUI)/FastDigest® Bsh1236I, MluI/FastDigest® MluI

PspXI VC↓TCGAGB VCTCGATCGAGBBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]

PsuI (BstYI)/FastDigest® PsuI R↓GATCY RGATCGATCY[Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest® ClaI (Bsu15I), [DpnI/FastDigest® DpnI], [MboI/FastDigest® MboI], TaqI/FastDigest® TaqI

PsyI (Tth111I)/FastDigest® PsyI GACN↓NNGTC GACNNNNGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)PsyI (Tth111I)/FastDigest® PsyI* GACN↓ANGTC GACNAANGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)PsyI (Tth111I)/FastDigest® PsyI* GACN↓CNGTC GACNCCNGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)PsyI (Tth111I)/FastDigest® PsyI* GACN↓GNGTC GACNGGNGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)PsyI (Tth111I)/FastDigest® PsyI* GACN↓TNGTC GACNTTNGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)

SalI/FastDigest® SalI G↓TCGAC GTCGATCGACBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]

FastDigest® SanDI (KflI)* GG↓GACCC GGGACGACCC [FaqI (BsmFI)/FastDigest® BsmFI (FaqI)]SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI) GC↓NGC GCNNGC Cac8ISatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)*

GC↓AGC GCAAGC Cac8I

SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)*

GC↓CGC GCCCGC Cac8I, SmuI (FauI), [SsiI (AciI)/FastDigest® AciI (SsiI)]


GC↓GGC GCGGGC Cac8I


GC↓TGC GCTTGC Cac8I

SgrAI CR↓CCGGYG CRCCGGCCGGYGBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI), [Cfr10I (BsrFI)/FastDigest® BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest® EagI (Eco52I), [HpaII/FastDigest® HpaII], [MspI (HpaII)/FastDigest® MspI]

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SgrDI CG↓TCGACG CGTCGATCGACGBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, [Hpy99I], MboI/FastDigest® MboI, PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]

SgsI (AscI)/FastDigest® AscI (SgsI) GG↓CGCGCC GGCGCGCGCGCC[Bsh1236I (BstUI)/FastDigest® Bsh1236I], [Cac8I], [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I)], MauBI/FastDigest® MauBI, [PauI (BssHII)/FastDigest® BssHII (PteI)]

SmoI (SmlI)* C↓TCGAG CTCGATCGAGBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]

SmoI (SmlI)* C↓TTAAG CTTAATTAAGPacI/FastDigest® PacI (TTAAT↓TAA), [FastDigest® MseI (SaqAI)], TasI (Tsp509I)/FastDigest® Tsp509I (TasI), [Tru1I (MseI)/FastDigest® Tru1I]

SsiI (AciI)/FastDigest® AciI (SsiI) C↓CGC CCGCGCBsh1236I (BstUI)/FastDigest® Bsh1236I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/FastDigest® HinP1I (Hin6I), [SsiI (AciI)/FastDigest® AciI (SsiI)]

SspDI (KasI) G↓GCGCC GGCGCGCGCCBsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, [HhaI/FastDigest® HhaI], [Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I)], PauI (BssHII)/FastDigest® BssHII (PteI)

TaqI/FastDigest® TaqI T↓CGA TCGCGA Bsh1236I (BstUI)/FastDigest® Bsh1236I, Hpy188III, Bsp68I (NruI)/FastDigest® NruI (RruI)TasI (Tsp509I)/FastDigest® Tsp509I (TasI)

↓AATT AATTAATTFastDigest® MseI (SaqAI), [TasI (Tsp509I)/FastDigest® Tsp509I (TasI)], Tru1I (MseI)/FastDigest® Tru1I, VspI (AseI)/FastDigest® AseI (VspI)

TatI/FastDigest® TatI W↓GTACW WGTACGTACW[Csp6I (CviQI)/FastDigest® Csp6I], Eco105I (SnaBI)/FastDigest® SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), [RsaI/FastDigest® RsaI], SetI, TaiI (MaeII)/FastDigest® TaiI

Tru1I (MseI)/FastDigest® Tru1I T↓TAA TTATAA AanI (PsiI)/FastDigest® PsiI (AanI), FaiITseI G↓CWGC GCWGCWGC [SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)], [TseI]

TseI* G↓CAGC GCAGCAGC[BseXI (BbvI)/FastDigest® BseXI], EcoP15I, [Lsp1109I (BbvI)/FastDigest® BbvI (Lsp1109I)], [SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)], [TseI]

TseI* G↓CTGC GCTGCTGC [SatI (Fnu4HI)/FastDigest® Fnu4HI (SatI)], [TseI]VspI (AseI)/FastDigest® AseI (VspI) AT↓TAAT ATTATAAT AanI (PsiI)/FastDigest® PsiI (AanI), FaiI

XapI (ApoI)/FastDigest® XapI R↓AATTY RAATTAATTYFastDigest® MseI (SaqAI), [TasI (Tsp509I)/FastDigest® Tsp509I (TasI)], Tru1I (MseI)/FastDigest® Tru1I, VspI (AseI)/FastDigest® AseI (VspI)

XbaI/FastDigest® XbaI T↓CTAGA TCTAGCTAGA AluI/FastDigest® AluI, CviJI, [FspBI (BfaI)/FastDigest® BfaI (FspBI)], SetI

XhoI/FastDigest® XhoI C↓TCGAG CTCGATCGAGBsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest® Sau3AI (Bsp143I), DpnI/FastDigest® DpnI, MboI/FastDigest® MboI, PvuI/FastDigest® PvuI, [TaqI/FastDigest® TaqI]

XmaJI (AvrII)/FastDigest® AvrII (XmaJI) C↓CTAGG CCTAGCTAGG AluI/FastDigest® AluI, CviJI, [FspBI (BfaI)/FastDigest® BfaI (FspBI)], SetIXmiI (AccI)/FastDigest® AccI (XmiI)* GT↓ATAC GTATATAC [FaiI]XmiI (AccI)/FastDigest® AccI (XmiI)* GT↓CGAC GTCGCGAC Bsh1236I (BstUI)/FastDigest® Bsh1236I, Hpy188III, Bsp68I (NruI)/FastDigest® NruI (RruI)XmiI (AccI)/FastDigest® AccI (XmiI)* GT↓CTAC GTCTCTAC Alw26I (BsmAI)/FastDigest® Alw26I

Table 1.24. Newly generated recognition sites resulting from removal of 3’-overhang and self-ligation.

Recognition Sites Resulting from Removal of 3’-overhang and Self-ligation

Restriction enzyme




AasI (DrdI)/FastDigest® DrdI (AasI) GACNNNN↓NNGTC GACNNNNGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)AdeI (DraIII)/FastDigest® DraIII (AdeI)

CACNNN↓GTG CACGTGEco72I (PmlI)/FastDigest® PmlI (Eco72I), MaeII, Ppu21I (BsaAI)/FastDigest® BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest® TaiI

AgsI TTS↓AA TTAA FastDigest® MseI (SaqAI), Tru1I (MseI)/FastDigest® Tru1IBglI/FastDigest® BglI GCCNNNN↓NGGC GCCNNGGC BseDI (BsaJI)/FastDigest® BsaJI (BseDI)Bsh1285I (BsiEI)/FastDigest® BsiEI (Bsh1285I)

CGRY↓CG CGCG Bsh1236I (BstUI)/FastDigest® Bsh1236I

CaiI (AlwNI)/FastDigest® AlwNI (CaiI)

CAGNNN↓CTG CAGCTG AluI/FastDigest® AluI, CviJI, MspA1I, PvuII/FastDigest® PvuII, SetI

Cfr42I (SacII) CCGC↓GG CCGG HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspIEam1105I (AhdI)/FastDigest® Eam1105I

GACNNN↓NNGTC GACNNNNGTC BoxI (PshAI)/FastDigest® PshAI (BoxI)

FseI GGCCGG↓CC GGCC [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [CviJI]Hpy188I TCN↓GA TCGA TaqI/FastDigest® TaqIPacI/FastDigest® PacI (TTAAT↓TAA)

TTAAT↓TAA TTATAA AanI (PsiI)/FastDigest® PsiI (AanI), FaiI

PvuI/FastDigest® PvuI CGAT↓CG CGCG Bsh1236I (BstUI)/FastDigest® Bsh1236ISdaI (SbfI)/FastDigest® SbfI (SdaI)

CCTGCA↓GG CCGG HpaII/FastDigest® HpaII, MspI (HpaII)/FastDigest® MspI

SfaAI (AsiSI)/FastDigest® AsiSI (SfaAI)

GCGAT↓CGC GCGCGCBsh1236I (BstUI)/FastDigest® Bsh1236I, Cac8I, HhaI/FastDigest® HhaI, Hin6I (HinP1I)/Fast-Digest® HinP1I (Hin6I), PauI (BssHII)/FastDigest® BssHII (PteI)

SfiI/FastDigest® SfiI GGCCNNNN↓NGGCC GGCCNNGGCC BseDI (BsaJI)/FastDigest® BsaJI (BseDI), [BsuRI (HaeIII)/FastDigest® HaeIII (BsuRI)], [CviJI]TaaI (HpyCH4III)/FastDigest® TaaI ACN↓GT ACGT MaeII, SetI, TaiI (MaeII)/FastDigest® TaiI

Note• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas conventional enzymes are shown in orange.

Single letter codeR = G or A; K = G or T; B = C, G or T; M = A or CY = C or T; S = C or G; D = A, G or T; V = A, C or G;W = A or T; H = A, C or T; N = G, A, T or C.

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Selection GuidesAlphabetic List of Commercially Available Restriction Enzymes

Table 1.25. Commercially available restriction enzymes.



Note[ ] Enzymes have different cleavage specificities (neoschizomers).m Isoschizomers with different sensitivity to methylation.* Restriction enzymes produced by Fermentas are shown in bold.• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas Conventional enzymes are shown in orange.

Enzyme Specificity 5’→3’ FastDigest® enzyme Page Conventional enzyme PageCommercially available isos-chizomers from other vendors

AanI TTA↓TAA FastDigest® PsiI (AanI) 55 AanI (PsiI) 80 PsiIAarI CACCTGC(4/8)↓ AarI 80AasI GACNNNN↓NNGTC FastDigest® DrdI (AasI) 34 AasI (DrdI) 80 DrdI, DseDIAatI AGG↓CCT FastDigest® StuI (Eco147I) 64 Eco147I (StuI) 115 PceI, SseBI, StuIAatII GACGT↓C FastDigest® AatII 11 AatII 81 [ZraIm]AbsI CC↓TCGAGGAcc16I TGC↓GCA FastDigest® FspI (NsbI) 39 NsbI (FspI) 133 AviII, FspIAcc36I ACCTGC(4/8)↓ FastDigest® BspMI (BveI) 29 BveI (BspMI) 104 BfuAI, BspMI

Acc65I G↓GTACCFastDigest® Acc65I, [FastDigest® KpnIm](GGTAC↓C)

1143

Acc65I (Asp718I), [KpnIm](GGTAC↓C)

81124

Asp718Im

AccB1I G↓GYRCC FastDigest® BanI (BshNI) 19 BshNI (BanI) 97 BanI, BspT107IAccB7I CCANNNN↓NTGG FastDigest® PflMI (Van91I) 54 Van91I (PflMI) 154 BasI, PflMIAccBSI CCGCTC(-3/-3)↓ FastDigest® BsrBI (MbiI) 30 MbiI (BsrBI) 126 BsrBIAccI GT↓MKAC FastDigest® AccI (XmiI) 11 XmiI (AccI) 157 AccI, FblIAccII CG↓CG FastDigest® Bsh1236I 25 Bsh1236I (BstUI) 97 BspFNI, BstFNI, BstUI, MvnIAccIII T↓CCGGA FastDigest® Kpn2Im 44 Kpn2I (BspEI)m 124 Aor13HI, BseAIm, Bsp13I, BspEIm, MroIm

AciI CCGC(-3/-1)↓ FastDigest® AciI (SsiI) 12 SsiI (AciI) 149 AciI, BspACIAclI AA↓CGTT FastDigest® AclI (Psp1406I) 12 Psp1406I (AclI) 139 AclIAclWI GGATC(4/5)↓ BspPI (AlwI) 101 AlwIAcoI Y↓GGCCR CfrI (EaeI) 105 EaeIAcsI R↓AATTY FastDigest® XapI 68 XapI (ApoI) 155 ApoIAcuI CTGAAG(16/14)↓ FastDigest® AcuI (Eco57I) 12 Eco57I (AcuI) 112 AcuIAcvI CAC↓GTG FastDigest® PmlI (Eco72I) 54 Eco72I (PmlI) 113 BbrPI, PmaCI, PmlI, PspCIAcyI GR↓CGYC FastDigest® BsaHI (Hin1I) 24 Hin1I (BsaHI) 120 BsaHI, BssNI, BstACI, Hsp92IAdeI CACNNN↓GTG FastDigest® DraIII (AdeI) 34 AdeI (DraIII) 82 DraIII

AfaI GT↓AC[FastDigest® Csp6Im](G↓TAC), FastDigest® RsaI

3258

[Csp6I (CviQI)m](G↓TAC), RsaI

107141

[CviQIm], [RsaNI]

AfeI AGC↓GCT FastDigest® AfeI (Eco47III) 13 Eco47III (AfeI) 111 AfeI, Aor51HIAfiI CCNNNNN↓NNGG FastDigest® BslI (BseLI) 26 BseLI (BslI) 95 Bsc4I, BslIAflII C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AflII) 101 AflII, BfrI, Bst98I, BstAFI, MspCI, Vha464IAflIII A↓CRYGTAgeI A↓CCGGT FastDigest® AgeI (BshTI) 13 BshTI (AgeI) 98 AgeI, AsiGI, CspAIm, PinAIAgsI TTS↓AAAhdI GACNNN↓NNGTC FastDigest® Eam1105I 35 Eam1105I (AhdI) 109 AspEI, BmeRI, DriI, EclHKIAhlI A↓CTAGT FastDigest® SpeI (BcuI) 63 BcuI (SpeI) 88 SpeIAjiI CACGTC(-3/-3)↓ AjiI (BmgBI) 82 BmgBI, BtrI

AjnI ↓CCWGG [FastDigest® MvaI](CC↓WGG) 48EcoRIIm, [MvaI (BstNI)](CC↓WGG)

116130

[BseBI], [Bst2UI], [BstNI], [BstOI], Psp6I, PspGIm

AjuI ↓(6/11)CCAA(N)7TTC(12/7)↓ FastDigest® AjuI 14 AjuI 83AleI CACNN↓NNGTG FastDigest® AleI (OliI) 14 OliI (AleI) 134 AleIAlfI ↓(10/12)GCA(N)6TGC(12/10)↓ AlfI 83AloI ↓(7/12)GAAC(N)6TCC(12/7)↓ AloI 84AluI AG↓CT FastDigest® AluI 14 AluI 84 AluBIAluBI AG↓CT FastDigest® AluI 14 AluI 84Alw21I GWGCW↓C FastDigest® Alw21I 15 Alw21I (BsiHKAI) 84 Bbv12I, BsiHKAIAlw26I GTCTC(1/5)↓ FastDigest® Alw26I 15 Alw26I (BsmAI) 85 BsmAIm, BstMAIAlw44I G↓TGCAC FastDigest® ApaLI (Alw44I) 16 Alw44I (ApaLI) 85 ApaLIm, VneIAlwI GGATC(4/5)↓ BspPI (AlwI) 101 AclWIAlwNI CAGNNN↓CTG FastDigest® AlwNI (CaiI) 15 CaiI (AlwNI) 104 AlwNIAma87I C↓YCGRG FastDigest® AvaI (Eco88I) 17 Eco88I (AvaI) 113 AvaI, [BmeT110I], BsiHKCI, BsoBIAor13HI T↓CCGGA FastDigest® Kpn2I 44 Kpn2I (BspEI) 124 AccIII, BseAI, Bsp13I, BspEI, MroIAor51HI AGC↓GCT FastDigest® AfeI (Eco47III) 13 Eco47III (AfeI) 111 AfeI

ApaI GGGCC↓CFastDigest® ApaI, [FastDigest® Bsp120I](G↓GGCCC)

1628

ApaI, [Bsp120I (PspOMI)](G↓GGCCC)

8599

[PspOMI]

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ApaLI G↓TGCAC FastDigest® ApaLI (Alw44I)m 16 Alw44I (ApaLI)m 85 ApaLI, VneIApeKI G↓CWGC TseIApoI R↓AATTY FastDigest® XapI 68 XapI (ApoI) 155 AcsIArsI ↓(8/13)GAC(N)6TTYG(11/6)↓AscI GG↓CGCGCC FastDigest® AscI (SgsI) 16 SgsI (AscI) 147 AscI, PalAIAseI AT↓TAAT FastDigest® AseI (VspI) 17 VspI (AseI) 154 AseI, PshBIAsiGI A↓CCGGT FastDigest® AgeI (BshTI) 13 BshTI (AgeI) 98 AgeI, CspAI, PinAIAsiSI GCGAT↓CGC FastDigest® AsiSI (SfaAI) 17 SfaAI (AsiSI) 145 AsiSI, RgaI, SgfIAsp700I GAANN↓NNTTC FastDigest® PdmI 53 PdmI (XmnI) 136 MroXI, XmnI

Asp718I G↓GTACCFastDigest® Acc65Im, [FastDigest® KpnIm](GGTAC↓C)

1143

Acc65I (Asp718I)m, [KpnIm](GGTAC↓C)

81124

AspA2I C↓CTAGG FastDigest® AvrII (XmaJI) 18 XmaJI (AvrII) 156 AvrII, BlnIAspEI GACNNN↓NNGTC FastDigest® Eam1105I 35 Eam1105I (AhdI) 109 AhdI, BmeRI, DriI, EclHKIAspI GACN↓NNGTC FastDigest® PsyI 57 PsyI (Tth111I) 140 PflFI, Tth111I

AspLEI GCG↓CFastDigest® HhaI, [FastDigest® HinP1I (Hin6I)](G↓CGC)

4042

HhaI, [Hin6I (HinP1I)](G↓CGC)

120121

BstHHI, CfoI, [HinP1I], [HspAI]

AspS9I G↓GNCC FastDigest® Sau96I (Cfr13I) 60 Cfr13I (Sau96I) 106 [BmgT120I], PspPI, Sau96IAssI AGT↓ACT FastDigest® ScaI 61 ScaI 144 BmcAI, ZrmIAsuC2I CC↓SGG FastDigest® NciI (BcnI) 49 BcnI (NciI) 88 BpuMI, NciIAsuHPI GGTGA(8/7)↓ HphI 123

AsuII TT↓CGAA FastDigest® Bsp119Im 27 Bsp119I (BstBI)m 99Bpu14I, BspT104Im, BstBIm, Csp45I, NspVm, SfuI

AsuNHI G↓CTAGC[FastDigest® BmtI (BspOI)](GCTAG↓C), FastDigest® NheI

2250

[BspOI (BmtI)](GCTAG↓C), NheI

100132

[BmtI]

AvaI C↓YCGRG FastDigest® AvaI (Eco88I)m 17 Eco88I (AvaI)m 113Ama87I, AvaI, [BmeT110Im], BsiHKCI, BsoBIm

AvaII G↓GWCC FastDigest® AvaII (Eco47I) 18 Eco47I (AvaII) 111 AvaII, Bme18I, SinI, VpaK11BIAviII TGC↓GCA FastDigest® FspI (NsbI) 39 NsbI (FspI) 133 Acc16I, FspIAvrII C↓CTAGG FastDigest® AvrII (XmaJI) 18 XmaJI (AvrII) 156 AspA2I, AvrII, BlnIAxyI CC↓TNAGG FastDigest® Bsu36I (Eco81I) 32 Eco81I (Bsu36I) 113 Bse21I, Bsu36IBaeGI GKGCM↓C FastDigest® Bme1580I (BseSI) 21 BseSI (Bme1580I) 96 BstSLIBaeI ↓(10/15)AC(N)4GTAYC(12/7)↓BalI TGG↓CCA FastDigest® MscI (MlsI) 47 MlsI (MscI) 128 MluNI, MscI, Msp20IBamHI G↓GATCC FastDigest® BamHI 18 BamHI 86BanI G↓GYRCC FastDigest® BanI (BshNI) 19 BshNI (BanI) 97 AccB1I, BanI, BspT107IBanII GRGCY↓C Eco24I (BanII) 110 EcoT38I, FriOI

BanIII AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103Bsa29I, BseCI, BshVI, BspDI, BspXI, BsuTUI, ClaI

BarI ↓(7/12)GAAG(N)6TAC(12/7)↓BasI CCANNNN↓NTGG FastDigest® PflMI (Van91I) 54 Van91I (PflMI) 154 AccB7I, PflMIBauI CACGAG(-5/-1)↓ BauI (BssSI) 87 BssSI, Bst2BI

BbeI GGCGC↓C [FastDigest® EheI](GGC↓GCC) 38[EheI (SfoI)](GGC↓GCC), [SspDI (KasI)](G↓GCGCC)

117150

[DinI], [EgeI], [KasI], [Mly113I], [NarI], [SfoIm]

BbrPI CAC↓GTG FastDigest® PmlI (Eco72I) 54 Eco72I (PmlI) 113 AcvI, PmaCI, PmlI, PspCIBbsI GAAGAC(2/6)↓ FastDigest® BbsI (BpiI) 19 BpiI (BbsI) 92 BbsI, BpuAI, BstV2IBbuI GCATG↓C FastDigest® SphI (PaeI) 64 PaeI (SphI) 134 SphIBbv12I GWGCW↓C FastDigest® Alw21I 15 Alw21I (BsiHKAI) 84 BsiHKAIBbvCI CCTCAGC(-5/-2)↓

BbvI GCAGC(8/12)↓ FastDigest® BbvI (Lsp1109I), FastDigest® BseXI

1925

Lsp1109I (BbvI), BseXI (BbvI)

12596

BbvI, BstV1I

BccI CCATC(4/5)↓BceAI ACGGC(12/14)↓BcgI ↓(10/12)CGA(N)6TGC(12/10)↓BciVI GTATCC(6/5)↓ BfuI (BciVI) 90BclI T↓GATCA FastDigest® BclI 20 BclI 87 FbaI, Ksp22IBcnI CC↓SGG FastDigest® NciI (BcnI) 49 BcnI (NciI) 88 AsuC2I, BpuMI, NciIBcuI A↓CTAGT FastDigest® SpeI (BcuI) 63 BcuI (SpeI) 88 AhlI, SpeIBdaI ↓(10/12)TGA(N)6TCA(12/10)↓ BdaI 88BfaI C↓TAG FastDigest® BfaI (FspBI) 20 FspBI (BfaI) 119 BfaI, MaeI, XspIBfiI ACTGGG(5/4)↓ BfiI (BmrI) 89 BmrI, BmuIBfmI C↓TRYAG FastDigest® SfcI (BfmI) 62 BfmI (SfcI) 89 BstSFI, SfcIBfrI C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AflII) 101 AflII, Bst98I, BstAFI, MspCI, Vha464IBfuAI ACCTGC(4/8)↓ FastDigest® BspMI (BveI) 29 BveI (BspMI) 104 Acc36I, BspMI

