topo ta cloning - molbio.mgh.harvard.edu · iv kit contents and storage shipping and storage the...
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TOPOª TA Cloning¨
Version E
18071425-0184
TOPOª TA Cloning¨
five-minute cloning of Taq polymerase-amplified PCR products
Catalog nos. K4500-01, K4500-40, K4550-01, K4550-40 (pCR¨2.1-TOPO)
Catalog nos. K4600-01, K4600-40, K4650-01, K4650-40 (pCR¨II-TOPO)
U.S. Headquarters: European Headquarters:Invitrogen Corporation Invitrogen BV1600 Faraday Avenue PO Box 2312, CH GroningenCarlsbad, CA 92008 The NetherlandsTel: (800) 955-6288 Tel: +31 (0) 50 5299 299Fax: (760) 603-7201 Fax: +31 (0) 50 5299 280E-mail: [email protected] E-mail: [email protected]
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Table of Contents
Kit Contents and Storage ............................................................................................. ivMethods .......................................................................................................................... 1
Overview....................................................................................................................................... 1
Using TOPOª TA Cloning¨ ........................................................................................................ 3
Map of pCR¨2.1-TOPO................................................................................................................ 6
Map of pCR¨II-TOPO .................................................................................................................. 7
TOPOª TA Cloning¨ Control Reactions..................................................................................... 8
Appendix ........................................................................................................................ 11Purifying PCR Products ................................................................................................................ 11
Addition of 3« A-Overhangs Post-Amplification ......................................................................... 12
Recipes .......................................................................................................................................... 13
Technical Service .......................................................................................................................... 14
References..................................................................................................................................... 16
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Kit Contents and Storage
Shipping andStorage
The TOPOª TA Cloning¨ Kit is shipped on dry ice. Each kit contains a box withTOPOª TA Cloning¨ reagents (Box 1) and a box with TOP10 or TOP10F« One Shotª
competent cells (Box 2). Store Box 1 at -20¡C and Box 2 at -80¡C.
Types of TOPOª
TA Cloning¨ KitsTwo different types of TOPOª TA Cloning¨ Kits are available in two different packsizes. Each type is available with either TOP10 or TOP10F« One Shotª competent cells(see next page for genotypes of the strains).
Product Pack Size One Shotª Cells Catalog no.
TOPOª TA Cloning¨ Kit
(containing pCR¨2.1-TOPO)
20 TOP10 K4500-01
40 TOP10 K4500-40
20 TOP10F« K4550-01
40 TOP10F« K4550-40
TOPOª TA Cloning¨ Kit Dual Promoter
(containing pCR¨II-TOPO)
20 TOP10 K4600-01
40 TOP10 K4600-40
20 TOP10F« K4650-01
40 TOP10F« K4650-40
TOPOª TACloning¨ Reagents
TOPOª TA Cloning¨ reagents (Box 1) are listed below. Please note that Taqpolymerase must be supplied by the user. Store Box 1 at -20¡C.
Item Concentration Amount
pCR¨2.1-TOPO or
pCR¨II-TOPO
10 ng/µl plasmid DNA in:
50% glycerol
50ÊmM Tris-HCl, pH 7.4 (at 25¡C)
1 mM EDTA
1 mM DTT
0.1% Triton X-100
100 µg/ml BSAphenol red
1 tube of 20 µl
10X PCR Buffer 100ÊmM Tris-HCl, pH 8.3 (at 42¡C)
500 mM KCl
25 mM MgCl2
0.01% gelatin
100 µl
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Kit Contents and Storage, continued
TOPOª TA Cloning¨ Reagents, continued
50 mM dNTPs 12.5 mM dATP
12.5 mM dCTP
12.5 mM dGTP
12.5 mM dTTP
neutralized at pH 8.0 in water
10 µl
M13 Forward (-20) Primer 0.1ʵg/µl in TE Buffer 20 µl
M13 Reverse Primer 0.1ʵg/µl in TE Buffer 20 µl
Control Template 0.1ʵg/µl in TE Buffer 10ʵl
Amplification Primer 1 0.1ʵg/µl in TE Buffer 10ʵl
Amplification Primer 2 0.1ʵg/µl in TE Buffer 10ʵl
Sterile Water -- 1 ml
One Shotª
ReagentsThe table below describes the items included in the One Shotª competent cell kit. Storeat -80¡C.
Item Composition Amount
SOC Medium
(may be stored at +4¡C orroom temperature)
2% Tryptone
0.5% Yeast Extract
10ÊmM NaCl
2.5ÊmM KCl
10ÊmM MgCl2
10ÊmM MgSO4
20ÊmM glucose
6 ml
b-mercaptoethanol 0.5ÊM 50ʵl
TOP10 or TOP10F« cells -- 21 x 50ʵl
pUC18 Control DNA 10Êng/µl 10ʵl
Genotypes ofE.Êcoli Strains
TOP10: Use this strain for general cloning and blue/white screening without IPTG.Please note that this strain cannot be used for single-strand rescue of DNA.