BfuCI ↓GATCFastDigest® Sau3AI (Bsp143I), FastDigest® MboIm

6044

Bsp143I (Sau3AI), MboIm

99127

BssMI, [BstKTIm], BstMBI, DpnIIm, Kzo9I, NdeIIm, Sau3AIm

BfuI GTATCC(6/5)↓ BfuI (BciVI) 90 BciVIBglI GCCNNNN↓NGGC FastDigest® BglI 20 BglI 90

209


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BglII A↓GATCT FastDigest® BglII 21 BglII 90BisI GC↓NGC [BlsI], GluIBlnI C↓CTAGG FastDigest® AvrII (XmaJI) 18 XmaJI (AvrII) 156 AspA2I, AvrIIBlpI GC↓TNAGC FastDigest® BlpI (Bpu1102I) 21 Bpu1102I (BlpI) 93 BlpI, Bsp1720I, CelIIBlsI GCN↓GC [BisI], [GluI]BmcAI AGT↓ACT FastDigest® ScaI 61 ScaI 144 AssI, ZrmI

Bme1390I CC↓NGG FastDigest® ScrFI (Bme1390I) 61 Bme1390I (ScrFI) 91BmrFI, [BssKI], [BstSCI], MspR9I, ScrFI, [StyD4I]

Bme1580I GKGCM↓C FastDigest® Bme1580I (BseSI) 21 BseSI (Bme1580I) 96 BaeGI, BstSLIBme18I G↓GWCC FastDigest® AvaII (Eco47I) 18 Eco47I (AvaII) 111 AvaII, SinI, VpaK11BIBmeRI GACNNN↓NNGTC FastDigest® Eam1105I 35 Eam1105I (AhdI) 109 AhdI, AspEI, DriI, EclHKIBmeT110I CY↓CGRG [FastDigest® AvaI (Eco88I)m](C↓YCGRG) 17 [Eco88I (AvaI)m](C↓YCGRG) 113 [Ama87I], [AvaIm], [BsiHKCI], [BsoBI]BmgBI CACGTC(-3/-3)↓ AjiI (BmgBI) 82 BtrIBmgT120I GG↓NCC [FastDigest® Sau96I (Cfr13I)](G↓GNCC) 60 [Cfr13I (Sau96I)](G↓GNCC) 106 [AspS9I], [PspPI], [Sau96I]BmiI GGN↓NCC FastDigest® NlaIV (BspLI) 51 BspLI (NlaIV) 100 NlaIV, PspN4IBmrFI CC↓NGG FastDigest® ScrFI (Bme1390I) 61 Bme1390I (ScrFI) 91 [BssKI], [BstSCI], MspR9I, ScrFI, [StyD4I]BmrI ACTGGG(5/4)↓ BfiI (BmrI) 89 BmuI

BmtI GCTAG↓CFastDigest® BmtI (BspOI), [FastDigest® NheIm](G↓CTAGC)

2250

BspOI (BmtI), [NheIm](G↓CTAGC)

100132

[AsuNHI], BmtI

BmuI ACTGGG(5/4)↓ BfiI (BmrI) 89 BmrIBoxI GACNN↓NNGTC FastDigest® PshAI (BoxI) 55 BoxI (PshAI) 91 BstPAI, PshAIBpiI GAAGAC(2/6)↓ FastDigest® BbsI (BpiI) 19 BpiI (BbsI) 92 BbsI, BpuAI, BstV2IBplI ↓(8/13)GAG(N)5CTC(13/8)↓ FastDigest® BplI 22 BplI 92BpmI CTGGAG(16/14)↓ FastDigest® BpmI (GsuI)m 22 GsuI (BpmI)m 119 BpmIBpu10I CCTNAGC(-5/-2)↓ FastDigest® Bpu10I 23 Bpu10I 93Bpu1102I GC↓TNAGC FastDigest® BlpI (Bpu1102I) 21 Bpu1102I (BlpI) 93 BlpI, Bsp1720I, CelIIBpu14I TT↓CGAA FastDigest® Bsp119I 27 Bsp119I (BstBI) 99 AsuII, BspT104I, BstBI, Csp45I, NspV, SfuIBpuAI GAAGAC(2/6)↓ FastDigest® BbsI (BpiI) 19 BpiI (BbsI) 92 BbsI, BstV2IBpuEI CTTGAG(16/14)↓BpuMI CC↓SGG FastDigest® NciI (BcnI) 49 BcnI (NciI) 88 AsuC2I, NciIBpvUI CGAT↓CG FastDigest® PvuI 57 PvuI 141 MvrI, Ple19I

Bsa29I AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, BseCI, BshVI, BspDI, BspXI, BsuTUI, ClaI

BsaAI YAC↓GTR FastDigest® BsaAI (Ppu21I) 23 Ppu21I (BsaAI) 138 BsaAI, BstBAIBsaBI GATNN↓NNATC FastDigest® BsaBI (BseJI) 23 BseJI (BsaBI) 94 BsaBI, Bse8IBsaHI GR↓CGYC FastDigest® BsaHI (Hin1I)m 24 Hin1I (BsaHI)m 120 AcyI, BsaHI, BssNI, BstACI, Hsp92IBsaI GGTCTC(1/5)↓ FastDigest® Eco31I 36 Eco31I (BsaI) 110 Bso31I, BspTNIBsaJI C↓CNNGG FastDigest® BsaJI (BseDI) 24 BseDI (BsaJI) 94 BsaJI, BssECIBsaMI GAATGC(1/-1)↓ FastDigest® Mva1269I 49 Mva1269I (BsmI) 131 BsmI, PctIBsaWI W↓CCGGWBsaXI ↓(9/12)AC(N)5CTCC(10/7)↓Bsc4I CCNNNNN↓NNGG FastDigest® BslI (BseLI) 26 BseLI (BslI) 95 AfiI, BslIBse118I R↓CCGGY FastDigest® BsrFI (Cfr10I) 30 Cfr10I (BsrFI) 105 BsrFI, BssAIBse1I ACTGG(1/-1)↓ FastDigest® BseNI 25 BseNI (BsrI) 96 BsrI, BsrSIBse21I CC↓TNAGG FastDigest® Bsu36I (Eco81I) 32 Eco81I (Bsu36I) 113 AxyI, Bsu36IBse3DI GCAATG(2/0)↓ FastDigest® BsrDI (BseMI) 30 BseMI (BsrDI) 95 BsrDIBse8I GATNN↓NNATC FastDigest® BsaBI (BseJI) 23 BseJI (BsaBI) 94 BsaBIBseAI T↓CCGGA FastDigest® Kpn2Im 44 Kpn2I (BspEI)m 124 AccIIIm, Aor13HI, Bsp13I, BspEIm, MroIm

BseBI CC↓WGG FastDigest® MvaI 48[EcoRII](↓CCWGG), MvaI (BstNI)

116130

[AjnI], Bst2UI, BstNI, BstOI, [Psp6I], [PspGI]

BseCI AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BshVI, BspDI, BspXI, BsuTUI, ClaI

BseDI C↓CNNGG FastDigest® BsaJI (BseDI) 24 BseDI (BsaJI) 94 BsaJI, BssECI

BseGI GGATG(2/0)↓ FastDigest® BseGI, [FastDigest® FokI](GGATG(9/13)↓)

2438

BseGI (BtsCI) 94 BstF5I, BtsCI, [FokI]

BseJI GATNN↓NNATC FastDigest® BsaBI (BseJI) 23 BseJI (BsaBI) 94 BsaBI, Bse8IBseLI CCNNNNN↓NNGG FastDigest® BslI (BseLI) 26 BseLI (BslI) 95 AfiI, Bsc4I, BslIBseMI GCAATG(2/0)↓ FastDigest® BsrDI (BseMI) 30 BseMI (BsrDI) 95 Bse3DI, BsrDIBseMII CTCAG(10/8)↓ FastDigest® BspCNI (BseMII) 29 BseMII (BspCNI) 95 [BspCNI]BseNI ACTGG(1/-1)↓ FastDigest® BseNI 25 BseNI (BsrI) 96 Bse1I, BsrI, BsrSIBsePI G↓CGCGC FastDigest® BssHII (PteI) 31 PauI (BssHII) 135 BssHIIBseRI GAGGAG(10/8)↓BseSI GKGCM↓C FastDigest® Bme1580I (BseSI) 21 BseSI (Bme1580I) 96 BaeGI, BstSLI

BseXI GCAGC(8/12)↓ FastDigest® BseXI, FastDigest® BbvI (Lsp1109I)

2519

BseXI (BbvI), Lsp1109I (BbvI)

96125

BbvI, BstV1I

BseX3I C↓GGCCG FastDigest® EagI (Eco52I) 34 Eco52I (EagI) 111 BstZI, EagI, EclXIBseYI CCCAGC(-5/-1)↓ [FastDigest® PspFI] (CCCAGC(-1/-5)↓) 56 [GsaI]

210

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BsgI GTGCAG(16/14)↓Bsh1236I CG↓CG FastDigest® Bsh1236I 25 Bsh1236I (BstUI) 97 AccII, BspFNI, BstFNI, BstUI, MvnIBsh1285I CGRY↓CG FastDigest® BsiEI (Bsh1285I) 26 Bsh1285I (BsiEI) 97 BsiEIm, BstMCIBshFI GG↓CC FastDigest® HaeIII (BsuRI) 40 BsuRI (HaeIII) 103 BsnI, BspANI, HaeIII, PhoIBshNI G↓GYRCC FastDigest® BanI (BshNI) 19 BshNI (BanI) 97 AccB1I, BanI, BspT107IBshTI A↓CCGGT FastDigest® AgeI (BshTI) 13 BshTI (AgeI) 98 AgeI, AsiGI, CspAIm, PinAI

BshVI AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BseCI, BspDI, BspXI, BsuTUI, ClaI

BsiEI CGRY↓CG FastDigest® BsiEI (Bsh1285I)m 26 Bsh1285I (BsiEI)m 97 BsiEI, BstMCIBsiHKAI GWGCW↓C FastDigest® Alw21I 15 Alw21I (BsiHKAI) 84 Bbv12IBsiHKCI C↓YCGRG FastDigest® AvaI (Eco88I) 17 Eco88I (AvaI) 113 Ama87I, AvaI, [BmeT110I], BsoBI

BsiSI C↓CGGFastDigest® HpaIIm, FastDigest® MspI

4248

HpaIIm, MspI (HpaII)

122129

HapIIm

BsiWI C↓GTACG FastDigest® BsiWI (Pfl23II) 26 Pfl23II (BsiWI) 137 BsiWI, PspLIBslFI GGGAC(10/14)↓ FastDigest® BsmFI (FaqI) 27 FaqI (BsmFI) 118 BsmFIBslI CCNNNNN↓NNGG FastDigest® BslI (BseLI) 26 BseLI (BslI) 95 AfiI, Bsc4I, BslIBsmAI GTCTC(1/5)↓ FastDigest® Alw26Im 15 Alw26I (BsmAI)m 85 BstMAIBsmBI CGTCTC(1/5)↓ FastDigest® BsmBI (Esp3I) 27 Esp3I (BsmBI) 117 BsmBIBsmFI GGGAC(10/14)↓ FastDigest® BsmFI (FaqI) 27 FaqI (BsmFI) 118 BslFI, BsmFIBsmI GAATGC(1/-1)↓ FastDigest® Mva1269I 49 Mva1269I (BsmI) 131 BsaMI, PctIBsnI GG↓CC FastDigest® HaeIII (BsuRI) 40 BsuRI (HaeIII) 103 BshFI, BspANI, HaeIII, PhoIBso31I GGTCTC(1/5)↓ FastDigest® Eco31I 36 Eco31I (BsaI) 110 BsaI, BspTNIBsoBI C↓YCGRG FastDigest® AvaI (Eco88I)m 17 Eco88I (AvaI)m 113 Ama87I, AvaIm, [BmeT110I], BsiHKCI

Bsp119I TT↓CGAA FastDigest® Bsp119I 27 Bsp119I (BstBI) 99AsuIIm, Bpu14I, BspT104I, BstBI, Csp45I, NspV, SfuIm

Bsp120I G↓GGCCC[FastDigest® ApaI](GGGCC↓C), FastDigest® Bsp120I

1628

[ApaI](GGGCC↓C), Bsp120I (PspOMI)

8599

PspOMI

Bsp1286I GDGCH↓C FastDigest® Bsp1286I (SduI) 28 SduI (Bsp1286I) 145 Bsp1286I, MhlIBsp1407I T↓GTACA FastDigest® Bsp1407I 28 Bsp1407I (BsrGI) 100 BsrGI, BstAUI

Bsp143I ↓GATCFastDigest® Sau3AI (Bsp143I), FastDigest® MboIm

6044


99127

BfuCI, BssMI, [BstKTIm], BstMBI, DpnIIm, Kzo9I, NdeIIm, Sau3AIm

Bsp13I T↓CCGGA FastDigest® Kpn2I 44 Kpn2I (BspEI) 124 AccIII, Aor13HI, BseAI, BspEI, MroIBsp1720I GC↓TNAGC FastDigest® BlpI (Bpu1102I) 21 Bpu1102I (BlpI) 93 BlpI, CelIIBsp19I C↓CATGG FastDigest® NcoI 50 NcoI 131Bsp68I TCG↓CGA FastDigest® NruI (RruI) 52 Bsp68I (NruI) 98 BtuMI, NruIBspACI CCGC(-3/-1)↓ FastDigest® AciI (SsiI) 12 SsiI (AciI) 149 AciIBspANI GG↓CC FastDigest® HaeIII (BsuRI) 40 BsuRI (HaeIII) 103 BshFI, BsnI, HaeIII, PhoI

BspCNI CTCAG(9/7)↓ [FastDigest® BspCNI (BseMII)](CTCAG(10/8)↓)

29 [BseMII (BspCNI)](CTCAG(10/8)↓) 95 BspCNI

BspDI AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BseCI, BshVI, BspXI, BsuTUI, ClaI

BspEI T↓CCGGA FastDigest® Kpn2Im 44 Kpn2I (BspEI)m 124 AccIIIm, Aor13HI, BseAIm, Bsp13I, MroIm

BspFNI CG↓CG FastDigest® Bsh1236I 25 Bsh1236I (BstUI) 97 AccII, BstFNI, BstUI, MvnIBspHI T↓CATGA FastDigest® BspHI (PagI)m 29 PagI (BspHI)m 135 BspHI, CciI, RcaIBspLI GGN↓NCC FastDigest® NlaIV (BspLI) 51 BspLI (NlaIV) 95 BmiI, NlaIVm, PspN4IBspMAI CTGCA↓G FastDigest® PstI 56 PstI 140BspMI ACCTGC(4/8)↓ FastDigest® BspMI (BveI) 29 BveI (BspMI) 104 Acc36I, BfuAI, BspMI

BspOI GCTAG↓CFastDigest® BmtI (BspOI), [FastDigest® NheI](G↓CTAGC)

2250

BspOI (BmtI), [NheI](G↓CTAGC)

100132

[AsuNHI], BmtI

BspPI GGATC(4/5)↓ BspPI (AlwI) 101 AclWI, AlwIBspQI GCTCTTC(1/4)↓ FastDigest® SapI (LguI) 59 LguI (SapI) 125 PciSI, SapIBspT104I TT↓CGAA FastDigest® Bsp119I 27 Bsp119I (BstBI) 99 AsuIIm, Bpu14I, BstBI, Csp45I, NspV, SfuIm

BspT107I G↓GYRCC FastDigest® BanI (BshNI) 19 BshNI (BanI) 97 AccB1I, BanIBspTI C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AflII) 101 AflII, BfrI, Bst98I, BstAFI, MspCI, Vha464IBspTNI GGTCTC(1/5)↓ FastDigest® Eco31I 36 Eco31I (BsaI) 110 BsaI, Bso31I

BspXI AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BseCI, BshVI, BspDI, BsuTUI, ClaI

BsrBI CCGCTC(-3/-3)↓ FastDigest® BsrBI (MbiI)m 30 MbiI (BsrBI)m 126 AccBSI, BsrBIBsrDI GCAATG(2/0)↓ FastDigest® BsrDI (BseMI) 30 BseMI (BsrDI) 95 Bse3DI, BsrDIBsrFI R↓CCGGY FastDigest® BsrFI (Cfr10I) 30 Cfr10I (BsrFI) 105 Bse118I, BsrFI, BssAIm

BsrGI T↓GTACA FastDigest® Bsp1407I 28 Bsp1407I (BsrGI) 100 BstAUIBsrI ACTGG(1/-1)↓ FastDigest® BseNI 25 BseNI (BsrI) 96 Bse1I, BsrSIBsrSI ACTGG(1/-1)↓ FastDigest® BseNI 25 BseNI (BsrI) 96 Bse1I, BsrIBssAI R↓CCGGY FastDigest® BsrFI (Cfr10I)m 30 Cfr10I (BsrFI)m 105 Bse118I, BsrFIm

BssECI C↓CNNGG FastDigest® BsaJI (BseDI) 24 BseDI (BsaJI) 94 BsaJIBssHII G↓CGCGC FastDigest® BssHII (PteI) 31 PauI (BssHII) 135 BsePI, BssHIIBssKI ↓CCNGG [FastDigest® ScrFI (Bme1390I)](CC↓NGG) 61 [Bme1390I (ScrFI)](CC↓NGG) 91 [BmrFI], BstSCI, [MspR9I], [ScrFI], StyD4I

211


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BssMI ↓GATCFastDigest® Sau3AI (Bsp143I), FastDigest® MboI

6044

Bsp143I (Sau3AI), MboI

99127

BfuCI, [BstKTI], BstMBI, DpnII, Kzo9I, NdeII, Sau3AI

BssNAI GTA↓TAC FastDigest® BstZ17I (Bst1107I) 31 Bst1107I (BstZ17I) 102 BstZ17IBssNI GR↓CGYC FastDigest® BsaHI (Hin1I) 24 Hin1I (BsaHI) 120 AcyI, BsaHI, BstACI, Hsp92IBssSI CACGAG(-5/-1)↓ BauI (BssSI) 87 Bst2BIBssT1I C↓CWWGG FastDigest® StyI (Eco130I) 65 Eco130I (StyI) 114 EcoT14I, ErhI, StyIBst1107I GTA↓TAC FastDigest® BstZ17I (Bst1107I) 31 Bst1107I (BstZ17I) 102 BssNAI, BstZ17IBst2BI CACGAG(-5/-1)↓ BauI (BssSI) 87 BssSI

Bst2UI CC↓WGG FastDigest® MvaI 48[EcoRIIm](↓CCWGG), MvaI (BstNI)

116130

[AjnI], BseBI, BstNI, BstOI, [Psp6I], [PspGIm]

Bst4CI ACN↓GT FastDigest® TaaI 65 TaaI (HpyCH4III) 150 HpyCH4IIIBst6I CTCTTC(1/4)↓ FastDigest® EarI (Eam1104I) 35 Eam1104I (EarI) 109 EarIBst98I C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AflII) 101 AflII, BfrI, BstAFI, MspCI, Vha464IBstACI GR↓CGYC FastDigest® BsaHI (Hin1I) 24 Hin1I (BsaHI) 120 AcyI, BsaHI, BssNI, Hsp92IBstAFI C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AflII) 101 AflII, BfrI, Bst98I, MspCI, Vha464IBstAPI GCANNNN↓NTGCBstAUI T↓GTACA FastDigest® Bsp1407I 28 Bsp1407I (BsrGI) 100 BsrGIBstBAI YAC↓GTR FastDigest® BsaAI (Ppu21I) 23 Ppu21I (BsaAI) 138 BsaAI

BstBI TT↓CGAA FastDigest® Bsp119I 27 Bsp119I (BstBI) 99AsuIIm, Bpu14I, BspT104I, Csp45I, NspVm, SfuIm

BstC8I GCN↓NGC Cac8IBstDEI C↓TNAG FastDigest® DdeI (HpyF3I) 33 HpyF3I (DdeI) 123 DdeIBstDSI C↓CRYGG BtgIBstEII G↓GTNACC FastDigest® Eco91I 36 Eco91I (BstEII) 114 BstPI, EcoO65I, PspEIBstENI CCTNN↓NNNAGG FastDigest® EcoNI (XagI) 36 XagI (EcoNI) 155 EcoNI

BstF5I GGATG(2/0)↓ FastDigest® BseGI, [FastDigest® FokI](GGATG(9/13)↓)

2438

BseGI (BtsCI) 94 BtsCI, [FokI]

BstFNI CG↓CG FastDigest® Bsh1236I 25 Bsh1236I (BstUI) 97 AccII, BspFNI, BstUI, MvnIBstH2I RGCGC↓Y FastDigest® HaeII (BfoI) 41 HaeII

BstHHI GCG↓CFastDigest® HhaI, [FastDigest® HinP1I (Hin6I)](G↓CGC)

4042


120121

AspLEI, CfoI, [HinP1I], [HspAI]

BstKTI GAT↓C[FastDigest® Sau3AI (Bsp143I)m](↓GATC), [FastDigest® MboI](↓GATC)

6044

[Bsp143I (Sau3AI)m](↓GATC), [MboI](↓GATC)

99127

[BfuCIm], [BssMI], [BstMBI], [DpnII], [Kzo9Im], [NdeIIm], [Sau3AIm]

BstMAI GTCTC(1/5)↓ FastDigest® Alw26I 15 Alw26I (BsmAI) 85 BsmAI

BstMBI ↓GATCFastDigest® Sau3AI (Bsp143I), FastDigest® MboI

6044

Bsp143I (Sau3AI), MboI

99127

BfuCI, BssMI, [BstKTI], DpnII, Kzo9I, NdeII, Sau3AI

BstMCI CGRY↓CG FastDigest® BsiEI (Bsh1285I) 26 Bsh1285I (BsiEI) 97 BsiEIBstMWI GCNNNNN↓NNGC FastDigest® HpyF10VI 43 HpyF10VI (MwoI) 124 MwoI

BstNI CC↓WGG FastDigest® MvaI 48[EcoRIIm](↓CCWGG), MvaI (BstNI)

116130

[AjnI], BseBI, Bst2UI, BstOI, [Psp6I], [PspGIm]

BstNSI RCATG↓Y FastDigest® NspI (XceI) 53 XceI (NspI) 156 NspI

BstOI CC↓WGG FastDigest® MvaI 48[EcoRIIm](↓CCWGG), MvaI (BstNI)

116130

[AjnI], BseBI, Bst2UI, BstNI, [Psp6I], [PspGIm]

BstPAI GACNN↓NNGTC FastDigest® PshAI (BoxI) 55 BoxI (PshAI) 91 PshAIBstPI G↓GTNACC FastDigest® Eco91I 36 Eco91I (BstEII) 114 BstEII, EcoO65I, PspEIBstSCI ↓CCNGG [FastDigest® ScrFI (Bme1390I)](CC↓NGG) 61 [Bme1390I (ScrFI)](CC↓NGG) 91 [BmrFI], BssKI, [MspR9I], [ScrFI], StyD4IBstSFI C↓TRYAG FastDigest® SfcI (BfmI) 62 BfmI (SfcI) 89 SfcIBstSLI GKGCM↓C FastDigest® Bme1580I (BseSI) 21 BseSI (Bme1580I) 96 BaeGIBstSNI TAC↓GTA FastDigest® SnaBI (Eco105I) 63 Eco105I (SnaBI) 114 SnaBIBstUI CG↓CG FastDigest® Bsh1236I 25 Bsh1236I (BstUI) 97 AccII, BspFNI, BstFNI, MvnI

BstV1I GCAGC(8/12)↓ FastDigest® BbvI (Lsp1109I), FastDigest® BseXI

1925


12596

BbvI

BstV2I GAAGAC(2/6)↓ FastDigest® BbsI (BpiI) 19 BpiI (BbsI) 92 BbsI, BpuAIBstX2I R↓GATCY FastDigest® PsuI 56 PsuI (BstYI) 140 BstYI, MflI, XhoIIBstXI CCANNNNN↓NTGG FastDigest® BstXI 31 BstXI 102BstYI R↓GATCY FastDigest® PsuI 56 PsuI (BstYI) 140 BstX2I, MflIm, XhoIIBstZ17I GTA↓TAC FastDigest® BstZ17I (Bst1107I) 31 Bst1107I (BstZ17I) 102 BssNAI, BstZ17IBstZI C↓GGCCG FastDigest® EagI (Eco52I) 34 Eco52I (EagI) 111 BseX3I, EagI, EclXI

Bsu15I AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BseCI, BshVI, BspDI, BspXI, BsuTUI, ClaI

Bsu36I CC↓TNAGG FastDigest® Bsu36I (Eco81I) 32 Eco81I (Bsu36I) 113 AxyI, Bse21I, Bsu36IBsuRI GG↓CC FastDigest® HaeIII (BsuRI) 40 BsuRI (HaeIII) 103 BshFI, BsnI, BspANI, HaeIII, PhoI

BsuTUI AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BseCI, BshVI, BspDI, BspXI, ClaI

BtgI C↓CRYGG BstDSIBtgZI GCGATG(10/14)↓BtrI CACGTC(-3/-3)↓ AjiI (BmgBI) 82 BmgBI

212

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BtsCI GGATG(2/0)↓ FastDigest® BseGI, [FastDigest® FokI](GGATG(9/13)↓)

2438

BseGI (BtsCI) 94 BstF5I, [FokI]

BtsI GCAGTG(2/0)↓BtuMI TCG↓CGA FastDigest® NruI (RruI) 52 Bsp68I (NruI) 98 NruIBveI ACCTGC(4/8)↓ FastDigest® BspMI (BveI) 29 BveI (BspMI) 104 Acc36I, BfuAI, BspMICac8I GCN↓NGC BstC8ICaiI CAGNNN↓CTG FastDigest® AlwNI (CaiI) 15 CaiI (AlwNI) 104 AlwNICciI T↓CATGA FastDigest® BspHI (PagI) 29 PagI (BspHI) 135 BspHI, RcaICciNI GC↓GGCCGC FastDigest® NotI 52 NotI 133CelII GC↓TNAGC FastDigest® BlpI (Bpu1102I) 21 Bpu1102I (BlpI) 93 BlpI, Bsp1720I

CfoI GCG↓CFastDigest® HhaI, [FastDigest® HinP1I (Hin6I)](G↓CGC)

4042


120121

AspLEI, BstHHI, [HinP1I], [HspAI]

Cfr10I R↓CCGGY FastDigest® BsrFI (Cfr10I) 30 Cfr10I (BsrFI) 105 Bse118I, BsrFI, BssAIm

Cfr13I G↓GNCC FastDigest® Sau96I (Cfr13I) 60 Cfr13I (Sau96I) 106 AspS9I, [BmgT120I], PspPI, Sau96ICfr42I CCGC↓GG Cfr42I (SacII) 106 KspI, SacII, Sfr303I, SgrBI, SstII

Cfr9I C↓CCGGG [FastDigest® SmaIm](CCC↓GGG) 63Cfr9I (XmaI), [SmaIm](CCC↓GGG)

105147

TspMIm, XmaCI, XmaIm

CfrI Y↓GGCCR CfrI (EaeI) 105 AcoI, EaeI

ClaI AT↓CGAT FastDigest® ClaI (Bsu15I) 32 Bsu15I (ClaI) 103BanIII, Bsa29I, BseCI, BshVI, BspDI, BspXI, BsuTUI, ClaI

CpoI CG↓GWCCG FastDigest® RsrII (CpoI) 58 CpoI (RsrII) 106 CspI, Rsr2I, RsrIICseI GACGC(5/10)↓ FastDigest® HgaI (CseI) 40 CseI (HgaI) 107 HgaI

Csp45I TT↓CGAA FastDigest® Bsp119I 27 Bsp119I (BstBI) 99AsuII, Bpu14I, BspT104I, BstBI, NspVm, SfuI

Csp6I G↓TACFastDigest® Csp6I, [FastDigest® RsaIm](GT↓AC)

3258

Csp6I (CviQI), [RsaIm](GT↓AC)

107141

[AfaIm], CviQI, RsaNI

CspAI A↓CCGGT FastDigest® AgeI (BshTI)m 13 BshTI (AgeI)m 98 AgeIm, AsiGI, PinAICspCI ↓(11/13)CAA(N)5GTGG(12/10)↓CspI CG↓GWCCG FastDigest® RsrII (CpoI) 58 CpoI (RsrII) 106 Rsr2I, RsrIICviAII C↓ATG [FastDigest® NlaIII (Hin1II)](CATG↓) 51 [Hin1II (NlaIII)](CATG↓) 120 [FaeI], [FatIm][Hsp92II], [NlaIII]CviJI RG↓CY CviKI-1CviKI-1 RG↓CY CviJI

CviQI G↓TACFastDigest® Csp6I, [FastDigest® RsaIm](GT↓AC)

3258

Csp6I (CviQI), [RsaIm](GT↓AC)

107141

[AfaIm], RsaNI

DdeI C↓TNAG FastDigest® DdeI (HpyF3I) 33 HpyF3I (DdeI) 123 BstDEI, DdeI

DinI GGC↓GCC FastDigest® EheI 117EheI (SfoI), [SspDI (KasI)](G↓GCGCC)

117150

[BbeI], EgeI, [KasI], [Mly113I], [NarI], SfoI

DpnI Gm6A↓TC FastDigest® DpnI 33 DpnI 108 MalI

DpnII ↓GATCFastDigest® Sau3AI (Bsp143I)m, FastDigest® MboIm

6044

[Bsp143I (Sau3AI)m], MboIm

99127

BfuCIm, BssMI, [BstKTIm], BstMBI, Kzo9Im, NdeII, Sau3AIm

DraI TTT↓AAA FastDigest® DraI 33 DraI 108DraII RG↓GNCCY FastDigest® EcoO109I 37 EcoO109I (DraII) 115DraIII CACNNN↓GTG FastDigest® DraIII (AdeI) 34 AdeI (DraIII) 82 DraIIIDrdI GACNNNN↓NNGTC FastDigest® DrdI (AasI) 34 AasI (DrdI) 80 DrdI, DseDIDriI GACNNN↓NNGTC FastDigest® Eam1105I 35 Eam1105I (AhdI) 109 AhdI, AspEI, BmeRI, EclHKIDseDI GACNNNN↓NNGTC FastDigest® DrdI (AasI) 34 AasI (DrdI) 80 DrdIEaeI Y↓GGCCR CfrI (EaeI) 105 AcoIEagI C↓GGCCG FastDigest® EagI (Eco52I) 34 Eco52I (EagI) 111 BseX3I, BstZI, EagI, EclXIEam1104I CTCTTC(1/4)↓ FastDigest® EarI (Eam1104I) 35 Eam1104I (EarI) 109 Bst6I, EarIEam1105I GACNNN↓NNGTC FastDigest® Eam1105I 35 Eam1105I (AhdI) 109 AhdI, AspEI, BmeRI, DriI, EclHKIEarI CTCTTC(1/4)↓ FastDigest® EarI (Eam1104I) 35 Eam1104I (EarI) 109 Bst6I, EarIEciI GGCGGA(11/9)↓

Ecl136II GAG↓CTCFastDigest® Ecl136II, [FastDigest® SacIm](GAGCT↓C)

3558

Ecl136II (EcoICRI), [SacIm](GAGCT↓C)

109142

Eco53kIm, EcoICRI, [Psp124BI], [SstIm]

EclHKI GACNNN↓NNGTC FastDigest® Eam1105I 35 Eam1105I (AhdI) 109 AhdI, AspEI, BmeRI, DriIEclXI C↓GGCCG FastDigest® EagI (Eco52I) 34 Eco52I (EagI) 111 BseX3I, BstZI, EagIEco105I TAC↓GTA FastDigest® SnaBI (Eco105I) 63 Eco105I (SnaBI) 114 BstSNI, SnaBIEco130I C↓CWWGG FastDigest® StyI (Eco130I) 65 Eco130I (StyI) 114 BssT1I, EcoT14I, ErhI, StyIEco147I AGG↓CCT FastDigest® StuI (Eco147I) 64 Eco147I (StuI) 115 AatI, PceI, SseBI, StuIEco24I GRGCY↓C Eco24I (BanII) 110 BanII, EcoT38I, FriOIEco31I GGTCTC(1/5)↓ FastDigest® Eco31I 36 Eco31I (BsaI) 110 BsaI, Bso31I, BspTNIEco32I GAT↓ATC FastDigest® EcoRV (Eco32I) 37 Eco32I (EcoRV) 110 EcoRVEco47I G↓GWCC FastDigest® AvaII (Eco47I) 18 Eco47I (AvaII) 111 AvaII, Bme18I, SinI, VpaK11BIEco47III AGC↓GCT FastDigest® AfeI (Eco47III) 13 Eco47III (AfeI) 111 AfeI, Aor51HIEco52I C↓GGCCG FastDigest® EagI (Eco52I) 34 Eco52I (EagI) 111 BseX3I, BstZI, EagI, EclXI

Eco53kI GAG↓CTCFastDigest® Ecl136IIm, [FastDigest® SacIm](GAGCT↓C)

3558

Ecl136II (EcoICRI)m, [SacIm](GAGCT↓C)

109142

EcoICRI, [Psp124BI], [SstI]

Eco57I CTGAAG(16/14)↓ FastDigest® AcuI (Eco57I) 12 Eco57I (AcuI) 112 AcuI

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Eco57MI CTGRAG(16/14)↓ Eco57MI 112Eco72I CAC↓GTG FastDigest® PmlI (Eco72I) 54 Eco72I (PmlI) 113 AcvI, BbrPI, PmaCI, PmlI, PspCIEco81I CC↓TNAGG FastDigest® Bsu36I (Eco81I) 32 Eco81I (Bsu36I) 113 AxyI, Bse21I, Bsu36I

Eco88I C↓YCGRG FastDigest® AvaI (Eco88I) 17 Eco88I (AvaI) 113Ama87I, AvaIm, [BmeT110Im], BsiHKCI, BsoBIm

Eco91I G↓GTNACC FastDigest® Eco91I 36 Eco91I (BstEII) 114 BstEII, BstPI, EcoO65I, PspEI

EcoICRI GAG↓CTCFastDigest® Ecl136II, [FastDigest® SacI](GAGCT↓C)

3558

Ecl136II (EcoICRI), [SacI](GAGCT↓C)

109142

Eco53kI, [Psp124BI], [SstI]

EcoNI CCTNN↓NNNAGG FastDigest® EcoNI (XagI) 36 XagI (EcoNI) 155 BstENI, EcoNIEcoO109I RG↓GNCCY FastDigest® EcoO109I 37 EcoO109I (DraII) 115 DraIIEcoO65I G↓GTNACC FastDigest® Eco91I 36 Eco91I (BstEII) 114 BstEII, BstPI, PspEIEcoP15I CAGCAG(25/27)↓EcoRI G↓AATTC FastDigest® EcoRI 37 EcoRI 116

EcoRII ↓CCWGG [FastDigest® MvaIm](CC↓WGG) 48EcoRII, [MvaI (BstNI)m](CC↓WGG)

116130

AjnIm, [BseBI], [Bst2UIm], [BstNIm], [BstOIm], Psp6I, PspGI

EcoRV GAT↓ATC FastDigest® EcoRV (Eco32I) 37 Eco32I (EcoRV) 110 EcoRVEcoT14I C↓CWWGG FastDigest® StyI (Eco130I) 65 Eco130I (StyI) 114 BssT1I, ErhI, StyIEcoT22I ATGCA↓T FastDigest® NsiI (Mph1103I) 52 Mph1103I (NsiI) 129 NsiIm, Zsp2IEcoT38I GRGCY↓C Eco24I (BanII) 110 BanII, FriOI

EgeI GGC↓GCC FastDigest® EheI 38EheI (SfoI), [SspDI (KasI)](G↓GCGCC)

117150

[BbeI], DinI, [KasI], [Mly113I], [NarI], SfoI

EheI GGC↓GCC FastDigest® EheI 38EheI (SfoI), [SspDI (KasI)](G↓GCGCC)

117150

[BbeI], DinI, EgeI, [KasI], [Mly113I], [NarI], SfoIm

ErhI C↓CWWGG FastDigest® StyI (Eco130I) 65 Eco130I (StyI) 114 BssT1I, EcoT14I, StyIEsp3I CGTCTC(1/5)↓ FastDigest® BsmBI (Esp3I) 27 Esp3I (BsmBI) 117 BsmBIFaeI CATG↓ FastDigest® NlaIII (Hin1II) 51 Hin1II (NlaIII) 120 [CviAII], [FatI], Hsp92II, NlaIIIFaiI YA↓TRFaqI GGGAC(10/14)↓ FastDigest® BsmFI (FaqI) 27 FaqI (BsmFI) 118 BslFI, BsmFIFalI ↓(8/13)AAG(N)5CTT(13/8)↓FatI ↓CATG [FastDigest® NlaIII (Hin1II)](CATG↓) 51 [Hin1II (NlaIII)](CATG↓) 120 [CviAIIm], [FaeI], [Hsp92II], [NlaIIIm]FauI CCCGC(4/6)↓ SmuI (FauI) 148FauNDI CA↓TATG FastDigest® NdeI 50 NdeI 132FbaI T↓GATCA FastDigest® BclI 20 BclI 87 Ksp22IFblI GT↓MKAC FastDigest® AccI (XmiI) 11 XmiI (AccI) 157 AccIFnu4HI GC↓NGC FastDigest® Fnu4HI (SatI) 38 SatI (Fnu4HI) 143 Fnu4HI, Fsp4HI, ItaIm

FokI GGATG(9/13)↓ [FastDigest® BseGI](GGATG(2/0)↓), FastDigest® FokI

2438

[BseGI (BtsCI)](GGATG(2/0)↓) 94 [BstF5I], [BtsCI], FokI

FriOI GRGCY↓C Eco24I (BanII) 110 BanII, EcoT38IFseI GGCCGG↓CC RigIFsp4HI GC↓NGC FastDigest® Fnu4HI (SatI) 38 SatI (Fnu4HI) 143 Fnu4HI, ItaIm

FspAI RTGC↓GCAY FastDigest® FspAI 39 FspAI 118FspBI C↓TAG FastDigest® BfaI (FspBI) 20 FspBI (BfaI) 119 BfaI, MaeI, XspIFspI TGC↓GCA FastDigest® FspI (NsbI) 39 NsbI (FspI) 133 Acc16I, AviII, FspIGlaI GC↓GCGluI GC↓NGC BisI, [BlsI]GsaI CCCAGC(-1/-5)↓ FastDigest® PspFI 56 [BseYI]GsuI CTGGAG(16/14)↓ FastDigest® BpmI (GsuI) 22 GsuI (BpmI) 119 BpmIm

HaeII RGCGC↓Y FastDigest® HaeII (BfoI) 39 BstH2I, HaeIIHaeIII GG↓CC FastDigest® HaeIII (BsuRI) 40 BsuRI (HaeIII) 103 BshFI, BsnI, BspANI, HaeIII, PhoI

HapII C↓CGGFastDigest® HpaII, FastDigest® MspIm

4248

HpaII, MspI (HpaII)m

122129

BsiSIm

HgaI GACGC(5/10)↓ FastDigest® HgaI (CseI) 40 CseI (HgaI) 107 HgaI

HhaI GCG↓CFastDigest® HhaI, [FastDigest® HinP1I (Hin6I)](G↓CGC)

4042


120121

AspLEI, BstHHI, CfoI, [HinP1I], [HspAI]

Hin1I GR↓CGYC FastDigest® BsaHI (Hin1I) 24 Hin1I (BsaHI) 120 AcyI, BsaHIm, BssNI, BstACI, Hsp92IHin1II CATG↓ FastDigest® NlaIII (Hin1II) 51 Hin1II (NlaIII) 120 [CviAII], FaeI, [FatI], Hsp92II, NlaIIIHin4I ↓(8/13)GAY(N)5VTC(13/8)↓ Hin4I 121

Hin6I G↓CGC[FastDigest® HhaI](GCG↓C), FastDigest® HinP1I (Hin6I)

4042

[HhaI](GCG↓C), Hin6I (HinP1I)

120121

[AspLEI], [BstHHI], [CfoI], HinP1I, HspAI

HincII GTY↓RAC FastDigest® HincII 41 HincII (HindII) 121 HindIIHindII GTY↓RAC FastDigest® HincII 41 HincII (HindII) 121HindIII A↓AGCTT FastDigest® HindIII 41 HindIII 122HinfI G↓ANTC FastDigest® HinfI 41 HinfI 122

HinP1I G↓CGC[FastDigest® HhaI](GCG↓C), FastDigest® HinP1I (Hin6I)

4042


120121

[AspLEI], [BstHHI], [CfoI], HinP1I, HspAI

HpaI GTT↓AAC FastDigest® HpaI (KspAI)m 42 KspAI (HpaI)m 125 HpaI

HpaII C↓CGGFastDigest® HpaII, FastDigest® MspIm

4248

HpaII, MspI (HpaII)m

122129

BsiSIm, HapII

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HphI GGTGA(8/7)↓ HphI 123 AsuHPIHpy166II GTN↓NAC FastDigest® Hpy8I 43 Hpy8I (MjaIV) 123Hpy188I TCN↓GAHpy188III TC↓NNGAHpy8I GTN↓NAC FastDigest® Hpy8I 43 Hpy8I (MjaIV) 123 Hpy166IIHpy99I CGWCG↓HpyAV CCTTC(6/5)↓HpyCH4III ACN↓GT FastDigest® TaaI 65 TaaI (HpyCH4III) 150 Bst4CIHpyCH4IV A↓CGT [FastDigest® TaiIm](ACGT↓) 66 [TaiI (MaeII)m](ACGT↓) 150 MaeIIHpyCH4V TG↓CAHpyF10VI GCNNNNN↓NNGC FastDigest® HpyF10VI 43 HpyF10VI (MwoI) 124 BstMWI, MwoIm

HpyF3I C↓TNAG FastDigest® DdeI (HpyF3I) 33 HpyF3I (DdeI) 123 BstDEI, DdeIHsp92I GR↓CGYC FastDigest® BsaHI (Hin1I) 24 Hin1I (BsaHI) 120 AcyI, BsaHI, BssNI, BstACIHsp92II CATG↓ FastDigest® NlaIII (Hin1II) 51 Hin1II (NlaIII) 120 [CviAII], FaeI, [FatI], NlaIII

HspAI G↓CGC[FastDigest® HhaI](GCG↓C), FastDigest® HinP1I (Hin6I)

4042


120121

[AspLEI], [BstHHI], [CfoI], HinP1I

ItaI GC↓NGC FastDigest® Fnu4HI (SatI)m 38 SatI (Fnu4HI)m 143 Fnu4HIm, Fsp4HIm

KasI G↓GCGCC [FastDigest® EheI](GGC↓GCC) 38[EheI (SfoI)](GGC↓GCC), SspDI (KasI)

117150

[BbeI], [DinI], [EgeI], [Mly113I], [NarI], [SfoIm]

Kpn2I T↓CCGGA FastDigest® Kpn2I 44 Kpn2I (BspEI) 124AccIIIm, Aor13HI, BseAIm, Bsp13I, BspEIm, MroI

KpnI GGTAC↓C[FastDigest® Acc65Im](G↓GTACC), FastDigest® KpnI

1143

[Acc65I (Asp718I)m](G↓GTACC), KpnI

81124

[Asp718Im]

Ksp22I T↓GATCA FastDigest® BclI 20 BclI 87 FbaIKspAI GTT↓AAC FastDigest® HpaI (KspAI) 42 KspAI (HpaI) 125 HpaIKspI CCGC↓GG Cfr42I (SacII) 106 SacII, Sfr303I, SgrBI, SstII

Kzo9I ↓GATCFastDigest® Sau3AI (Bsp143I), FastDigest® MboIm

6044


99127

BfuCI, BssMI, [BstKTIm], BstMBI, DpnIIm, NdeIIm, Sau3AI

LguI GCTCTTC(1/4)↓ FastDigest® SapI (LguI) 59 LguI (SapI) 125 BspQI, PciSI, SapI

Lsp1109I GCAGC(8/12)↓ FastDigest® BbvI (Lsp1109I), FastDigest® BseXI

1925


12596

BbvI, BstV1I

LweI GCATC(5/9)↓ FastDigest® SfaNI (BmsI) 62 LweI (SfaNI) 126 SfaNIMabI A↓CCWGGT FastDigest® SexAI (CsiI) 61 SexAIm