F- mcrA D(mrr-hsdRMS-mcrBC) F80lacZDM15 DlacC74 recA1 deoR araD139 D(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG
TOP10F«: This strain expresses the Lac repressor (lacIq gene). For blue/white screening,you will need to add IPTG to the plates to obtain expression from the lac promoter. Thisstrain contains the F episome and can be used for single-strand rescue of plasmid DNAcontaining an f1 origin.
F« {lacIq Tn10 (TetR)} mcrA D(mrr-hsdRMS-mcrBC) F80lacZDM15 DlacC74 recA1deoR araD139 D(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG
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1
Methods
Overview
Introduction TOPOª TA Cloning¨* provides a highly efficient, 5 minute, one-step cloning strategy("TOPOª Cloning") for the direct insertion of Taq polymerase-amplified PCR productsinto a plasmid vector. No ligase, post-PCR procedures, or PCR primers containingspecific sequences are required.
How It Works The plasmid vector (pCR¨II-TOPO or pCR¨2.1-TOPO) is supplied linearized with:
¥ Single 3« thymidine (T) overhangs for TA Cloning¨
¥ Topoisomerase covalently bound to the vector (referred to as "activated" vector)
Taq polymerase has a nontemplate-dependent terminal transferase activity which adds asingle deoxyadenosine (A) to the 3« ends of PCR products. The linearized vector suppliedin this kit has single, overhanging 3« deoxythymidine (T) residues. This allows PCRinserts to ligate efficiently with the vector.
TOPOª Cloning exploits the ligation activity of topoisomerase by providing an"activated", linearized TA vector using proprietary technology (Shuman, 1994). Ligationof the vector with a PCR product containing 3« A overhangs is very efficient and occursspontaneously within 5Êminutes at room temperature. The TOPOª Cloning Reaction canbe transformed into chemically competent cells (provided) or electroporated directly intoelectrocompetent cells.
+
3'
ÒActivatedÓ TOPOª Cloning Vector
5'
T
Vector
T Vector3'
5'
Topoisomerase I
Topoisomerase I
Taq-amplified PCR productwith 3«-A overhangs
A
A
3'5'
5'3'
Taq-amplifiedPCR Product
5 minutes at room temperature
Ligation complete-ready for transformation
APCRProduct
T
T
Vector Vector
Topoisomerase I
Topoisomerase I
A
*The Fine Print Covered under U.S. Patent 5,487,993; 5,766,891; and corresponding foreign patents. Other patents pending andlicensed exclusively to Invitrogen.
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Overview, continued
ExperimentalOutline
¥ Produce Your PCR Product
¥ Set Up TOPOª Cloning Reaction (Mix Together PCR Product and pCR¨TOPOVector)
¥ Incubate 5 Minutes at Room Temperature
¥ Transform TOPOª Cloning Reaction into One Shotª Competent Cells
¥ Select and Analyze 10 White Colonies For Insert
NOTE
Do not add 5« phosphates to your primers for PCR. The PCR product synthesized willnot ligate into pCR¨2.1-TOPO or pCR¨II-TOPO.
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Using TOPO™ TA Cloning®
Introduction The section below outlines the procedures for generating PCR products, TOPOª Cloningyour PCR product, and transforming the resulting construct into TOP10 One Shotª cells.
Materials Suppliedby the User
In addition to general microbiological supplies (i.e. plates, spreaders), you will need thefollowing reagents and equipment.
¥ Taq polymerase
¥ Thermocycler
¥ DNA template and primers for PCR product
¥ 42¡C water bath
¥ LB plates containing 50 µg/ml ampicillin or 50ʵg/ml kanamycin (two pertransformation)
¥ 40Êmg/ml X-gal in DMF (dimethylformamide)
¥ 100ÊmM IPTG in water (for use with TOP10F«)
¥ 37¡C shaking and non-shaking incubator
Producing PCRProducts
1. Set up the following 50ʵl PCR reaction. Use the cycling parameters suitable foryour primers and template and be sure to end with a 7 to 30 minute extension at72¡C to ensure that all PCR products are full length and 3« adenylated.
DNA Template 10-100Êng
10X PCR Buffer 5ʵl
50ÊmM dNTPs 0.5ʵl
Primers (~200Êng each) 1ʵM each
Sterile water add to a final volume of 49ʵl
Taq Polymerase (1Êunit/µl) 1Êunit
Total Volume 50ʵl
2. Check the PCR product by agarose gel electrophoresis. You should see a single,discrete band. If not, gel-purify your fragment before using TOPOª TA Cloning¨
(see page 11). Take special care to avoid sources of nuclease contamination.Alternatively, you may elect to optimize your PCR to eliminate multiple bands andsmearing (Innis et al., 1990).
Preparation For each transformation, you will need one vial of competent cells and two selective plates.
¥ Equilibrate a water bath to 42¡C.
¥ Thaw the vial of SOC medium from Box 2 and bring to room temperature.
¥ Warm LB plates containing 50ʵg/ml ampicillin OR 50ʵg/ml kanamycin at 37¡C for30Êminutes.