MaeI C↓TAG FastDigest® BfaI (FspBI) 20 FspBI (BfaI) 119 BfaI, XspIMaeII A↓CGT [FastDigest® TaiI](ACGT↓) 66 [TaiI (MaeII)](ACGT↓) 150 HpyCH4IVMaeIII ↓GTNACMalI GA↓TC FastDigest® DpnI 33 DpnI 108MauBI CG↓CGCGCG FastDigest® MauBI 44 MauBI 126MbiI CCGCTC(-3/-3)↓ FastDigest® BsrBI (MbiI) 30 MbiI (BsrBI) 126 AccBSI, BsrBIm

MboI ↓GATCFastDigest® Sau3AI (Bsp143I)m, FastDigest® MboI

6044

Bsp143I (Sau3AI)m, MboI

99127

BfuCIm, BssMI, [BstKTI], BstMBI, DpnIIm, Kzo9Im, NdeIIm, Sau3AIm

MboII GAAGA(8/7)↓ FastDigest® MboII 45 MboII 127MfeI C↓AATTG FastDigest® MfeI (MunI) 45 MunI (MfeI) 130 MfeIMflI R↓GATCY FastDigest® PsuIm 56 PsuI (BstYI)m 140 BstX2I, BstYIm, XhoIIm

MhlI GDGCH↓C FastDigest® Bsp1286I (SduI) 28 SduI (Bsp1286I) 145 Bsp1286IMlsI TGG↓CCA FastDigest® MscI (MlsI) 47 MlsI (MscI) 128 BalI, MluNI, MscI, Msp20IMluI A↓CGCGT FastDigest® MluI 45 MluI 128MluNI TGG↓CCA FastDigest® MscI (MlsI) 47 MlsI (MscI) 128 BalI, MscI, Msp20I

Mly113I GG↓CGCC [FastDigest® EheI](GGC↓GCC) 38[EheI (SfoI)](GGC↓GCC), [SspDI (KasI)](G↓GCGCC)

117150

[BbeI], [DinI], [EgeI], [KasI], NarI, [SfoI]

MlyI GAGTC(5/5)↓ FastDigest® MlyI (SchI) 46 SchI (MlyI) 144 MlyI, [PleIm], [PpsI]MmeI TCCRAC(20/18)↓MnlI CCTC(7/6)↓ FastDigest® MnlI 46 MnlI 128Mph1103I ATGCA↓T FastDigest® NsiI (Mph1103I) 52 Mph1103I (NsiI) 129 EcoT22I, NsiI, Zsp2IMreI CG↓CCGGCG FastDigest® MreI 46 MreI (Sse232I) 129MroI T↓CCGGA FastDigest® Kpn2I 44 Kpn2I (BspEI) 124 AccIIIm, Aor13HI, BseAIm, Bsp13I, BspEIm

MroNI G↓CCGGC [FastDigest® NaeI (PdiI)](GCC↓GGC) 49 [PdiI (NaeI)](GCC↓GGC) 136 [NaeI], NgoMIVMroXI GAANN↓NNTTC FastDigest® PdmI 53 PdmI (XmnI) 136 Asp700I, XmnIMscI TGG↓CCA FastDigest® MscI (MlsI) 47 MlsI (MscI) 128 BalI, MscI, MluNI, Msp20I

MseI T↓TAAFastDigest® MseI (SaqAI), FastDigest® Tru1I

4767

Tru1I (MseI) 152 MseI, Tru9I

MslI CAYNN↓NNRTG FastDigest® MslI (RseI) 47 RseI (MslI) 142 MslI, SmiMIMsp20I TGG↓CCA FastDigest® MscI (MlsI) 47 MlsI (MscI) 128 BalI, MluNI, MscIMspA1I CMG↓CKGMspCI C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AlfII) 101 AflII, BfrI, Bst98I, BstAFI, Vha464I

MspI C↓CGGFastDigest® HpaIIm, FastDigest® MspI

4248

HpaIIm, MspI (HpaII)

122129

BsiSI, HapIIm

MspR9I CC↓NGG FastDigest® ScrFI (Bme1390I) 61 Bme1390I (ScrFI) 91 BmrFI, [BssKI], [BstSCI], ScrFI, [StyD4I]

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MssI GTTT↓AAAC FastDigest® MssI 48 MssI (PmeI) 130 PmeIm

MunI C↓AATTG FastDigest® MfeI (MunI) 45 MunI (MfeI) 130 MfeIMva1269I GAATGC(1/-1)↓ FastDigest® Mva1269I 49 Mva1269I (BsmI) 131 BsaMI, BsmI, PctI

MvaI CC↓WGG FastDigest® MvaI 48[EcoRIIm](↓CCWGG), MvaI (BstNI)

116130

[AjnI], BseBI, Bst2UI, BstNI, BstOI, [Psp6I], [PspGIm]

MvnI CG↓CG FastDigest® Bsh1236I 25 Bsh1236I (BstUI) 97 AccII, BspFNI, BstFNI, BstUIMvrI CGAT↓CG FastDigest® PvuI 57 PvuI 141 BpvUI, Ple19IMwoI GCNNNNN↓NNGC FastDigest® HpyF10VIm 124 HpyF10VI (MwoI)m 124 BstMWINaeI GCC↓GGC FastDigest® NaeI (PdiI) 49 PdiI (NaeI) 136 [MroNI], NaeI, [NgoMIV]

NarI GG↓CGCC [FastDigest® EheI](GGC↓GCC) 38[EheI (SfoI)](GGC↓GCC), [SspDI (KasI)](G↓GCGCC)

117150

[BbeI], [DinI], [EgeI], [KasI], Mly113I, [SfoIm]

NciI CC↓SGG FastDigest® NciI (BcnI) 49 BcnI (NciI) 88 AsuC2I, BpuMI, NciINcoI C↓CATGG FastDigest® NcoI 50 NcoI 131 Bsp19INdeI CA↓TATG FastDigest® NdeI 50 NdeI 132 FauNDI

NdeII ↓GATCFastDigest® Sau3AI (Bsp143I)m, FastDigest® MboIm

6044

Bsp143I (Sau3AI)m, MboIm

99127

BfuCIm, BssMI, [BstKTIm], BstMBI, DpnII, Kzo9Im, Sau3AIm

NgoMIV G↓CCGGC [FastDigest® NaeI (PdiI)](GCC↓GGC) 49 [PdiI (NaeI)](GCC↓GGC) 136 MroNI, [NaeI]

NheI G↓CTAGCFastDigest® NheI, [FastDigest® BmtI (BspOI)](GCTAG↓C)

5022

NheI, [BspOI (BmtI)](GCTAG↓C)

132100

AsuNHI, [BmtI]m

NlaIII CATG↓ FastDigest® NlaIII (Hin1II) 51 Hin1II (NlaIII) 120 [CviAII], FaeI, [FatIm], Hsp92II, NlaIIINlaIV GGN↓NCC FastDigest® NlaIV (BspLI)m 51 BspLI (NlaIV)m 100 BmiI, NlaIV, PspN4INmeAIII GCCGAG(21/19)↓NmuCI ↓GTSAC FastDigest® NmuCI 51 NmuCI (Tsp45I) 133 Tsp45INotI GC↓GGCCGC FastDigest® NotI 52 NotI 133 CciNINruI TCG↓CGA FastDigest® NruI (RruI) 52 Bsp68I (NruI) 98 BtuMI, NruINsbI TGC↓GCA FastDigest® FspI (NsbI) 39 NsbI (FspI) 133 Acc16I, AviII, FspINsiI ATGCA↓T FastDigest® NsiI (Mph1103I) 52 Mph1103I (NsiI) 129 EcoT22Im, NsiI, Zsp2INspI RCATG↓Y FastDigest® NspI (XceI) 53 XceI (NspI) 156 BstNSI, NspI

NspV TT↓CGAA FastDigest® Bsp119I 27 Bsp119I (BstBI) 99AsuIIm, Bpu14I, BspT104I, BstBIm, Csp45Im, SfuIm

OliI CACNN↓NNGTG FastDigest® AleI (OliI) 14 OliI (AleI) 134 AleIPacI TTAAT↓TAA FastDigest® PacI 53 PacI 134PaeI GCATG↓C FastDigest® SphI (PaeI) 64 PaeI (SphI) 134 BbuI, SphIPaeR7I C↓TCGAG FastDigest® XhoI 69 XhoI 156 Sfr274I, SlaI, StrI, TliIPagI T↓CATGA FastDigest® BspHI (PagI) 29 PagI (BspHI) 135 CciI, BspHIm, RcaIPalAI GG↓CGCGCC FastDigest® AscI (SgsI) 16 SgsI (AscI) 147 AscIPasI CC↓CWGGG PasI 135PauI G↓CGCGC FastDigest® BssHII (PteI) 31 PauI (BssHII) 135 BsePI, BssHIIPceI AGG↓CCT FastDigest® StuI (Eco147I) 64 Eco147I (StuI) 115 AatI, SseBI, StuIPciI A↓CATGT PscI (PciI) 138PciSI GCTCTTC(1/4)↓ FastDigest® SapI (LguI) 59 LguI (SapI) 125 BspQI, SapIPctI GAATGC(1/-1)↓ FastDigest® Mva1269I 49 Mva1269I (BsmI) 131 BsaMI, BsmIPdiI GCC↓GGC FastDigest® NaeI (PdiI) 49 PdiI (NaeI) 136 [MroNI], NaeI, [NgoMIV]PdmI GAANN↓NNTTC FastDigest® PdmI 53 PdmI (XmnI) 136 Asp700I, MroXI, XmnIm

PfeI G↓AWTC FastDigest® TfiI (PfeI) 67 PfeI (TfiI) 136 TfiIPfl23II G↓GTACG FastDigest® BsiWI (Pfl23II) 26 Pfl23II (BsiWI) 137 BsiWI, PspLIPflFI GACN↓NNGTC FastDigest® PsyI 57 PsyI (Tth111I) 140 AspI, Tth111IPflMI CCANNNN↓NTGG FastDigest® PflMI (Van91I) 54 Van91I (PflMI) 154 AccB7I, BasI, PflMIPfoI T↓CCNGGA FastDigest® PfoI 54 PfoI 137PhoI GG↓CC FastDigest® HaeIII (BsuRI) 40 BsuRI (HaeIII) 103 BshFI, BsnI, BspANI, HaeIIIPinAI A↓CCGGT FastDigest® AgeI (BshTI) 13 BshTI (AgeI) 101 AgeI, AsiGI, CspAIPle19I CGAT↓CG FastDigest® PvuI 57 PvuI 141 BpvUI, MvrIPleI GAGTC(4/5)↓ [FastDigest® MlyI (SchI)](GAGTC(5/5)↓) 46 [SchI (MlyI)](GAGTC(5/5)↓) 144 [MlyIm], PpsIPmaCI CAC↓GTG FastDigest® PmlI (Eco72I) 54 Eco72I (PmlI) 113 AcvI, BbrPI, PmlI, PspCIPmeI GTTT↓AAAC FastDigest® MssIm 48 MssI (PmeI)m 130PmlI CAC↓GTG FastDigest® PmlI (Eco72I) 54 Eco72I (PmlI) 113 AcvI, BbrPI, PmaCI, PmlI, PspCIPpiI ↓(7/12)GAAC(N)5CTC(13/8)↓ PpiI 138PpsI GAGTC(4/5)↓ [FastDigest® MlyI (SchI)](GAGTC(5/5)↓) 46 [SchI (MlyI)](GAGTC(5/5)↓) 144 [MlyI], PleIPpu21I YAC↓GTR FastDigest® BsaAI (Ppu21I) 23 Ppu21I (BsaAI) 138 BsaAI, BstBAIPpuMI RG↓GWCCY FastDigest® PpuMI (Psp5II) 55 Psp5II (PpuMI) 139 PpuMI, PspPPIPscI A↓CATGT PscI (PciI) 138 PciIPshAI GACNN↓NNGTC FastDigest® PshAI (BoxI) 55 BoxI (PshAI) 91 BstPAI, PshAIPshBI AT↓TAAT FastDigest® AseI (VspI) 17 VspI (AseI) 154 AseIPsiI TTA↓TAA FastDigest® PsiI (AanI) 55 AanI (PsiI) 80 PsiI

Psp124BI GAGCT↓C[FastDigest® Ecl136II](GAG↓CTC), FastDigest® SacI

3558

[Ecl136II (EcoICRI)](GAG↓CTC), SacI

109142

[Eco53kI], [EcoICRI], SstI

Psp1406I AA↓CGTT FastDigest® AclI (Psp1406I) 12 Psp1406I (AclI) 139 AclI

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Psp5II RG↓GWCCY FastDigest® PpuMI (Psp5II) 55 Psp5II (PpuMI) 139 PpuMI, PspPPI

Psp6I ↓CCWGG [FastDigest® MvaI](CC↓WGG) 48EcoRII, [MvaI (BstNI)](CC↓WGG)

116130

AjnI, [BseBI], [Bst2UI], [BstNI], [BstOI], PspGI

PspCI CAC↓GTG FastDigest® PmlI (Eco72I) 54 Eco72I (PmlI) 113 AcvI, BbrPI, PmaCI, PmlIPspEI G↓GTNACC FastDigest® Eco91I 36 Eco91I (BstEII) 114 BstEII, BstPI, EcoO65IPspFI CCCAGC(-1/-5)↓ FastDigest® PspFI 56 GsaI, [BseYI]

PspGI ↓CCWGG [FastDigest® MvaIm](CC↓WGG) 130EcoRII, [MvaI (BstNI)m](CC↓WGG)

116130

AjnIm, [BseBI], [Bst2UIm], [BstNIm], [BstOIm], Psp6I

PspLI C↓GTACG FastDigest® BsiWI (Pfl23II) 26 Pfl23II (BsiWI) 137 BsiWIPspN4I GGN↓NCC FastDigest® NlaIV (BspLI) 51 BspLI (NlaIV) 100 BmiI, NlaIV

PspOMI G↓GGCCC[FastDigest® ApaI](GGGCC↓C), FastDigest® Bsp120I

1628

[ApaI](GGGCC↓C), Bsp120I (PspOMI)

8599

PspPI G↓GNCC FastDigest® Sau96I (Cfr13I) 60 Cfr13I (Sau96I) 106 AspS9I, [BmgT120I], Sau96IPspPPI RG↓GWCCY FastDigest® PpuMI (Psp5II) 55 Psp5II (PpuMI) 139 PpuMIPspXI VC↓TCGAGBPsrI ↓(7/12)GAAC(N)6TAC(12/7)↓PstI CTGCA↓G FastDigest® PstI 56 PstI 140 BspMAIPsuI R↓GATCY FastDigest® PsuI 56 PsuI (BstYI) 140 BstX2I, BstYI, MflIm, XhoIIPsyI GACN↓NNGTC FastDigest® PsyI 57 PsyI (Tth111I) 140 AspI, PflFI, Tth111IPvuI CGAT↓CG FastDigest® PvuI 57 PvuI 141 BpvUI, MvrI, Ple19IPvuII CAG↓CTG FastDigest® PvuII 57 PvuII 141RcaI T↓CATGA FastDigest® BspHI (PagI) 29 PagI (BspHI) 135 BspHI, CciIRgaI GCGAT↓CGC FastDigest® AsiSI (SfaAI) 17 SfaAI (AsiSI) 145 AsiSI, SgfIRigI GGCCGG↓CC FseI

RsaI GT↓AC[FastDigest® Csp6Im](G↓TAC), FastDigest® RsaI

3258

[Csp6I (CviQI)m](G↓TAC), RsaI

107141

AfaI, [CviQIm], [RsaNI]

RsaNI G↓TACFastDigest® Csp6I, [FastDigest® RsaI](GT↓AC)

3258

Csp6I (CviQI), [RsaI](GT↓AC)

107141

[AfaI], CviQI

RseI CAYNN↓NNRTG FastDigest® MslI (RseI) 47 RseI (MslI) 142 MslI, SmiMIRsr2I CG↓GWCCG FastDigest® RsrII (CpoI) 58 CpoI (RsrII) 106 CspI, RsrIIRsrII CG↓GWCCG FastDigest® RsrII (CpoI) 58 CpoI (RsrII) 106 CspI, Rsr2I, RsrII

SacI GAGCT↓C[FastDigest® Ecl136IIm](GAG↓CTC), FastDigest® SacI

3558

[Ecl136II (EcoICRI)m](GAG↓CTC), SacI

109142

[Eco53kIm], [EcoICRI], Psp124BI, SstI

SacII CCGC↓GG Cfr42I (SacII) 106 KspI, Sfr303I, SgrBI, SstIISalI G↓TCGAC FastDigest® SalI 59 SalI 143SanDI GG↓GWCCC FastDigest® SanDI (KflI) 59 SanDISapI GCTCTTC(1/4)↓ FastDigest® SapI (LguI) 59 LguI (SapI) 125 BspQI, PciSI, SapISatI GC↓NGC FastDigest® Fnu4HI (SatI) 38 SatI (Fnu4HI) 143 Fnu4HI, Fsp4HI, ItaIm

Sau3AI ↓GATCFastDigest® Sau3AI (Bsp143I), FastDigest® MboIm

6044


99127

BfuCIm, BssMI, [BstKTIm], BstMBI, DpnIIm, Kzo9I, NdeIIm

Sau96I G↓GNCC FastDigest® Sau96I (Cfr13I) 60 Cfr13I (Sau96I) 106 AspS9I, [BmgT120I], PspPI, Sau96ISbfI CCTGCA↓GG FastDigest® SbfI (SdaI) 60 SdaI (SbfI) 144 SbfI, Sse8387IScaI AGT↓ACT FastDigest® ScaI 61 ScaI 144 AssI, BmcAI, ZrmISchI GAGTC(5/5)↓ FastDigest® MlyI (SchI) 46 SchI (MlyI) 144 MlyI, [PleI], [PpsI], SchI

ScrFI CC↓NGG FastDigest® ScrFI (Bme1390I) 61 Bme1390I (ScrFI) 91BmrFI, [BssKI], [BstSCI], MspR9I, ScrFI, [StyD4I]

SdaI CCTGCA↓GG FastDigest® SbfI (SdaI) 60 SdaI (SbfI) 144 SbfI, Sse8387ISduI GDGCH↓C FastDigest® Bsp1286I (SduI) 28 SduI (Bsp1286I) 145 Bsp1286I, MhlISetI ASST↓SexAI A↓CCWGGT FastDigest® SexAI (CsiI)m 61 MabI m, SexAISfaAI GCGAT↓CGC FastDigest® AsiSI (SfaAI) 17 SfaAI (AsiSI) 145 AsiSI, RgaI, SgfISfaNI GCATC(5/9)↓ FastDigest® SfaNI (BmsI) 62 LweI (SfaNI) 126SfcI C↓TRYAG FastDigest® SfcI (BfmI) 62 BfmI (SfcI) 89 BstSFI, SfcISfiI GGCCNNNN↓NGGCC FastDigest® SfiI 62 SfiI 146

SfoI GGC↓GCC FastDigest® EheIm 38EheI (SfoI)m, [SspDI (KasI)](G↓GCGCC)

117150

[BbeIm], DinI, EgeI, [KasIm], [Mly113I], [NarIm]

Sfr274I C↓TCGAG FastDigest® XhoI 69 XhoI 156 PaeR7I, SlaI, StrI, TliISfr303I CCGC↓GG Cfr42I (SacII) 106 KspI, SacII, SgrBI, SstIISgeI m5CNNG(9/13)↓ SgeI 146

SfuI TT↓CGAA FastDigest® Bsp119Im 27 Bsp119I (BstBI)m 99AsuII, Bpu14I, BspT104Im, BstBIm, Csp45I, NspVm

SgfI GCGAT↓CGC FastDigest® AsiSI (SfaAI) 17 SfaAI (AsiSI) 145 AsiSI, RgaISgrAI CR↓CCGGYGSgrBI CCGC↓GG Cfr42I (SacII) 106 KspI, SacII, Sfr303I, SstIISgrDI CG↓TCGACG SgrDI 147SgsI GG↓CGCGCC FastDigest® AscI (SgsI) 16 SgsI (AscI) 147 AscI, PalAISinI G↓GWCC FastDigest® AvaII (Eco47I) 18 Eco47I (AvaII) 111 AvaII, Bme18I, VpaK11BISlaI C↓TCGAG FastDigest® XhoI 69 XhoI 156 PaeR7I, Sfr274I, StrI, TliI

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SmaI CCC↓GGG FastDigest® SmaI 63[Cfr9I (XmaI)m](C↓CCGGG), SmaI

105147

[TspMI], [XmaCI], [XmaIm]

SmiI ATTT↓AAAT FastDigest® SwaI (SmiI) 65 SmiI (SwaI) 148 SwaISmiMI CAYNN↓NNRTG FastDigest® MslI (RseI) 47 RseI (MslI) 142 MslISmlI C↓TYRAG SmoI (SmlI) 148SmoI C↓TYRAG SmoI (SmlI) 148 SmlISmuI CCCGC(4/6)↓ SmuI (FauI) 148 FauISnaBI TAC↓GTA FastDigest® SnaBI (Eco105I) 63 Eco105I (SnaBI) 114 BstSNI, SnaBISpeI A↓CTAGT FastDigest® SpeI (BcuI) 63 BcuI (SpeI) 88 AhlI, SpeISphI GCATG↓C FastDigest® SphI (PaeI) 64 PaeI (SphI) 134 BbuI, SphISrfI GCCC↓GGGCSse8387I CCTGCA↓GG FastDigest® SbfI (SdaI) 60 SdaI (SbfI) 144 SbfISse9I ↓AATT FastDigest® Tsp509I (TasI) 68 TasI (Tsp509I) 151 Tsp509I, TspEISseBI AGG↓CCT FastDigest® StuI (Eco147I) 64 Eco147I (StuI) 115 AatI, PceI, StuISsiI CCGC(-3/-1)↓ FastDigest® AciI (SsiI) 12 SsiI (AciI) 149 AciI, BspACI

SspDI G↓GCGCC [FastDigest® EheI](GGC↓GCC) 38[EheI (SfoI)](GGC↓GCC), SspDI (KasI)

117150

[BbeI], [DinI], [EgeI], KasI, [Mly113I], [NarI], [SfoI]

SspI AAT↓ATT FastDigest® SspI 64 SspI 149

SstI GAGCT↓C[FastDigest® Ecl136IIm](GAG↓CTC), FastDigest® SacI

3558

[Ecl136II (EcoICRI)m](GAG↓CTC), SacI

109142

[Eco53kI], [EcoICRI], Psp124BI

SstII CCGC↓GG Cfr42I (SacII) 106 KspI, SacII, Sfr303I, SgrBIStrI C↓TCGAG FastDigest® XhoI 69 XhoI 156 PaeR7I, Sfr274I, SlaI, TliIStuI AGG↓CCT FastDigest® StuI (Eco147I) 64 Eco147I (StuI) 115 AatI, PceI, SseBI, StuIStyD4I ↓CCNGG [FastDigest® ScrFI (Bme1390I)](CC↓NGG) 61 [Bme1390I (ScrFI)](CC↓NGG) 91 [BmrFI], BssKI, BstSCI, [MspR9I], [ScrFI]StyI C↓CWWGG FastDigest® StyI (Eco130I) 65 Eco130I (StyI) 114 BssT1I, EcoT14I, ErhI, StyISwaI ATTT↓AAAT FastDigest® SwaI (SmiI) 65 SmiI (SwaI) 148 SwaITaaI ACN↓GT FastDigest® TaaI 150 TaaI (HpyCH4III) 150 Bst4CI, HpyCH4IIITaiI ACGT↓ FastDigest® TaiI 66 TaiI (MaeII) 150 [HpyCH4IVm], [MaeII]TaqI T↓CGA FastDigest® TaqI 66 TaqI 151

TaqIIGACCGA(11/9)↓,CACCCA(11/9)↓

TasI ↓AATT FastDigest® Tsp509I (TasI) 68 TasI (Tsp509I) 151 Sse9I, Tsp509I, TspEITatI W↓GTACW FastDigest® TatI 66 TatI 151TauI GCSG↓C FastDigest® TauI 67 TauI 152TfiI G↓AWTC FastDigest® TfiI (PfeI) 67 PfeI (TfiI) 136 TfiITliI C↓TCGAG FastDigest® XhoI 69 XhoI 156 PaeR7I, Sfr274I, SlaI, StrI

Tru1I T↓TAAFastDigest® MseI (SaqAI), FastDigest® Tru1I

4767

Tru1I (MseI) 152 MseI, Tru9I

Tru9I T↓TAAFastDigest® MseI (SaqAI), FastDigest® Tru1I

4767

Tru1I (MseI) 152 MseI

TscAI CASTG(2/-7)↓ FastDigest® TspRI (TscAI) 68 TscAI (TspRI) 153 TspRITseI G↓CWGC ApeKITsoI TARCCA(11/9)↓ TsoI 153Tsp45I ↓GTSAC FastDigest® NmuCI 51 NmuCI (Tsp45I) 133Tsp509I ↓AATT FastDigest® Tsp509I (TasI) 68 TasI (Tsp509I) 151 Sse9I, Tsp509I, TspEITspDTI ATGAA(11/9)↓TspEI ↓AATT FastDigest® Tsp509I (TasI) 68 TasI (Tsp509I) 151 Sse9I, Tsp509ITspGWI ACGGA(11/9)↓

TspMI C↓CCGGG [FastDigest® SmaI](CCC↓GGG) 63Cfr9I (XmaI)m, [SmaI](CCC↓GGG)

105147

XmaCI, XmaIm

TspRI CASTGNN(2/-7)↓ FastDigest® TspRI (TscAI) 68 TscAI (TspRI) 153 TspRITstI ↓(8/13)CAC(N)6TCC(12/7)↓ TstI 154Tth111I GACN↓NNGTC FastDigest® PsyI 57 PsyI (Tth111I) 140 AspI, PflFIVan91I CCANNNN↓NTGG FastDigest® PflMI (Van91I) 54 Van91I (PflMI) 154 AccB7I, BasI, PflMIVha464I C↓TTAAG FastDigest® AflII (BspTI) 13 BspTI (AflII) 101 AflII, BfrI, Bst98I, BstAFI, MspCIVneI G↓TGCAC FastDigest® ApaLI (Alw44I) 16 Alw44I (ApaLI) 85 ApaLIVpaK11BI G↓GWCC FastDigest® AvaII (Eco47I) 18 Eco47I (AvaII) 111 AvaII, Bme18I, SinIVspI AT↓TAAT FastDigest® AseI (VspI) 17 VspI (AseI) 154 AseI, PshBIXagI CCTNN↓NNNAGG FastDigest® EcoNI (XagI) 36 XagI (EcoNI) 155 BstENI, EcoNIXapI R↓AATTY FastDigest® XapI 68 XapI (ApoI) 155 AcsI, ApoIXbaI T↓CTAGA FastDigest® XbaI 69 XbaI 155XceI RCATG↓Y FastDigest® NspI (XceI) 53 XceI (NspI) 156 BstNSI, NspIXcmI CCANNNNN↓NNNNTGGXhoI C↓TCGAG FastDigest® XhoI 69 XhoI 156 PaeR7I, Sfr274I, SlaI, StrI, TliIXhoII R↓GATCY FastDigest® PsuI 56 PsuI (BstYI) 140 BstX2I, BstYI, MflIm,

XmaCI C↓CCGGG [FastDigest® SmaI](CCC↓GGG) 63Cfr9I (XmaI), [SmaI](CCC↓GGG)

105147

TspMI, XmaI

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XmaI C↓CCGGG [FastDigest® SmaIm](CCC↓GGG) 63Cfr9I (XmaI)m, [SmaIm](CCC↓GGG)

105147

TspMIm, XmaCI

XmaJI C↓CTAGG FastDigest® AvrII (XmaJI) 18 XmaJI (AvrII) 156 AspA2I, AvrII, BlnIXmiI GT↓MKAC FastDigest® AccI (XmiI) 11 XmiI (AccI) 157 AccI, FblIXmnI GAANN↓NNTTC FastDigest® PdmIm 53 PdmI (XmnI)m 136 Asp700I, MroXIXspI C↓TAG FastDigest® BfaI (FspBI) 20 FspBI (BfaI) 119 BfaI, MaeIZraI GAC↓GTC [FastDigest® AatIIm](GACGT↓C) 11 [AatIIm](GACGT↓C) 81ZrmI AGT↓ACT FastDigest® ScaI 144 ScaI 144 AssI, BmcAIZsp2I ATGCA↓T FastDigest® NsiI (Mph1103I) 52 Mph1103I (NsiI) 129 EcoT22I, NsiI

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Table 1.26. Recognition sequences of restriction enzymes.

Alphabetic List of Recognition Sequences



FastDigest® enzyme

Conventional enzyme

AA↓CGTT FastDigest® AclI (Psp1406I) Psp1406I (AclI)A↓AGCTT FastDigest® HindIII HindIII↓(8/13)AAG(N)5CTT(13/8)↓ FalIAAT↓ATT FastDigest® SspI SspI↓AATT FastDigest® Tsp509I (TasI) TasI (Tsp509I)A↓CATGT PscI (PciI)A↓CCGGT FastDigest® AgeI (BshTI) BshTI (AgeI)ACCTGC(4/8)↓* FastDigest® BspMI (BveI) BveI (BspMI)A↓CCWGGT FastDigest® SexAI (CsiI) SexAIA↓CGCGT FastDigest® MluI MluIACGGA(11/9)↓* TspGWIACGGC(12/14)↓* BceAIACGT↓ FastDigest® TaiI TaiI (MaeII)A↓CGT MaeIIACN↓GT FastDigest® TaaI TaaI (HpyCH4III)↓(10/15)AC(N)4GTAYC(12/7)↓* BaeI↓(9/12)AC(N)5CTCC(10/7)↓* BsaXIA↓CRYGT AflIIIA↓CTAGT FastDigest® SpeI (BcuI) BcuI (SpeI)ACTGG(1/-1)↓* FastDigest® BseNI BseNI (BsrI)ACTGGG(5/4)↓* BfiI (BmrI)A↓GATCT FastDigest® BglII BglIIAGC↓GCT FastDigest® AfeI (Eco47III) Eco47III (AfeI)AG↓CT FastDigest® AluI AluIAGG↓CCT FastDigest® StuI (Eco147I) Eco147I (StuI)AGT↓ACT FastDigest® ScaI ScaIASST↓ SetIAT↓CGAT FastDigest® ClaI (Bsu15I) Bsu15I (ClaI)ATGAA(11/9)↓* TspDTIATGCA↓T FastDigest® NsiI (Mph1103I) Mph1103I (NsiI)AT↓TAAT FastDigest® AseI (VspI) VspI (AseI)ATTT↓AAAT FastDigest® SwaI (SmiI) SmiI (SwaI)C↓(11/13)CAA(N)5GTGG(12/10)↓* CspCIC↓AATTG FastDigest® MfeI (MunI) MunI (MfeI)CACCCA(11/9)↓* TaqIICACCTGC(4/8)↓* AarICACGAG(-5/-1)↓* BauI (BssSI)CACGTC(-3/-3)↓* AjiI (BmgBI)CAC↓GTG FastDigest® PmlI (Eco72I) Eco72I (PmlI)CACNNN↓GTG FastDigest® DraIII (AdeI) AdeI (DraIII)CACNN↓NNGTG FastDigest® AleI (OliI) OliI (AleI)↓(8/13)CAC(N)6TCC(12/7)↓* TstI↓(0/2)CACTGC* BtsICAGCAG(25/27)↓* EcoP15ICAG↓CTG FastDigest® PvuII PvuIICAGNNN↓CTG FastDigest® AlwNI (CaiI) CaiI (AlwNI)CASTG(2/-7)↓ FastDigest® TspRI (TscAI) TscAI (TspRI)CA↓TATG FastDigest® NdeI NdeI↓(13/9)CATCC* FastDigest® FokI FokI↓(0/2)CATCC* FastDigest® BseGI BseGI (BtsCI)↓(14/10)CATCGC* BtgZICATG↓ FastDigest® NlaIII (Hin1II) Hin1II (NlaIII)↓CATG FatIC↓ATG CviAII↓(0/2)CATTGC* FastDigest® BsrDI (BseMI) BseMI (BsrDI)CAYNN↓NNRTG FastDigest® MslI (RseI) RseI (MslI)↓(6/11)CCAA(N)7TTC(12/7)↓* FastDigest® AjuI AjuI↓(10/12)CCAC(N)5TTG(13/11)↓* CspCI


FastDigest® enzyme

Conventional enzyme

↓(-1/1)CCAGT* FastDigest® BseNI BseNI (BsrI)CCANNNNN↓NNNNTGG XcmICCANNNNN↓NTGG FastDigest® BstXI BstXICCANNNN↓NTGG FastDigest® PflMI (Van91I) Van91I (PflMI)CCATC(4/5)↓* BccIC↓CATGG FastDigest® NcoI NcoICCCAGC(-5/-1)↓* BseYICCCAGC(-1/-5)↓* FastDigest® PspFI GsaI↓(4/5)CCCAGT* BfiI (BmrI)CCCGC(4/6)↓* SmuI (FauI)C↓CCGGG Cfr9I (XmaI)CCC↓GGG FastDigest® SmaI SmaICC↓CWGGG PasICCGC(-3/-1)↓* FastDigest® AciI (SsiI) SsiI (AciI)CCGC↓GG Cfr42I (SacII)CCGCTC(-3/-3)↓* FastDigest® BsrBI (MbiI) MbiI (BsrBI)

C↓CGGFastDigest® HpaIIFastDigest® MspI

HpaIIMspI (HpaII)

↓CCNGG StyD4ICC↓NGG FastDigest® ScrFI (Bme1390I) Bme1390I (ScrFI)C↓CNNGG FastDigest® BsaJI (BseDI) BseDI (BsaJI)CCNNNNN↓NNGG FastDigest® BslI (BseLI) BseLI (BslI)C↓CRYGG BtgICC↓SGG FastDigest® NciI (BcnI) BcnI (NciI)C↓CTAGG FastDigest® AvrII (XmaJI) XmaJI (AvrII)CCTC(7/6)↓* FastDigest® MnlI MnlICCTCAGC(-5/-2)↓* BbvCICC↓TCGAGG AbsICCTGCA↓GG FastDigest® SbfI (SdaI) SdaI (SbfI)CCTNAGC(-5/-2)↓* FastDigest® Bpu10I Bpu10ICC↓TNAGG FastDigest® Bsu36I (Eco81I) Eco81I (Bsu36I)CCTNN↓NNNAGG FastDigest® EcoNI (XagI) XagI (EcoNI)CCTTC(6/5)↓* HpyAV↓CCWGG EcoRIICC↓WGG FastDigest® MvaI MvaI (BstNI)C↓CWWGG FastDigest® StyI (Eco130I) Eco130I (StyI)↓(10/12)CGA(N)6TGC(12/10)↓* BcgICGAT↓CG FastDigest® PvuI PvuICG↓CCGGCG FastDigest® MreI MreI (Sse232I)CG↓CG FastDigest® Bsh1236I Bsh1236I (BstUI)CG↓CGCGCG FastDigest® MauBI MauBIC↓GGCCG FastDigest® EagI (Eco52I) Eco52I (EagI)CG↓GWCCG FastDigest® RsrII (CpoI) CpoI (RsrII)CGRY↓CG FastDigest® BsiEI (Bsh1285I) Bsh1285I (BsiEI)C↓GTACG FastDigest® BsiWI (Pfl23II) Pfl23II (BsiWI)CG↓TCGACG SgrDICGTCTC(1/5)↓* FastDigest® BsmBI (Esp3I) Esp3I (BsmBI)CGWCG↓ Hpy99ICMG↓CKG MspA1Im5CNNG(9/13)↓ SgeI↓(6/11)CRAA(N)6GTC(13/18)↓* ArsICR↓CCGGYG SgrAIC↓TAG FastDigest® BfaI (FspBI) FspBI (BfaI)↓(14/16)CTCAAG* BpuEICTCAG(9/7)↓* BspCNICTCAG(10/8)↓* FastDigest® BspCNI (BseMII) BseMII (BspCNI)↓(14/16)CTCCAG* FastDigest® BpmI (GsuI) GsuI (BpmI)↓(8/10)CTCCTC* BseRIC↓TCGAG FastDigest® XhoI XhoI

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FastDigest® enzyme

Conventional enzyme

↓(19/21)CTCGGC* NmeAIIICTCGTG(-5/-1)↓* BauI (BssSI)CTCTTC(1/4)↓* FastDigest® EarI (Eam1104I) Eam1104I (EarI)CTGAAG(16/14)↓* FastDigest® AcuI (Eco57I) Eco57I (AcuI)↓(7/9)CTGAG* BspCNI↓(8/10)CTGAG* FastDigest® BspCNI (BseMII) BseMII (BspCNI)↓(14/16)CTGCAC* BsgICTGCA↓G FastDigest® PstI PstI↓(27/25)CTGCTG* EcoP15ICTGGAG(16/14)↓* FastDigest® BpmI (GsuI) GsuI (BpmI)CTGRAG(16/14)↓* Eco57MIC↓TNAG FastDigest® DdeI (HpyF3I) HpyF3I (DdeI)C↓TRYAG FastDigest® SfcI (BfmI) BfmI (SfcI)C↓TTAAG FastDigest® AflII (BspTI) BspTI (AflII)↓(14/16)CTTCAG* FastDigest® AcuI (Eco57I) Eco57I (AcuI)CTTGAG(16/14)↓* BpuEI↓(14/16)CTYCAG* Eco57MIC↓TYRAG SmoI (SmlI)C↓YCGRG FastDigest® AvaI (Eco88I) Eco88I (AvaI)CY↓CGRG BmeT110IG↓(7/12)GAAC(N)5CTC(13/8)↓* PpiI↓(7/12)GAAC(N)6TAC(12/7)↓* PsrI↓(7/12-13)GAAC(N)6TCC(12-13/7)↓* AloIGAAGA(8/7)↓* FastDigest® MboII MboIIGAAGAC(2/6)↓* FastDigest® BbsI (BpiI) BpiI (BbsI)↓(4/1)GAAGAG* FastDigest® EarI (Eam1104I) Eam1104I (EarI)↓(4/1)GAAGAGC* FastDigest® SapI (LguI) LguI (SapI)↓(5/6)GAAGG* HpyAV↓(7/12)GAAG(N)6TAC(12/7)↓* BarI↓(7/12)GAA(N)7TTGG(11/6)↓* FastDigest® AjuI AjuIGAANN↓NNTTC FastDigest® PdmI PdmI (XmnI)GAATGC(1/-1)↓* FastDigest® Mva1269I Mva1269I (BsmI)G↓AATTC FastDigest® EcoRI EcoRI↓(8/13-14)GAB(N)5RTC(13-14/8)↓* Hin4IGACCGA(11/9)↓* TaqIIGACGC(5/10)↓* FastDigest® HgaI (CseI) CseI (HgaI)GAC↓GTC ZraIGACGT↓C FastDigest® AatII AatIIGACGTG(-3/-3)↓* AjiI (BmgBI)GACN↓NNGTC FastDigest® PsyI PsyI (Tth111I)GACNN↓NNGTC FastDigest® PshAI (BoxI) BoxI (PshAI)GACNNN↓NNGTC FastDigest® Eam1105I Eam1105I (AhdI)GACNNNN↓NNGTC FastDigest® DrdI (AasI) AasI (DrdI)↓(8/13)GAC(N)6TTYG(11/6)↓* ArsI↓(5/5)GACTC* FastDigest® MlyI (SchI) SchI (MlyI)↓(5/4)GACTC* PleI↓(5/1)GAGAC* FastDigest® Alw26I Alw26I (BsmAI)↓(5/1)GAGACC* FastDigest® Eco31I Eco31I (BsaI)↓(5/1)GAGACG* FastDigest® BsmBI (Esp3I) Esp3I (BsmBI)GAGCGG(-3/-3)↓* FastDigest® BsrBI (MbiI) MbiI (BsrBI)GAGCT↓C FastDigest® SacI SacIGAG↓CTC FastDigest® Ecl136II Ecl136II (EcoICRI)↓(6/7)GAGG* FastDigest® MnlI MnlIGAGGAG(10/8)↓* BseRI↓(8/13)GAG(N)5CTC(13/8)↓ FastDigest® BplI BplI↓(8/13)GAG(N)5GTTC(12/7)↓* PpiIGAGTC(5/5)↓* FastDigest® MlyI (SchI) SchI (MlyI)GAGTC(4/5)↓* PleIG↓ANTC FastDigest® HinfI HinfIGAT↓ATC FastDigest® EcoRV (Eco32I) Eco32I (EcoRV)


FastDigest® enzyme

Conventional enzyme

↓GATCFastDigest® Sau3AI (Bsp143I)FastDigest® MboI

Bsp143I (Sau3AI)MboI

Gm6A↓TC FastDigest® DpnI DpnIGAT↓C BstKTI↓(5/4)GATCC* BspPI (AlwI)↓(9/5)GATGC* FastDigest® SfaNI (BmsI) LweI (SfaNI)↓(5/4)GATGG* BccIGATNN↓NNATC FastDigest® BsaBI (BseJI) BseJI (BsaBI)G↓AWTC FastDigest® TfiI (PfeI) PfeI (TfiI)↓(8/13-14)GAY(N)5VTC(13-14/8)↓* Hin4IGCAATG(2/0)↓* FastDigest® BsrDI (BseMI) BseMI (BsrDI)

GCAGC(8/12)↓*FastDigest® BbvI (Lsp1109I)FastDigest® BseXI

Lsp1109I (BbvI)BseXI (BbvI)

↓(8/4)GCAGGT* FastDigest® BspMI (BveI) BveI (BspMI)↓(8/4)GCAGGTG* AarIGCAGTG(2/0)↓* BtsI↓(10/12)GCA(N)6TCG(12/10)↓* BcgI↓(10/12)GCA(N)6TGC(12/10)↓ AlfIGCANNNN↓NTGC BstAPIGCATC(5/9)↓* FastDigest® SfaNI (BmsI) LweI (SfaNI)GCATG↓C FastDigest® SphI (PaeI) PaeI (SphI)↓(-1/1)GCATTC* FastDigest® Mva1269I Mva1269I (BsmI)GCCC↓GGGC SrfIGCCGAG(21/19)↓* NmeAIIIG↓CCGGC NgoMIVGCC↓GGC FastDigest® NaeI (PdiI) PdiI (NaeI)↓(14/12)GCCGT* BceAIGCCNNNN↓NGGC FastDigest® BglI BglIGCGAT↓CGC FastDigest® AsiSI (SfaAI) SfaAI (AsiSI)GCGATG(10/14)↓* BtgZIG↓CGC FastDigest® HinP1I (Hin6I) Hin6I (HinP1I)GCG↓C FastDigest® HhaI HhaIGC↓CG GlaIG↓CGCGC FastDigest® BssHII (PteI) PauI (BssHII)GCGG(-3/-1)↓* FastDigest® AciI (SsiI) SsiI (AciI)GC↓GGCCGC FastDigest® NotI NotI↓(6/4)GCGGG* SmuI (FauI)↓(10/5)GCGTC* FastDigest® HgaI (CseI) CseI (HgaI)GC↓NGC FastDigest® Fnu4HI (SatI) SatI (Fnu4HI)GC↓NGC BisIGCN↓GC BlsIGCN↓NGC Cac8IGCNNNNN↓NNGC FastDigest® HpyF10VI HpyF10VI (MwoI)GCSG↓C FastDigest® TauI TauIGCTAG↓C FastDigest® BmtI (BspOI) BspOI (BmtI)G↓CTAGC FastDigest® NheI NheIGCTCTTC(1/4)↓* FastDigest® SapI (LguI) LguI (SapI)GCTGAGG(-5/-2)↓* BbvCI

↓(12/8)GCTGC*FastDigest® BbvI (Lsp1109I)FastDigest® BseXI


GCTGGG(-5/-1)↓* BseYIGCTGGG(-1/-5)↓* FastDigest® PspFI GsaIGC↓TNAGC FastDigest® BlpI (Bpu1102I) Bpu1102I (BlpI)GCTNAGG(-5/-2)↓* FastDigest® Bpu10I Bpu10IG↓CWGC TseIGDGCH↓C FastDigest® Bsp1286I (SduI) SduI (Bsp1286I)↓(7/10)GGAG(N)5GT(12/9)↓* BsaXI↓(7/12)GGA(N)6GTG(13/8)↓* TstI↓(7/12-13)GGA(N)6GTTC(12-13/7)↓* AloI↓(5/6)GGATAC* BfuI (BciVI)GGATC(4/5)↓* BspPI (AlwI)

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Note* Asymetric sequences.• Fermentas FastDigest® enzymes are shown in ruby.• Fermentas conventional enzymes are shown in orange.



FastDigest® enzyme

Conventional enzyme

G↓GATCC FastDigest® BamHI BamHIGGATG(9/13)↓* FastDigest® FokI FokIGGATG(2/0)↓* FastDigest® BseGI BseGI (BtsCI)GG↓CC FastDigest® HaeIII (BsuRI) BsuRI (HaeIII)GGCCGG↓CC FseIGGCCNNNN↓NGGCC FastDigest® SfiI SfiIGGC↓GCC FastDigest® EheI EheI (SfoI)GG↓CGCC NarIG↓GCGCC SspDI (KasI)GGCGC↓C BbeIGG↓CGCGCC FastDigest® AscI (SgsI) SgsI (AscI)GGCGGA(11/9)↓* EciIGGGAC(10/14)↓* FastDigest® BsmFI (FaqI) FaqI (BsmFI)G↓GGCCC FastDigest® Bsp120I Bsp120I (PspOMI)GGGCC↓C FastDigest® ApaI ApaIGG↓GWCCC FastDigest® SanDI (KflI) SanDIG↓GNCC FastDigest® Sau96I (Cfr13I) Cfr13I (Sau96I)GG↓NCC BmgT120IGGN↓NCC FastDigest® NlaIV (BspLI) BspLI (NlaIV)GGTAC↓C FastDigest® KpnI KpnIG↓GTACC FastDigest® Acc65I Acc65I (Asp718I)GGTCTC(1/5)↓* FastDigest® Eco31I Eco31I (BsaI)GGTGA(8/7)↓* HphIG↓GTNACC FastDigest® Eco91I Eco91I (BstEII)G↓GWCC FastDigest® AvaII (Eco47I) Eco47I (AvaII)G↓GYRCC FastDigest® BanI (BshNI) BshNI (BanI)GKGCM↓C FastDigest® Bme1580I (BseSI) BseSI (Bme1580I)GR↓CGYC FastDigest® BsaHI (Hin1I) Hin1I (BsaHI)GRGCY↓C Eco24I (BanII)↓(7/12)GRTAC(N)4GT(15/10)↓* BaeIGT↓AC FastDigest® RsaI RsaIG↓TAC FastDigest® Csp6I Csp6I (CviQI)↓(7/12)GTA(N)6CTTC(12/7)↓* BarI↓(7/12)GTA(N)6GTTC(12/7)↓* PsrIGTA↓TAC FastDigest® BstZ17I (Bst1107I) Bst1107I (BstZ17I)GTATCC(6/5)↓* BfuI (BciVI)↓(14/10)GTCCC* FastDigest® BsmFI (FaqI) FaqI (BsmFI)G↓TCGAC FastDigest® SalI SalIGTCTC(1/5)↓* FastDigest® Alw26I Alw26I (BsmAI)↓(6/2)GTCTTC* FastDigest® BbsI (BpiI) BpiI (BbsI)G↓TGCAC FastDigest® ApaLI (Alw44I) Alw44I (ApaLI)GTGCAG(16/14)↓* BsgIGT↓MKAC FastDigest® AccI (XmiI) XmiI (AccI)↓GTNAC MaeIIIGTN↓NAC FastDigest® Hpy8I Hpy8I (MjaIV)↓GTSAC FastDigest® NmuCI NmuCI (Tsp45I)GTT↓AAC FastDigest® HpaI (KspAI) KspAI (HpaI)GTTT↓AAAC FastDigest® MssI MssI (PmeI)↓(18/20)GTYGGA* MmeIGTY↓RAC FastDigest® HincII HincII (HindII)GWGCW↓C FastDigest® Alw21I Alw21I (BsiHKAI)RR↓AATTY FastDigest® XapI XapI (ApoI)RCATG↓Y FastDigest® NspI (XceI) XceI (NspI)R↓CCGGY FastDigest® BsrFI (Cfr10I) Cfr10I (BsrFI)R↓GATCY FastDigest® PsuI PsuI (BstYI)RGCGC↓Y FastDigest® HaeII (BfoI) HaeIIRG↓CY CviJIRG↓GNCCY FastDigest® EcoO109I EcoO109I (DraII)RG↓GWCCY FastDigest® PpuMI (Psp5II) Psp5II (PpuMI)RTGC↓GCAY FastDigest® FspAI FspAI


FastDigest® enzyme

Conventional enzyme

TTAC↓GTA FastDigest® SnaBI (Eco105I) Eco105I (SnaBI)TARCCA(11/9)↓* TsoI↓(7/8)TCACC* HphIT↓CATGA FastDigest® BspHI (PagI) PagI (BspHI)↓(9/11)TCCGCC* EciIT↓CCGGA FastDigest® Kpn2I Kpn2I (BspEI)↓(9/11)TCCGT* TspGWI↓(9/11)TCGGTC* TaqIIT↓CCNGGA FastDigest® PfoI PfoITCCRAC(20/18)↓* MmeIT↓CGA FastDigest® TaqI TaqITCG↓CGA FastDigest® NruI (RruI) Bsp68I (NruI)TCN↓GA Hpy188ITC↓NNGA Hpy188IIIT↓CTAGA FastDigest® XbaI XbaI↓(7/8)TCTTC* FastDigest® MboII MboII↓(10/12)TGA(N)6TCA(12/10)↓ BdaIT↓GATCA FastDigest® BclI BclITG↓CA HpyCH4VTGC↓GCA FastDigest® FspI (NsbI) NsbI (FspI)TGG↓CCA FastDigest® MscI (MlsI) MlsI (MscI)↓(9/11)TGGGTG* TaqII↓(9/11)TGGYTA* FastDigest® MscI (MlsI) TsoIT↓GTACA FastDigest® Bsp1407I Bsp1407I (BsrGI)

T↓TAAFastDigest® MseI (SaqAI)FastDigest® Tru1I

Tru1I (MseI)

TTAAT↓TAA FastDigest® PacI PacITTA↓TAA FastDigest® PsiI (AanI) AanI (PsiI)↓(9/11)TTCAT* TspDTITT↓CGAA FastDigest® Bsp119I Bsp119I (BstBI)TTS↓AA AgsITTT↓AAA FastDigest® DraI DraIVVC↓TCGAGB PspXIWW↓CCGGW BsaWIW↓GTACW FastDigest® TatI TatIYYAC↓GTR FastDigest® BsaAI (Ppu21I) Ppu21I (BsaAI)YA↓TR FaiIY↓GGCCR CfrI (EaeI)

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Enzyme Recognizing 2 bp Long TargetsTable 1.27. Enzyme recognizing 2 bp long targets.

Alphabetic List of Enzymes Recognizing 5 bp Long Targets




FastDigest® enzyme

Conventional enzyme

m5CNNG(9/13)↓ SgeI

Alphabetic List of Enzymes Recognizing 4 bp Long TargetsTable 1.28. Enzymes recognizing 4 bp long targets.Specificity 5’→3’

FastDigest® enzyme

Conventional enzyme

↓AATT FastDigest® Tsp509I (TasI) TasI (Tsp509I)ACGT↓ FastDigest® TaiI TaiI (MaeII)A↓CGT MaeIIACN↓GT FastDigest® TaaI TaaI (HpyCH4III)AG↓CT FastDigest® AluI AluIASST↓ SetICATG↓ FastDigest® NlaIII (Hin1II) Hin1II (NlaIII)↓CATG FatIC↓ATG CviAII CCGC(-3/-1)↓* FastDigest® AciI (SsiI) SsiI (AciI)


HpaIIMspI (HpaII)

↓CCNGG StyD4ICC↓NGG FastDigest® ScrFI (Bme1390I) Bme1390I (ScrFI)C↓CNNGG FastDigest® BsaJI (BseDI) BseDI (BsaJI)CCNNNNN↓NNGG FastDigest® BslI (BseLI) BseLI (BslI)CCTC(7/6)↓* FastDigest® MnlI MnlICG↓CG FastDigest® Bsh1236I Bsh1236I (BstUI)C↓TAG FastDigest® BfaI (FspBI) FspBI (BfaI)C↓TNAG FastDigest® DdeI (HpyF3I) HpyF3I (DdeI)↓(6/7)GAGG* FastDigest® MnlI MnlIG↓ANTC FastDigest® HinfI HinfI

↓GATCFastDigest® MboIFastDigest® Sau3AI (Bsp143I)

MboIBsp143I (Sau3AI)

Gm6A↓TC FastDigest® DpnI DpnIGAT↓C BstKTIG↓CGC FastDigest® HinP1I (Hin6I) Hin6I (HinP1I)GCG↓C FastDigest® HhaI HhaIGC↓GC GlaIGCGG(-3/-1)↓* FastDigest® AciI (SsiI) SsiI (AciI)GC↓NGC FastDigest® Fnu4HI (SatI) SatI (Fnu4HI)GC↓NGC BisIGCN↓GC BlsIGCN↓NGC Cac8IGCNNNNN↓NNGC FastDigest® HpyF10VI HpyF10VI (MwoI)GG↓CC FastDigest® HaeIII (BsuRI) BsuRI (HaeIII)G↓GNCC FastDigest® Sau96I (Cfr13I) Cfr13I (Sau96I)GG↓NCC BmgT120IGGN↓NCC FastDigest® NlaIV (BspLI) BspLI (NlaIV)GT↓AC FastDigest® RsaI RsaIG↓TAC FastDigest® Csp6I Csp6I (CviQI)↓GTNAC MaeIIIGTN↓NAC FastDigest® Hpy8I Hpy8I (MjaIV)RG↓CY CviJIT↓CGA FastDigest® TaqI TaqITCN↓GA Hpy188ITC↓NNGA Hpy188IIITG↓CA HpyCH4V


Tru1I (MseI)

YA↓TR FaiI


FastDigest® enzyme

Conventional enzyme

ACGGA(11/9)↓* TspGWIACGGC(12/14)↓* BceAIACTGG(1/-1)↓* FastDigest® BseNI BseNI (BsrI)ATGAA(11/9)↓* TspDTICASTG(2/-7)↓ FastDigest® TspRI (TscAI) TscAI (TspRI)↓(13/9)CATCC* FastDigest® FokI FokI↓(0/2)CATCC* FastDigest® BseGI BseGI (BtsCI)↓(-1/1)CCAGT* FastDigest® BseNI BseNI (BsrI)CCATC(4/5)↓* BccICCCGC(4/6)↓* SmuI (FauI)CC↓SGG FastDigest® NciI (BcnI) BcnI (NciI)CCTTC(6/5)↓* HpyAV↓CCWGG EcoRIICC↓WGG FastDigest® MvaI MvaI (BstNI)CGWCG↓ Hpy99ICTCAG(9/7)↓* BspCNICTCAG(10/8)↓* FastDigest® BspCNI (BseMII) BseMII (BspCNI)↓(7/9)CTGAG* BspCNI↓(8/10)CTGAG* FastDigest® BspCNI (BseMII) BseMII (BspCNI)GAAGA(8/7)↓* FastDigest® MboII MboII↓(5/6)GAAGG* HpyAVGACGC(5/10)↓* FastDigest® HgaI (CseI) CseI (HgaI)↓(5/5)GACTC* FastDigest® MlyI (SchI) SchI (MlyI)↓(5/4)GACTC* PleI↓(5/1)GAGAC* FastDigest® Alw26I Alw26I (BsmAI)GAGTC(5/5)↓* FastDigest® MlyI (SchI) SchI (MlyI)GAGTC(4/5)↓* PleI↓(5/4)GATCC* BspPI (AlwI)↓(9/5)GATGC* FastDigest® SfaNI (BmsI) LweI (SfaNI)↓(5/4)GATGG* BccIG↓AWTC FastDigest® TfiI (PfeI) PfeI (TfiI)

GCAGC(8/12)↓*FastDigest® BbvI (Lsp1109I)FastDigest® BseXI


GCATC(5/9)↓* FastDigest® SfaNI (BmsI) LweI (SfaNI)↓(14/12)GCCGT* BceAI↓(6/4)GCGGG* SmuI (FauI)↓(10/5)GCGTC* FastDigest® HgaI (CseI) CseI (HgaI)GCSG↓C FastDigest® TauI TauI

↓(12/8)GCTGC*FastDigest® BbvI (Lsp1109I)FastDigest® BseXI


G↓CWGC TseIGGATC(4/5)↓* BspPI (AlwI)GGATG(9/13)↓* FastDigest® FokI FokIGGATG(2/0)↓* FastDigest® BseGI BseGI (BtsCI)GGGAC(10/14)↓* FastDigest® BsmFI (FaqI) FaqI (BsmFI)GGTGA(8/7)↓* HphIG↓GWCC FastDigest® AvaII (Eco47I) Eco47I (AvaII)↓(14/10)GTCCC* FastDigest® BsmFI (FaqI) FaqI (BsmFI)GTCTC(1/5)↓* FastDigest® Alw26I Alw26I (BsmAI)↓GTSAC FastDigest® NmuCI NmuCI (Tsp45I)↓(7/8)TCACC* HphI↓(9/11)TCCGT* TspGWI↓(7/8)TCTTC* FastDigest® MboII MboII↓(9/11)TTCAT* TspDTITTS↓AA AgsI

Table 1.29. Enzymes recognizing 5 bp long targets.

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FastDigest® enzyme

Conventional enzyme

AA↓CGTT FastDigest® AclI (Psp1406I) Psp1406I (AclI)A↓AGCTT FastDigest® HindIII HindIII↓(8/13)AAG(N)5CTT(13/8)↓ FalIAAT↓ATT FastDigest® SspI SspIA↓CATGT PscI (PciI)A↓CCGGT FastDigest® AgeI (BshTI) BshTI (AgeI)ACCTGC(4/8)↓* FastDigest® BspMI (BveI) BveI (BspMI)A↓CGCGT FastDigest® MluI MluI↓(9/12)AC(N)5CTCC(10/7)↓* BsaXIA↓CRYGT AflIIIA↓CTAGT FastDigest® SpeI (BcuI) BcuI (SpeI)ACTGGG(5/4)↓* BfiI (BmrI)A↓GATCT FastDigest® BglII BglIIAGC↓GCT FastDigest® AfeI (Eco47III) Eco47III (AfeI)AGG↓CCT FastDigest® StuI (Eco147I) Eco147I (StuI)AGT↓ACT FastDigest® ScaI ScaIAT↓CGAT FastDigest® ClaI (Bsu15I) Bsu15I (ClaI)ATGCA↓T FastDigest® NsiI (Mph1103I) Mph1103I (NsiI)AT↓TAAT FastDigest® AseI (VspI) VspI (AseI)C↓AATTG FastDigest® MfeI (MunI) MunI (MfeI)CACCCA(11/9)↓* TaqIICACGAG(-5/-1)↓* BauI (BssSI)CACGTC(-3/-3)↓* AjiI (BmgBI)CAC↓GTG FastDigest® PmlI (Eco72I) Eco72I (PmlI)CACNNN↓GTG FastDigest® DraIII (AdeI) AdeI (DraIII)CACNN↓NNGTG FastDigest® AleI (OliI) OliI (AleI)↓(8/13)CAC(N)6TCC(12/7)↓* TstI↓(0/2)CACTGC* BtsICAGCAG(25/27)↓* EcoP15ICAG↓CTG FastDigest® PvuII PvuIICAGNNN↓CTG FastDigest® AlwNI (CaiI) CaiI (AlwNI)CA↓TATG FastDigest® NdeI NdeI↓(14/10)CATCGC* BtgZI↓(0/2)CATTGC* FastDigest® BsrDI (BseMI) BseMI (BsrDI)CAYNN↓NNRTG FastDigest® MslI (RseI) RseI (MslI)CCANNNNN↓NNNNTGG XcmICCANNNNN↓NTGG FastDigest® BstXI BstXICCANNNN↓NTGG FastDigest® PflMI (Van91I) Van91I (PflMI)C↓CATGG FastDigest® NcoI NcoICCCAGC(-5/-1)↓* BseYICCCAGC(-1/-5)↓* FastDigest® PspFI GsaI↓(4/5)CCCAGT* BfiI (BmrI)C↓CCGGG Cfr9I (XmaI)CCC↓GGG FastDigest® SmaI SmaICCGC↓GG Cfr42I (SacII)CCGCTC(-3/-3)↓* FastDigest® BsrBI (MbiI) MbiI (BsrBI)C↓CRYGG BtgIC↓CTAGG FastDigest® AvrII (XmaJI) XmaJI (AvrII)CCTNAGC(-5/-2)↓* FastDigest® Bpu10I Bpu10ICC↓TNAGG FastDigest® Bsu36I (Eco81I) Eco81I (Bsu36I)CCTNN↓NNNAGG FastDigest® EcoNI (XagI) XagI (EcoNI)C↓CWWGG FastDigest® StyI (Eco130I) Eco130I (StyI)↓(10/12)CGA(N)6TGC(12/10)↓* BcgICGAT↓CG FastDigest® PvuI PvuIC↓GGCCG FastDigest® EagI (Eco52I) Eco52I (EagI)CGRY↓CG FastDigest® BsiEI (Bsh1285I) Bsh1285I (BsiEI)C↓GTACG FastDigest® BsiWI (Pfl23II) Pfl23II (BsiWI)CGTCTC(1/5)↓* FastDigest® BsmBI (Esp3I) Esp3I (BsmBI)CMG↓CKG MspA1I↓(14/16)CTCAAG* BpuEI↓(14/16)CTCCAG* FastDigest® BpmI (GsuI) GsuI (BpmI)↓(8/10)CTCCTC* BseRIC↓TCGAG FastDigest® XhoI XhoI↓(19/21)CTCGGC* NmeAIII


FastDigest® enzyme

Conventional enzyme

CTCGTG(-5/-1)↓* BauI (BssSI)CTCTTC(1/4)↓* FastDigest® EarI (Eam1104I) Eam1104I (EarI)CTGAAG(16/14)↓* FastDigest® AcuI (Eco57I) Eco57I (AcuI)↓(14/16)CTGCAC* BsgICTGCA↓G FastDigest® PstI PstI↓(27/25)CTGCTG* EcoP15ICTGGAG(16/14)↓* FastDigest® BpmI (GsuI) GsuI (BpmI)CTGRAG(16/14)↓* Eco57MIC↓TRYAG FastDigest® SfcI (BfmI) BfmI (SfcI)C↓TTAAG FastDigest® AflII (BspTI) BspTI (AflII)↓(14/16)CTTCAG* FastDigest® AcuI (Eco57I) Eco57I (AcuI)CTTGAG(16/14)↓* BpuEI↓(14/16)CTYCAG* Eco57MIC↓TYRAG SmoI (SmlI)C↓YCGRG FastDigest® AvaI (Eco88I) Eco88I (AvaI)CY↓CGRG BmeT110IGAAGAC(2/6)↓* FastDigest® BbsI (BpiI) BpiI (BbsI)↓(4/1)GAAGAG* FastDigest® EarI (Eam1104I) Eam1104I (EarI)GAANN↓NNTTC FastDigest® PdmI PdmI (XmnI)GAATGC(1/-1)↓* FastDigest® Mva1269I Mva1269I (BsmI)G↓AATTC FastDigest® EcoRI EcoRI↓(8/13-14)GAB(N)5RTC(13-14/8)↓* Hin4IGACCGA(11/9)↓* TaqIIGAC↓GTC ZraIGACGT↓C FastDigest® AatII AatIIGACGTG(-3/-3)↓* AjiI (BmgBI)GACN↓NNGTC FastDigest® PsyI PsyI (Tth111I)GACNN↓NNGTC FastDigest® PshAI (BoxI) BoxI (PshAI)GACNNN↓NNGTC FastDigest® Eam1105I Eam1105I (AhdI)GACNNNN↓NNGTC FastDigest® DrdI (AasI) AasI (DrdI)↓(5/1)GAGACC* FastDigest® Eco31I Eco31I (BsaI)↓(5/1)GAGACG* FastDigest® BsmBI (Esp3I) Esp3I (BsmBI)GAGCGG(-3/-3)↓* FastDigest® BsrBI (MbiI) MbiI (BsrBI)GAGCT↓C FastDigest® SacI SacIGAG↓CTC FastDigest® Ecl136II Ecl136II (EcoICRI)GAGGAG(10/8)↓* BseRI↓(8/13)GAG(N)5CTC(13/8)↓ FastDigest® BplI BplIGAT↓ATC FastDigest® EcoRV (Eco32I) Eco32I (EcoRV)GATNN↓NNATC FastDigest® BsaBI (BseJI) BseJI (BsaBI)(8/13-14)GAY(N)5VTC(13-14/8)↓* Hin4IGCAATG(2/0)↓* FastDigest® BsrDI (BseMI) BseMI (BsrDI)↓(8/4)GCAGGT* FastDigest® BspMI (BveI) BveI (BspMI)GCAGTG(2/0)↓* BtsI↓(10/12)GCA(N)6TCG(12/10)↓* BcgI↓(10/12)GCA(N)6TGC(12/10)↓ AlfIGCANNNN↓NTGC BstAPIGCATG↓C FastDigest® SphI (PaeI) PaeI (SphI)↓(-1/1)GCATTC* FastDigest® Mva1269I Mva1269I (BsmI)GCCGAG(21/19)↓* NmeAIIIG↓CCGGC NgoMIVGCC↓GGC FastDigest® NaeI (PdiI) PdiI (NaeI)GCCNNNN↓NGGC FastDigest® BglI BglIGCGATG(10/14)↓* BtgZIG↓CGCGC FastDigest® BssHII (PfeI) PauI (BssHII)GCTAG↓C FastDigest® BmtI (BspOI) BspOI (BmtI)G↓CTAGC FastDigest® NheI NheIGCTGGG(-5/-1)↓* BseYIGCTGGG(-1/-5)↓* FastDigest® PspFI GsaIGC↓TNAGC FastDigest® BlpI (Bpu1102I) Bpu1102I (BlpI)GCTNAGG(-5/-2)↓* FastDigest® Bpu10I Bpu10IGDGCH↓C FastDigest® Bsp1286I (SduI) SduI (Bsp1286I)↓(7/10)GGAG(N)5GT(12/9)↓* BsaXI↓(7/12)GGA(N)6GTG(13/8)↓* TstI↓(5/6)GGATAC* BfuI (BciVI)

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Single letter codeR = G or A; K = G or T; B = C, G or T; M = A or C;Y = C or T; S = C or G; D = A, G or T; V = A, C or G.W = A or T; H = A, C or T; N = G, A, T or C;


FastDigest® enzyme

Conventional enzyme

G↓GATCC FastDigest® BamHI BamHIGGC↓GCC FastDigest® EheI EheI (SfoI)GG↓CGCC NarIG↓GCGCC SspDI (KasI)GGCGC↓C BbeIGGCGGA(11/9)↓* EciIG↓GGCCC FastDigest® Bsp120I Bsp120I (PspOMI)GGGCC↓C FastDigest® ApaI ApaIGGTAC↓C FastDigest® KpnI KpnIG↓GTACC FastDigest® Acc65I Acc65I (Asp718I)GGTCTC(1/5)↓* FastDigest® Eco31I Eco31I (BsaI)G↓GTNACC FastDigest® Eco91I Eco91I (BstEII)G↓GYRCC FastDigest® BanI (BshNI) BshNI (BanI)GKGCM↓C FastDigest® Bme1580I (BseSI) BseSI (Bme1580I)GR↓CGYC FastDigest® BsaHI (Hin1I) Hin1I (BsaHI)GRGCY↓C Eco24I (BanII)GTA↓TAC FastDigest® BstZ17I (Bst1107I) Bst1107I (BstZ17I)GTATCC(6/5)↓* BfuI (BciVI)G↓TCGAC FastDigest® SalI SalI↓(6/2)GTCTTC* FastDigest® BbsI (BpiI) BpiI (BbsI)G↓TGCAC FastDigest® ApaLI (Alw44I) Alw44I (ApaLI)GTGCAG(16/14)↓* BsgIGT↓MKAC FastDigest® AccI (XmiI) XmiI (AccI)GTT↓AAC FastDigest® HpaI (KspAI) KspAI (HpaI)↓(18/20)GTYGGA* MmeIGTY↓RAC FastDigest® HincII HincII (HindII)GWGCW↓C FastDigest® Alw21I Alw21I (BsiHKAI)R↓AATTY FastDigest® XapI XapI (ApoI)RCATG↓Y FastDigest® NspI (XceI) XceI (NspI)R↓CCGGY FastDigest® BsrFI (Cfr10I) Cfr10I (BsrFI)R↓GATCY FastDigest® PsuI PsuI (BstYI)RGCGC↓Y FastDigest® HaeII (BfoI) HaeIIRG↓GNCCY FastDigest® EcoO109I EcoO109I (DraII)TAC↓GTA FastDigest® SnaBI (Eco105I) Eco105I (SnaBI)TARCCA(11/9)↓* TsoIT↓CATGA FastDigest® BspHI (PagI) PagI (BspHI)↓(9/11)TCCGCC* EciIT↓CCGGA FastDigest® Kpn2I Kpn2I (BspEI)T↓CCNGGA FastDigest® PfoI PfoITCCRAC(20/18)↓* MmeITCG↓CGA FastDigest® NruI (RruI) Bsp68I (NruI)↓(9/11)TCGGTC* TaqIIT↓CTAGA FastDigest® XbaI XbaI↓(10/12)TGA(N)6TCA(12/10)↓ BdaIT↓GATCA FastDigest® BclI BclITGC↓GCA FastDigest® FspI (NsbI) NsbI (FspI)TGG↓CCA FastDigest® MscI (MlsI) MlsI (MscI)↓(9/11)TGGGTG* TaqII↓(9/11)TGGYTA* TsoIT↓GTACA FastDigest® Bsp1407I Bsp1407I (BsrGI)TTA↓TAA FastDigest® PsiI (AanI) AanI (PsiI)TT↓CGAA FastDigest® Bsp119I Bsp119I (BstBI)TTT↓AAA FastDigest® DraI DraIW↓CCGGW BsaWIW↓GTACW FastDigest® TatI TatIYAC↓GTR FastDigest® BsaAI (Ppu21I) Ppu21I (BsaAI)Y↓GGCCR CfrI (EaeI)





FastDigest® enzyme

Conventional enzyme

A↓CCWGGT FastDigest® SexAI (CsiI) SexAI↓(10/15)AC(N)4GTAYC(12/7)↓* BaeI↓(11/13)CAA(N)5GTGG(12/10)↓* CspCICACCTGC(4/8)↓* AarI↓(6/11)CCAA(N)7TTC(12/7)↓* FastDigest® AjuI AjuI↓(10/12)CCAC(N)5TTG(13/11)↓* CspCICC↓CWGGG PasICCTCAGC(-5/-2)↓* BbvCICG↓GWCCG FastDigest® RsrII (CpoI) CpoI (RsrII)↓(6/11)CRAA(N)6GTC(13/8)↓* ArsI↓(7/12)GAAC(N)5CTC(13/8)↓* PpiI↓(7/12)GAAC(N)6TAC(12/7)↓* PsrI↓(7/12-13)GAAC(N)6TCC(12-13/7)↓* AloI↓(4/1)GAAGAGC* FastDigest® SapI (LguI) LguI (SapI)↓(7/12)GAAG(N)6TAC(12/7)↓* BarI↓(7/12)GAA(N)7TTGG(11/6)↓* FastDigest® AjuI AjuI↓(8/13)GAC(N)6TTYG(11/6)↓* ArsI↓(8/13)GAG(N)5GTTC(12/7)↓* PpiI↓(8/4)GCAGGTG* AarIGCTCTTC(1/4)↓* FastDigest® SapI (LguI) LguI (SapI)GCTGAGG(-5/-2)↓* BbvCI↓(7/12-13)GGA(N)6GTTC(12-13/7)↓* AloIGG↓GWCCC FastDigest® SanDI (KflI) SanDI↓(7/12)GRTACNNNNGT(15/10)↓* BaeI↓(7/12)GTA(N)6CTTC(12/7)↓* BarI↓(7/12)GTA(N)6GTTC(12/7)↓* PsrIRG↓GWCCY FastDigest® PpuMI (Psp5II) Psp5II (PpuMI)

Specificity 5’→3’FastDigest® enzyme

Conventional enzyme

ATTT↓AAAT FastDigest® SwaI (SmiI) SmiI (SwaI)CC↓TCGAGG AbsICCTGCA↓GG FastDigest® SbfI (SdaI) SdaI (SbfI)CG↓CCGGCG FastDigest® MreI MreI (Sse232I)CG↓CGCGCG FastDigest® MauBI MauBICG↓TCGACG SgrDICR↓CCGGYG SgrAIGCCC↓GGGC SrfIGCGAT↓CGC FastDigest® AsiSI (SfaAI) SfaAI (AsiSI)GC↓GGCCGC FastDigest® NotI NotIGGCCGG↓CC FseIGGCCNNNN↓NGGCC FastDigest® SfiI SfiIGG↓CGCGCC FastDigest® AscI (SgsI) SgsI (AscI)GTTT↓AAAC FastDigest® MssI MssI (PmeI)RTGC↓GCAY FastDigest® FspAI FspAITTAAT↓TAA FastDigest® PacI PacIVC↓TCGAGB PspXI


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Table 1.33. Commercial restriction enzymes generating 5’-protruding ends.

Commercial Restriction Enzymes Generating 5’-protruding Ends



FastDigest® enzyme

Conventional enzyme

5’-protruding end (5’→ 3’)1 nt 2 nt 3 nt 4 nt 5 nt

AA↓CGTT FastDigest® AclI (Psp1406I) Psp1406I (AclI) CGA↓AGCTT FastDigest® HindIII HindIII AGCT↓AATT FastDigest® Tsp509I (TasI) TasI (Tsp509I) AATTA↓CATGT PscI (PciI) CATGA↓CCGGT FastDigest® AgeI (BshTI) BshTI (AgeI) CCGGACCTGC(4/8)↓* FastDigest® BspMI (BveI) BveI (BspMI) NNNNA↓CCWGGT FastDigest® SexAI (CsiI) SexAI CCWGGA↓CGCGT FastDigest® MluI MluI CGCGACGGC(12/14)↓* BceAI NNA↓CGT MaeII CGA↓CRYGT AflIII CRYGA↓CTAGT FastDigest® SpeI (BcuI) BcuI (SpeI) CTAGA↓GATCT FastDigest® BglII BglII GATCAT↓CGAT FastDigest® ClaI (Bsu15I) Bsu15I (ClaI) CGAT↓TAAT FastDigest® AseI (VspI) VspI (AseI) TAC↓AATTG FastDigest® MfeI (MunI) MunI (MfeI) AATTCACCTGC(4/8)↓* AarI NNNNCACGAG(-5/-1)↓* BauI (BssSI) ACGACAGCAG(25/27)↓* EcoP15I NNCA↓TATG FastDigest® NdeI NdeI TA↓(13/9)CATCC* FastDigest® FokI FokI NNNN↓(14/10)CATCGC* BtgZI NNNN↓CATG FatI CATGC↓ATG CviAII ATCCATC(4/5)↓* BccI NC↓CATGG FastDigest® NcoI NcoI CATGCCCAGC(-5/-1)↓* BseYI CCAGCCCGC(4/6)↓* SmuI (FauI) NNC↓CCGGG Cfr9I (XmaI) CCGGCC↓CWGGG PasI CWGCCGC(-3/-1)↓* FastDigest® AciI (SsiI) SsiI (AciI) CG


HpaIIMspI (HpaII)

CG

↓CCNGG StyD4I CCNGGCC↓NGG FastDigest® ScrFI (Bme1390I) Bme1390I (ScrFI) NC↓CNNGG FastDigest® BsaJI (BseDI) BseDI (BsaJI) CNNGC↓CRYGG BtgI CRYGCC↓SGG FastDigest® NciI (BcnI) BcnI (NciI) SC↓CTAGG FastDigest® AvrII (XmaJI) XmaJI (AvrII) CTAGCCTCAGC(-5/-2)↓* BbvCI TCACC↓TCGAGG AbsI TCGACCTNAGC(-5/-2)↓* FastDigest® Bpu10I Bpu10I TNACC↓TNAGG FastDigest® Bsu36I (Eco81I) Eco81I (Bsu36I) TNACCTNN↓NNNAGG FastDigest® EcoNI (XagI) XagI (EcoNI) N↓CCWGG EcoRII CCWGGCC↓WGG FastDigest® MvaI MvaI (BstNI) WC↓CWWGG FastDigest® StyI (Eco130I) Eco130I (StyI) CWWGCG↓CCGGCG FastDigest® MreI MreI (Sse232I) CCGGCG↓CGCGCG FastDigest® MauBI MauBI CGCGC↓GGCCG FastDigest® EagI (Eco52I) Eco52I (EagI) GGCCCG↓GWCCG FastDigest® RsrII (CpoI) CpoI (RsrII) GWCC↓GTACG FastDigest® BsiWI (Pfl23II) Pfl23II (BsiWI) GTAC5mCNNG(9/13)↓ SgeI NNNNCG↓TCGACG SgrDI TCGACGTCTC(1/5)↓* FastDigest® BsmBI (Esp3I) Esp3I (BsmBI) NNNNCR↓CCGGYG SgrAI CCGG

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FastDigest® enzyme

Conventional enzyme


C↓TAG FastDigest® BfaI (FspBI) FspBI (BfaI) TAC↓TCGAG FastDigest® XhoI XhoI TCGACTCGTG(-5/-1)↓* BauI (BssSI) TCGTCTCTTC(1/4)↓* FastDigest® EarI (Eam1104I) Eam1104I (EarI) NNN↓(27/25)CTGCTG* EcoP15I NNC↓TNAG FastDigest® DdeI (HpyF3I) HpyF3I (DdeI) TNAC↓TRYAG FastDigest® SfcI (BfmI) BfmI (SfcI) TRYAC↓TTAAG FastDigest® AflII (BspTI) BspTI (AflII) TTAAC↓TYRAG SmoI (SmlI) TYRAC↓YCGRG FastDigest® AvaI (Eco88I) Eco88I (AvaI) YCGRCY↓CGRG BmeT110I CGGAAGAC(2/6)↓* FastDigest® BbsI (BpiI) BpiI (BbsI) NNNN↓(4/1)GAAGAG* FastDigest® EarI (Eam1104I) Eam1104I (EarI) NNN↓(4/1)GAAGAGC* FastDigest® SapI (LguI) LguI (SapI) NNNG↓AATTC FastDigest® EcoRI EcoRI AATTGACGC(5/10)↓* FastDigest® HgaI (CseI) CseI (HgaI) NNNNNGACN↓NNGTC FastDigest® PsyI PsyI (Tth111I) N↓(5/4)GACTC* PleI N↓(5/1)GAGAC* FastDigest® Alw26I Alw26I (BsmAI) NNNN↓(5/1)GAGACC* FastDigest® Eco31I Eco31I (BsaI) NNNN↓(5/1)GAGACG* FastDigest® BsmBI (Esp3I) Esp3I (BsmBI) NNNNGAGTC(4/5)↓* PleI NG↓ANTC FastDigest® HinfI HinfI ANT

↓GATCFastDigest® MboIFastDigest® Sau3AI (Bsp143I)

MboIBsp143I (Sau3AI)

GATC

↓(5/4)GATCC* BspPI (AlwI) N↓(9/5)GATGC* FastDigest® SfaNI (BmsI) LweI (SfaNI) NNNN↓(5/4)GATGG* BccI NG↓AWTC FastDigest® TfiI (PfeI) PfeI (TfiI) AWT

GCAGC(8/12)↓*FastDigest® BbvI (Lsp1109I)FastDigest® BseXI (BbvI)


NNNN

↓(8/4)GCAGGT* FastDigest® BspMI (BveI) BveI (BspMI) NNNN↓(8/4)GCAGGTG* AarI NNNNGCATC(5/9)↓* FastDigest® SfaNI (BmsI) LweI (SfaNI) NNNNG↓CCGGC NgoMIV CCGG↓(14/12)GCCGT* BceAI NNGCGATG(10/14)↓* BtgZI NNNNG↓CGC FastDigest® HinP1I (Hin6I) Hin6I (HinP1I) CGG↓CGCGC FastDigest® BssHII (PteI) PauI (BssHII) CGCGGCGG(-3/-1)↓* FastDigest® AciI (SsiI) SsiI (AciI) CGGC↓GGCCGC FastDigest® NotI NotI GGCC↓(6/4)GCGGG* SmuI (FauI) NN↓(10/5)GCGTC* FastDigest® HgaI (CseI) CseI (HgaI) NNNNNGC↓NGC FastDigest® Fnu4HI (SatI) SatI (Fnu4HI) NG↓CTAGC FastDigest® NheI NheI CTAGGCTCTTC(1/4)↓* FastDigest® SapI (LguI) LguI (SapI) NNNGCTGAGG(-5/-2)↓* BbvCI TGA

↓(12/8)GCTGC*FastDigest® BbvI (Lsp1109I)FastDigest® BseXI (BbvI)


NNNN

GCTGGG(-5/-1)↓* BseYI CTGGGC↓TNAGC FastDigest® BlpI (Bpu1102I) Bpu1102I (BlpI) TNAGCTNAGG(-5/-2)↓* FastDigest® Bpu10I Bpu10I TNAG↓CWGC TseI CWGGGATC(4/5)↓* BspPI (AlwI) NG↓GATCC FastDigest® BamHI BamHI GATCGGATG(9/13)↓* FastDigest® FokI FokI NNNNGG↓CGCC NarI CGG↓GCGCC SspDI (KasI) GCGCGG↓CGCGCC FastDigest® AscI (SgsI) SgsI (AscI) CGCGGGGAC(10/14)↓* FastDigest® BsmFI (FaqI) FaqI (BsmFI) NNNN

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FastDigest® enzyme

Conventional enzyme


G↓GGCCC FastDigest® Bsp120I Bsp120I (PspOMI) GGCCGG↓GWCCC FastDigest® SanDI (KflI) SanDI GWCG↓GNCC FastDigest® Sau96I (Cfr13I) Cfr13I (Sau96I) GNCGG↓NCC BmgT120I NG↓GTACC FastDigest® Acc65I Acc65I (Asp718I) GTACGGTCTC(1/5)↓* FastDigest® Eco31I Eco31I (BsaI) NNNNG↓GTNACC FastDigest® Eco91I Eco91I (BstEII) GTNACG↓GWCC FastDigest® AvaII (Eco47I) Eco47I (AvaII) GWCG↓GYRCC FastDigest® BanI (BshNI) BshNI (BanI) GYRCGR↓CGYC FastDigest® BsaHI (Hin1I) Hin1I (BsaHI) CGG↓TAC FastDigest® Csp6I Csp6I (CviQI) TA↓(14/10)GTCCC* FastDigest® BsmFI (FaqI) FaqI (BsmFI) NNNNG↓TCGAC FastDigest® SalI SalI TCGAGTCTC(1/5)↓* FastDigest® Alw26I Alw26I (BsmAI) NNNN↓(6/2)GTCTTC* FastDigest® BbsI (BpiI) BpiI (BbsI) NNNNG↓TGCAC FastDigest® ApaLI (Alw44I) Alw44I (ApaLI) TGCAGT↓MKAC FastDigest® AccI (XmiI) XmiI (AccI) MK↓GTNAC MaeIII GTNAC↓GTSAC FastDigest® NmuCI NmuCI (Tsp45I) GTSACR↓AATTY FastDigest® XapI XapI (ApoI) AATTR↓CCGGY FastDigest® BsrFI (Cfr10I) Cfr10I (BsrFI) CCGGR↓GATCY FastDigest® PsuI PsuI (BstYI) GATCRG↓GNCCY FastDigest® EcoO109I EcoO109I (DraII) GNCRG↓GWCCY FastDigest® PpuMI (Psp5II) Psp5II (PpuMI) GWCT↓CATGA FastDigest® BspHI (PagI) PagI (BspHI) CATGT↓CCGGA FastDigest® Kpn2I Kpn2I (BspEI) CCGGT↓CCNGGA FastDigest® PfoI PfoI CCNGGT↓CGA FastDigest® TaqI TaqI CGTC↓NNGA Hpy188III NNT↓CTAGA FastDigest® XbaI XbaI CTAGT↓GATCA FastDigest® BclI BclI GATCT↓GTACA FastDigest® Bsp1407I Bsp1407I (BsrGI) GTAC


Tru1I (MseI) TA

TT↓CGAA FastDigest® Bsp119I Bsp119I (BstBI) CGVC↓TCGAGB PspXI TCGAW↓CCGGW BsaWI CCGGW↓GTACW FastDigest® TatI TatI GTACY↓GGCCR CfrI (EaeI) GGCC



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Commercial Restriction Enzymes Generating 3’-protruding Ends

Recognition sequenceFastDigest® enzyme

Conventional enzyme

3’-protruding end (5’→ 3’)1 nt 2 nt 3 nt 4 nt 5 nt 9 nt

ACGGA(11/9)↓* TspGWI NNACGT↓ FastDigest® TaiI TaiI (MaeII) ACGTACN↓GT FastDigest® TaaI TaaI (HpyCH4III) NACTGG(1/-1)↓* FastDigest® BseNI BseNI (BsrI) GNACTGGG(5/4)↓* BfiI (BmrI) NASST↓ SetI ASSTATGAA(11/9)↓* TspDTI NNATGCA↓T FastDigest® NsiI (Mph1103I) Mph1103I (NsiI) TGCACACCCA(11/9)↓* TaqII NNCACNNN↓GTG FastDigest® DraIII (AdeI) AdeI (DraIII) NNN↓(0/2)CACTGC* BtsI NNCAGNNN↓CTG FastDigest® AlwNI (CaiI) CaiI (AlwNI) NNNCASTG(2/-7)↓ FastDigest® TspRI (TscAI) TscAI (TspRI) NNCASTGNN↓(0/2)CATCC* FastDigest® BseGI BseGI (BtsCI) NNCATG↓ FastDigest® NlaIII (Hin1II) Hin1II (NlaIII) CATG↓(0/2)CATTGC* FastDigest® BsrDI (BseMI) BseMI (BsrDI) NN↓(-1/1)CCAGT* FastDigest® BseNI BseNI (BsrI) NCCCANNNNN↓NNNNTGG XcmI NCCANNNNN↓NTGG FastDigest® BstXI BstXI NNNNCCANNNN↓NTGG FastDigest® PflMI (Van91I) Van91I (PflMI) NNNCCCAGC(-1/-5)↓* FastDigest® PspFI GsaI CCAG↓(4/5)CCCAGT* BfiI (BmrI) NCCGC↓GG Cfr42I (SacII) GCCCNNNNN↓NNGG FastDigest® BslI (BseLI) BseLI (BslI) NNNCCTC(7/6)↓* FastDigest® MnlI MnlI NCCTGCA↓GG FastDigest® SbfI (SdaI) SdaI (SbfI) TGCACCTTC(6/5)↓* HpyAV NCGAT↓CG FastDigest® PvuI PvuI ATCGRY↓CG FastDigest® BsiEI (Bsh1285I) Bsh1285I (BsiEI) RYCGWCG↓ Hpy99I CGWCG↓(14/16)CTCAAG* BpuEI NNCTCAG(9/7)↓* BspCNI NNCTCAG(10/8)↓* FastDigest® BspCNI (BseMII) BseMII (BspCNI) NN↓(14/16)CTCCAG* FastDigest® BpmI (GsuI) GsuI (BpmI) NN↓(8/10)CTCCTC* BseRI NN↓(19/21)CTCGGC* NmeAIII NNCTGAAG(16/14)↓* FastDigest® AcuI (Eco57I) Eco57I (AcuI) NN↓(7/9)CTGAG* BspCNI NN↓(8/10)CTGAG* FastDigest® BspCNI (BseMII) BseMII (BspCNI) NN↓(14/16)CTGCAC* BsgI NNCTGCA↓G FastDigest® PstI PstI TGCACTGGAG(16/14)↓* FastDigest® BpmI (GsuI) GsuI (BpmI) NNCTGRAG(16/14)↓* Eco57MI NN↓(14/16)CTTCAG* FastDigest® AcuI (Eco57I) Eco57I (AcuI) NNCTTGAG(16/14)↓* BpuEI NN↓(14/16)CTYCAG* Eco57MI NNGAAGA(8/7)↓* FastDigest® MboII MboII N↓(5/6)GAAGG* HpyAV NGAATGC(1/-1)↓* FastDigest® Mva1269I Mva1269I (BsmI) CNGACCGA(11/9)↓* TaqII NNGACGT↓C FastDigest® AatII AatII ACGTGACNNN↓NNGTC FastDigest® Eam1105I Eam1105I (AhdI) NGACNNNN↓NNGTC FastDigest® DrdI (AasI) AasI (DrdI) NNGAGCT↓C FastDigest® SacI SacI AGCT↓(6/7)GAGG* FastDigest® MnlI MnlI NGAGGAG(10/8)↓* BseRI NNGAT↓C BstKTI ATGCAATG(2/0)↓* FastDigest® BsrDI (BseMI) BseMI (BsrDI) NNGCAGTG(2/0)↓* BtsI NN

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Conventional enzyme

3’-protruding end (5’→ 3’)1 nt 2 nt 3 nt 4 nt 5 nt 9 nt

GCANNNN↓NTGC BstAPI NNNGCATG↓C FastDigest® SphI (PaeI) PaeI (SphI) CATG↓(-1/1)GCATTC* FastDigest® Mva1269I Mva1269I (BsmI) NGGCCGAG(21/19)↓* NmeAIII NNGCCNNNN↓NGGC FastDigest® BglI BglI NNNGCGAT↓CGC FastDigest® AsiSI (SfaAI) SfaAI (AsiSI) ATGCG↓C FastDigest® HhaI HhaI CGGCNNNNN↓NNGC FastDigest® HpyF10VI HpyF10VI (MwoI) NNNGCSG↓C FastDigest® TauI TauI CSGGCTAG↓C FastDigest® BmtI (BspOI) BspOI (BmtI) CTAGGCTGGG(-1/-5)↓* FastDigest® PspFI GsaI CTGGGDGCH↓C FastDigest® Bsp1286I (SduI) SduI (Bsp1286I) DGCH↓(5/6)GGATAC* BfuI (BciVI) NGGATG(2/0)↓* FastDigest® BseGI BseGI (BtsCI) NNGGCCGG↓CC FseI CCGGGGCCNNNN↓NGGCC FastDigest® SfiI SfiI NNNGGCGC↓C BbeI GCGCGGCGGA(11/9)↓* EciI NNGGGCC↓C FastDigest® ApaI ApaI GGCCGGTAC↓C FastDigest® KpnI KpnI GTACGGTGA(8/7)↓* HphI N

GKGCM↓CFastDigest® Bme1580I (BseSI)

BseSI (Bme1580I) KGCM

GRGCY↓C Eco24I (BanII) RGCYGTATCC(6/5)↓* BfuI (BciVI) NGTGCAG(16/14)↓* BsgI NN↓(18/20)GTYGGA* MmeI NNGWGCW↓C FastDigest® Alw21I Alw21I (BsiHKAI) WGCWRCATG↓Y FastDigest® NspI (XceI) XceI (NspI) CATGRGCGC↓Y FastDigest® HaeII (BfoI) HaeII GCGCTARCCA(11/9)↓* TsoI NN↓(7/8)TCACC* HphI N↓(9/11)TCCGCC* EciI NN↓(9/11)TCCGT* TspGWI NNTCCRAC(20/18)↓* MmeI NN↓(9/11)TCGGTC* TaqII NNTCN↓GA Hpy188I N↓(7/8)TCTTC* FastDigest® MboII MboII N↓(9/11)TGGGTG* TaqII NN↓(9/11)TGGYTA* TsoI NNTTAAT↓TAA FastDigest® PacI PacI AT↓(9/11)TTCAT* TspDTI NNTTS↓AA AgsI S

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Table 1.35. Commercial restriction enzymes generating blunt ends.

Commercial Restriction Enzymes Generating Blunt Ends

Table 1.36. Commercial restriction enzymes cleaving DNA on the both sides of their recognition sequence.

Commercial Restriction Enzymes Cleaving DNA on the Both Sides of their Recognition Sequence

Reccognition sequence

FastDigest®

enzymeConventional enzyme

AAT↓ATT FastDigest® SspI SspIAGC↓GCT FastDigest® AfeI (Eco47III) Eco47III (AfeI)AG↓CT FastDigest® AluI AluIAGG↓CCT FastDigest® StuI (Eco147I) Eco147I (StuI)AGT↓ACT FastDigest® ScaI ScaIATTT↓AAAT FastDigest® SwaI (SmiI) SmiI (SwaI)CACGTC(-3/-3)↓* AjiI (BmgBI)CAC↓GTG FastDigest® PmlI (Eco72I) Eco72I (PmlI)CACNN↓NNGTG FastDigest® AleI (OliI) OliI (AleI)CAG↓CTG FastDigest® PvuII PvuIICAYNN↓NNRTG FastDigest® MslI (RseI) RseI (MslI)CCC↓GGG FastDigest® SmaI SmaICCGCTC(-3/-3)↓* FastDigest® BsrBI (MbiI) MbiI (BsrBI)CG↓CG FastDigest® Bsh1236I Bsh1236I (BstUI)CMG↓CKG MspA1IGAANN↓NNTTC FastDigest® PdmI PdmI (XmnI)GAC↓GTC ZraIGACGTG(-3/-3)↓* AjiI (BmgBI)GACNN↓NNGTC FastDigest® PshAI (BoxI) BoxI (PshAI)↓(5/5)GACTC* FastDigest® MlyI (SchI) SchI (MlyI)GAGCGG(-3/-3)↓* FastDigest® BsrBI (MbiI) MbiI (BsrBI)GAG↓CTC FastDigest® Ecl136II Ecl136II (EcoICRI)GAGTC(5/5)↓* FastDigest® MlyI (SchI) SchI (MlyI)GAT↓ATC FastDigest® EcoRV (Eco32I) Eco32I (EcoRV)GA↓TC FastDigest® DpnI DpnIGATNN↓NNATC FastDigest® BsaBI (BseJI) BseJI (BsaBI)GCCC↓GGGC SrfIGCC↓GGC FastDigest® NaeI (PdiI) PdiI (NaeI)GC↓GC GlaIGCN↓NGC Cac8IGG↓CC FastDigest® HaeIII (BsuRI) BsuRI (HaeIII)GGC↓GCC FastDigest® EheI EheI (SfoI)GGN↓NCC FastDigest® NlaIV (BspLI) BspLI (NlaIV)GT↓AC FastDigest® RsaI RsaIGTA↓TAC FastDigest® BstZ17I (Bst1107I) Bst1107I (BstZ17I)GTN↓NAC FastDigest® Hpy8I Hpy8I (MjaIV)GTT↓AAC FastDigest® HpaI (KspAI) KspAI (HpaI)GTTT↓AAAC FastDigest® MssI MssI (PmeI)GTY↓RAC FastDigest® HincII HincII (HindII)RG↓CY CviJIRTGC↓GCAY FastDigest® FspAI FspAITAC↓GTA FastDigest® SnaBI (Eco105I) Eco105I (SnaBI)TCG↓CGA FastDigest® NruI (RruI) Bsp68I (NruI)TG↓CA HpyCH4VTGC↓GCA FastDigest® FspI (NsbI) NsbI (FspI)TGG↓CCA FastDigest® MscI (MlsI) MlsI (MscI)TTA↓TAA FastDigest® PsiI (AanI) AanI (PsiI)TTT↓AAA FastDigest® DraI DraIYAC↓GTR FastDigest® BsaAI (Ppu21I) Ppu21I (BsaAI)YA↓TR FaiI




Conventional enzyme

↓(8/13)AAG(N)5CTT(13/8)↓ FalI↓(9/12)AC(N)5CTCC(10/7)↓ BsaXI↓(10/15)AC(N)4GTAYC(12/7)↓ BaeI↓(11/13)CAA(N)5GTGG(12/10)↓ CspCI↓(8/13)CAC(N)6TCC(12/7)↓ TstI↓(6/11)CCAA(N)7TTC(12/7)↓ FastDigest® AjuI AjuI↓(10/12)CCAC(N)5TTG(13/11)↓ CspCI↓(10/12)CGA(N)6TGC(12/10)↓ BcgI↓(6/11)CRAA(N)6GTC(13/8)↓ ArsI↓(7/12)GAA(N)7TTGG(11/6)↓ FastDigest® AjuI AjuI↓(7/12)GAAC(N)5CTC(13/8)↓ PpiI↓(7/12)GAAC(N)6TAC(12/7)↓ PsrI↓(7/12-13)GAAC(N)6TCC(12-13/7)↓ AloI↓(7/12)GAAG(N)6TAC(12/7)↓ BarI↓(8/13-14)GAB(N)5RTC(13-14/8)↓ Hin4I↓(8/13)GAC(N)6TTYG(11/6)↓ ArsI↓(8/13)GAG(N)5CTC(13/8)↓ FastDigest® BplI BplI↓(8/13)GAG(N)5GTTC(12/7)↓ PpiI↓(8/13-14)GAY(N)5VTC(13-14/8)↓ Hin4I↓(10/12)GCA(N)6TCG(12/10)↓ BcgI↓(10/12)GCA(N)6TGC(12/10)↓ AlfI↓(7/12)GGA(N)6GTG(13/8)↓ TstI↓(7/12-13)GGA(N)6GTTC(12-13/7)↓ AloI↓(7/10)GGAG(N)5GT(12/9)↓ BsaXI↓(7/12)GRTAC(N)4GT(15/10)↓ BaeI↓(7/12)GTA(N)6CTTC(12/7)↓ BarI↓(7/12)GTA(N)6GTTC(12/7)↓ PsrI↓(10/12)TGA(N)6TCA(12/10)↓ BdaI

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Troubleshooting Guide

3 Diffuse DNA bands on gel

3.1 Gel shift

3.2 Contaminated

reagents

1 Incomplete

digestion, no digestion

Assess enzyme activity with control DNA

1.1 Inactive enzyme Active

enzyme

1.3 Unsuitable or contaminated

DNA

1.4 Water

contains impurities

2 Unexpected

cleavage pattern

2.1 Star

activity

2.2 Contamination with another

RE

2.3 Contamination with another

DNA

2.4 Incomplete DNA

digestion

2.5 Gel shift

2.6 Unexpected

sites in template DNA

1.2.1 Suboptimal di-

gestion protocol

1.2 Suboptimal

reaction conditions

1.2.2 Improper enzyme dilution

1.2.3 Improper reaction

assembly

1.2.4 Excess glycerol

1.2.5 Suboptimal DNA

concentration

Evaluate inhibition by

template DNA solution

No inhibition

1.3.1 Contaminants

in DNA solution

1.3.2 Absence of recognition

sites

1.3.3 Cleavage

blocked by methylation

Digestion problem

1.3.4 Structure of

DNA substrate

1.3.4.4 Site

preference

1.3.4.3 At least two

sites required

1.3.4.1 Supercoiled

plasmid DNA

1.3.4.2 Proximity of site to DNA

ends

Inhibition

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Table 1.37. Troubleshooting guide for DNA digestion.

Problem Possible cause and recommended solution

1. Incomplete digestion or no digestion

Assess enzyme activityThe restriction enzyme may lose activity due to improper storage or handling. Perform a digestion reaction with 1 µg of a standard control DNA, e.g. Lambda DNA (dam–, dcm–) (#SD0021).

1.1. Inactive enzyme. If the enzyme does not cut the control DNA:• Check the expiration date.• Verify that the enzyme has been stored at -20°C.• Check the temperature of your freezer. Do not allow the temperature go below -20°C as the enzyme may

freeze and multiple freeze thaw cycles (more than 3 cycles) may result in reduced enzyme activity.

1.2. Suboptimal reaction conditions. 1.2.1. Suboptimal digestion protocol. • Follow digestion protocol specified for the restriction enzyme and type of substrate DNA. All FastDigest®

enzymes are experimentally tested on Lambda DNA (or other control substrate DNA), plasmid and genomic DNA as well as PCR products. Please refer to Table 1.3 on p.71 for specific recommendations.

• For FastDigest® enzymes use FastDigest® or FastDigest® Green Buffer. All FastDigest® enzymes are 100% active in these buffers.

• For conventional restriction enzymes use the recommended reaction buffer supplied with the enzyme. For double digestions follow the recommendations of the DoubleDigest™ engine at www.fermentas.com/doubledigest.

• Use additives where required.• Perform the reaction at the optimal temperature specified for the restriction enzyme. For double digestions

with enzymes requiring different incubation temperatures perform sequential DNA cleavage: complete the first digestion reaction at the lower temperature, add the second enzyme and increase the digestion temperature for the second enzyme cleavage.

• Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation; the increase in salt concentration may reduce enzyme activity. For thermophilic enzymes use a heat block with a hot bonnet, e.g. a PCR cycler.

1.2.2. Improper dilution of conventional enzyme.• Dilute conventional restriction enzymes with Dilution Buffer for Restriction Enzymes (#B19). Restriction

enzymes diluted with this buffer are stable for at least 3-4 weeks at -20°C. • Never dilute enzymes in water or 10X reaction buffer. • Never dilute enzymes in 1X reaction buffer in the absence of DNA.• Use the recommended amount of FastDigest® enzymes, which are experimentally tested on four different

DNA substrates (see Table 1.3 on p.71). 1.2.3. Improper reaction assembly.• The restriction enzyme should always be the last component added to the reaction mixture.• The restriction enzyme may be inactivated if added directly to a 10X reaction buffer. 1.2.4. Excess glycerol in the reaction mixture.• The glycerol concentration in the reaction mixture should not exceed 5%. Thus, the volume of the restriction

enzyme added to the mixture should not exceed 1/10 of the total reaction volume.• Enzymes sensitive to high glycerol concentration include: Alw21I, BpiI, Bsp68I, BspTI, Eco32I, Eco91I, EcoRI,

Hin6I, HinfI, Mph1103I, Mva1269I and NcoI. 1.2.5. Suboptimal DNA concentration. The optimal range of DNA concentration in the reaction mixture is 0.02-0.1 µg/µl.

1.3. Unsuitable DNA template or contaminated DNA solution. If the enzyme is active in the control digest, assay the substrate DNA solution for inhibitory contaminants in

a mixing experiment with control template, e.g. Lambda DNA (dam–, dcm–) (#SD0021). Perform a control digest with two templates, control and sample, in one reaction mixture. Do not exceed the optimal DNA concentration in the reaction mixture (0.02-0.1 µg/µl).

• The sample template is contaminated if neither the control DNA nor sample DNA template is digested (see 1.3.1).• The sample template is not contaminated if the control DNA template is digested but the sample template

is not. Poor digestion of the experimental template is caused by errors in the DNA sequence (see 1.3.2), methylation effects (see 1.3.3) or structure of the DNA substrate (see 1.3.4).

Note Always ensure that the control DNA contains a recognition site for the enzyme present in the reaction. For example, there is no NotI recog-

nition site in lambda DNA.


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1.3.1. Contaminants in the DNA solution. • Template DNA may contain residual SDS, EDTA, proteins, salts or nucleases. Repurify the template using a

GeneJET™ PCR Purification Kit (#K0701) or by phenol/chloroform extraction and ethanol precipitation (see p.358). DNA A260/280 ratio should be 1.8-2.0. To remove EDTA and salts, wash the pellet with 70% cold ethanol.

• For reliable and reproducible plasmid miniprep purity, use the GeneJET™ Plasmid Miniprep Kit (#K0502).• For digestion of unpurified PCR products, dilute DNA at least 3-fold in the recommended 1X restriction

enzyme buffer see protocols on p.70 or p.160.• If the template DNA has been purified using silica or resin suspensions, remove all remaining particles by

centrifugation for 10 min at 10,000 rpm and ensure that no resin is carried over while transferring the DNA solution into a new tube.

1.3.2. The substrate DNA does not contain a recognition sequence for the restriction enzyme.• Re-check the DNA sequence and cloning strategy. • Determine if the restriction enzyme selected requires more than one site per target DNA for 100% activity

(see also 1.3.4.3).• Check literature for known site preferences for the restriction enzyme (see also 1.3.4.4).• If the recognition sequence was introduced by PCR primers, verify that the primer sequence contains the

recognition site. 1.3.3. Methylation effects. Restriction enzyme may be inhibited by methylation of the recognition site. • Identify which type of DNA methylation can occur on the recognition site and determine if the methylation

impairs or blocks DNA digestion with the enzyme. See Digestion of Methylated DNA on p.175 and useTables 1.13 and 1.20 on pp.176-181.

If methylation impairs or blocks DNA cleavage:• Propagate your plasmid in an E.coli dam–, dcm– strain (the E.coli GM2163 dam–, dcm– strain; #M0099, is

available upon request with the purchase of any Fermentas product),• Use the REsearch™ engine at www.fermentas.com/research or check the Fermentas catalog for the avail-

ability of a restriction enzyme isoschizomer not sensitive to DNA methylation.• Using of restriction enzyme which requires a methylated recognition sequence (DpnI or SgeI) for digestion

of unmethylated DNA will result in no DNA cleavage. Propagate your plasmid in E.coli dam+ or dcm+ strains (please refer to p.486 for genotype information of some common E.coli strains) to get DNA with methylated DpnI or SgeI recognition sequences. Alternatively, in the case of DpnI, the neoschizomers Bsp143I or MboI can be used to digest non-methylated DpnI recognition sites.

Note When PCR is carried out with standard dNTPs and non-methylated primers the resulting DNA product is not methylated.

1.3.4. Structure of substrate DNA. 1.3.4.1. Supercoiled plasmid DNA. • Use FastDigest® enzymes which are qualified for supercoiled DNA and provided with specific recommenda-

tions for each enzyme (see Table 1.3 on p.71). • For some conventional restriction enzymes, additional units are required to completely digest supercoiled

plasmids (e.g. 5-10 u (1 µl ) of restriction enzyme per 1 µg of DNA). Check notes in the catalog description of the enzyme or refer to the Certificate of Analysis.

1.3.4.2. Proximity of the recognition sequence to the DNA ends. Some restriction enzymes cleave DNA poorly if the recognition site is too close to the end of the DNA molecule. • For FastDigest® enzymes refer to Table 1.3 on p.71 or the product description to determine the effectiveness

of restriction enzyme cleavage at the ends of DNA. • For conventional restriction enzymes refer to Tables 1.10 (p.171) and 1.11 (p.172). • 1.3.4.3. Restriction enzyme requires at least two sites per DNA molecule for optimal activity. Some restriction enzymes such as AarI, BveI, Cfr10I, Cfr42I, Eco57I, EcoRII, LweI, SfiI require at least two

target sites per DNA molecule for efficient cleavage (for more details see Site Preferences by Restriction Enzymes on p.173). If there is only one recognition site per DNA molecule, add a DNA oligonucleotide containing the recognition site.

1.3.4.4. Site Preferences by Restriction Enzymes. The DNA sequence surrounding the recognition site may influence the efficiency of digestion. Some DNA sites

are cleaved slowly or not cleaved at all (for more details see p.173) due to the surrounding sequence. Use ad-ditional units (5-10 u) of conventional restriction enzyme per 1 µg of DNA or determine if an isoschizomer has superior cleavage efficiency (see Table 1.25 on p.207 or REsearch™ engine at www.fermentas.com/research).



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1.4. Water contains impurities.• Compare your results using commercially available nuclease free, molecular biology grade water, e.g. Water,

nuclease-free (#R0581). Check the quality of the water used in your lab. • Check the pH and conductivity of water. The pH of high quality water should be 5.5-6.0 with a resistance of >18 MΩ.• Centrifuge (10 min, 10,000 rpm) 1 ml of water and check if there is a visible pellet.• Determine if water contains nucleases or bacterial contamination (see 3.2 for control reactions).

2. Unexpected cleavage pattern

2.1. Star activity (relaxed specificity) of restriction enzyme (see p.174 for more details).• Use FastDigest® restriction enzymes. For these enzymes the incubation time without star activity is determined. • Reduce the amount of conventional restriction enzyme (use no more than 10 u).• Use the recommended reaction buffer.• Ensure that the glycerol concentration in the reaction mixture does not exceed 5%. • Reduce the incubation time. For FastDigest® enzymes – refer to Table 1.3 on p.71 for maximum incubation times.• Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation. The

resulting increase in glycerol concentration may cause star activity. 2.2. Contamination with another restriction enzyme. The restriction enzyme or buffer may be contaminated with another restriction enzyme due to improper

handling. Use a new tube of enzyme and/or buffer.2.3. Contamination with another substrate DNA. The sample DNA may contain a mixture of two or more different DNAs. Prepare new sample of DNA. • For plasmid DNA preparation pick one isolated colony of recombinant E.coli grow cells and purify plasmid

with GeneJET™ Plasmid Miniprep Kit (#K0503).• For PCR products: check the product purity on an agarose gel. If necessary, purify the PCR product prior to

digestion with GeneJET™ Gel Extraction Kit (#K0692) or Silica Bead DNA Gel Extraction Kit (#K0513).2.4. Incomplete DNA digestion (see 1). 2.5. Gel shift (see 3.1).2.6. Unexpected recognition sites in template DNA. Newly generated target sites in constructed DNA may be overlooked. Re-check your DNA sequence and

cloning strategy. Refer to Tables 1.21, 1.22, 1.23 or 1.24 for Newly Generated Recognition Sequences (pp.182-206) to identify all the cleavage sites present in the substrate DNA.

3. Diffused DNA bands

3.1. Gel shift. Enzyme that remains bound to the substrate DNA will affect the electrophoretic mobility of the digestion products.

FastDigest® Restriction Enzymes BspMI (BveI), HgaI (CseI), AcuI (Eco57I), FokI, MboII, TauI, TspRI (TscAI) and con-ventional restriction enzymes AarI, AloI, BdaI, BseXI, BveI, CseI, Eco57I, Eco57MI, EcoRII, FaqI, GsuI, Lsp1109I, LweI, MboII, MnlI, SchI, TauI, TscAI, TsoI and TstI are particularly prone to remaining bound to the substrate DNA. This will result in a band or smear above the expected band (see picture below). Heat the digested DNA for 10 min. at 65°C in the presence of 6X DNA Loading Dye & SDS Solution (#R1151) or 0.2% SDS prior to electrophoresis.

M – GeneRuler™ DNA Ladder Mix (#SM0331).1 – 0.5 µg λ DNA prepared for loading with 6X DNA Loading Dye (#R0611).2 – 0.5 µg λ DNA prepared for loading with 6X DNA Loading Dye & SDS Solution (#R1151).3 – 0.5 µg λ DNA digested with TsoI (#ER1991), probe prepared for loading with 6X DNA Loading Dye (#R0611).4 – 0.5 µg λ DNA digested with TsoI, probe prepared for loading with 6X DNA Loading Dye & SDS Solution (#R1151).

M 1 2 3 4

3.2. Contaminated reagents. Any restriction digestion reaction components may become contaminated with nucleases due to improper handling

or storage. Nuclease contamination causes DNA degradation, which appears as diffused DNA bands on a gel. Perform four control reactions in order to check for the nuclease contamination: I – without restriction enzyme, II – with a new vial of buffer, III – without restriction enzyme, with a new vial

of buffer, IV – with commercially available water e.g. Water, nuclease-free (#R0581).• Contaminated sample DNA (diffused bands in all controls). Prepare new DNA sample (re-purify DNA).• Contaminated enzyme (diffused bands in controls 2 and 4). The enzyme may become contaminated due to

improper handling. Use a new vial of enzyme.• Contaminated buffer (diffused bands in controls 1 and 4). Bacterial contamination of the reaction buffer will

cause DNA degradation. Use a new vial of buffer. Store all buffers at -20°C.• Contamination of both enzyme & buffer (diffused bands in controls 1, 2 and 4). Follow the recommendations given above.• Contaminated water (diffused bands in controls 1, 2 and 3). Bacterial or DNase contamination in improperly handled water

will cause DNA degradation. Use commercially available nuclease-free molecular biology grade water (e.g. #R0581).


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