¥ Spread 40ʵl of 40 mg/ml X-gal on each LB plate and incubate at 37¡C until ready for use.
¥ If using TOP10F« cells spread 40ʵl of 100 mM IPTG, in addition to X-gal, on each LBplate and incubate at 37¡C until ready for use. IPTG is required for blue/white screening.
¥ Thaw on ice 1 vial of One Shotª cells for each transformation. Place the b-mercapto-ethanol on ice.
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Using TOPO™ TA Cloning®, continued
TOPOª CloningReaction
In general, 0.5 to 2ʵl of a typical PCR sample (10Êng/µl) with an average insert length of400 to 1000Êbp will give the proper insert:vector ratio for TOPOª Cloning.
1. Set up the following 5ʵl TOPOª Cloning reaction.
Fresh PCR product 0.5 to 2ʵl
Sterile Water add to a final volume of 4ʵl
pCR¨-TOPO vector 1ʵl
Final Volume 5ʵl
2. Mix gently and incubate for 5 minutes at room temperature (~25¡C). For the bestpossible results, do not leave for more than 5 minutes or the transformationand cloning efficiencies will decrease. (See note on the next page.)
3. Briefly centrifuge and place tube on ice. Proceed immediately to One Shotª
Transformation Reaction, below.
Note on TOPOª
Cloning ReactionPlease note that after completion of the 5 minute, room temperature TOPOª Cloningreaction we have incubated the reaction tube on ice for up to 30 to 60 minutes prior totransformation. We have not seen more than a 50% decrease in the number of colonies.However, your results may vary depending on the nature of the insert.
One Shotª
TransformationReaction
Important: Please note that TOP10 One Shotª competent cells are supplied at atransformation efficiency of 1 x 109. TOP10F« One Shotª cells are supplied at atransformation efficiency of 1 x 108. Please pay careful attention to the platinginstructions in Step 8.
1. Add 2ʵl of 0.5ÊM b-mercaptoethanol to each vial of competent cells and mix bystirring gently with the pipette tip. DO NOT MIX BY PIPETTING UP ANDDOWN.
2. Add 2ʵl of the TOPOª Cloning reaction into a vial of One Shotª cells and mixgently.
3. Incubate on ice for 30 minutes.
4. Heat shock the cells for 30 seconds at 42¡C without shaking.
5. Immediately transfer the tubes to ice and incubate for 2Êminutes.
6. Add 250ʵl of room temperature SOC medium.
7. Cap the tube tightly and shake the tube horizontally at 37¡C for 30 minutes(ampicillin selection) or 1 hour (kanamycin selection). Place on ice.
8. If you are using TOP10 cells, spread 10-50 µl from each transformation ontoselective plates.
If you are using TOP10F« cells, spread 50-100ʵl from each transformation ontoselective plates.
NOTE: We recommend that you plate two volumes to ensure that one plate willhave well-spaced colonies. For plating small volumes, add 20 µl of SOC to alloweven spreading.
9. Incubate overnight at 37¡C.
10. An efficient TOPOª Cloning reaction will produce hundreds of colonies. Pick ~10white or light blue colonies for analysis. Do not pick dark blue colonies.
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Using TOPO™ TA Cloning®, continued
Analysis of PositiveClones
1. Take the 10 white or light blue colonies and culture them overnight in LB mediumcontaining 50ʵg/ml ampicillin or kanamycin.
2. Isolate plasmid DNA using your method of choice. If you need ultra-pure plasmidDNA for automated or manual sequencing, we recommend the S.N.A.P.ª MiniPrepKit (Catalog no. K1900-01).
3. Analyze the plasmids by restriction analysis (digest with EcoRÊI or refer to thevector map accompanying the manual for alternate sites) or by sequencing. M13Forward (-20) and M13 Reverse primers are included to help you sequence yourinsert. Please refer to the maps on page 6 (pCR¨2.1-TOPO) or page 7 (pCR¨II-TOPO) for sequence surrounding the TOPOª TA Cloning¨ site. For the fullsequence of either vector, please see our Web site or contact Technical Service(page 14).
If you need help with setting up restriction enzyme digests or DNA sequencing,please refer to general molecular biology texts (Ausubel et al., 1994; Sambrook etal., 1989).
AlternativeMethod of Analysis
You may wish to use PCR to directly analyze positive transformants. You may use theM13 Forward (-20) and the M13 Reverse primers as PCR primers. If this is the first timeyou have used this technique, we recommend that you perform restriction analysis inparallel to confirm that PCR gives you the correct result. False negative results can beobtained because of mispriming or contaminating template.
The following protocol is provided for your convenience. Other protocols are suitable.
1. Prepare a PCR cocktail consisting of PCR buffer, dNTPs, primers, and Taqpolymerase. Use a 20 µl reaction volume. Multiply by the number of colonies to beanalyzed (e.g.Ê10).
2. Pick 10 colonies and resuspend them individually in 20ʵl of the PCR cocktail.
3. Incubate the reaction for 10 minutes at 94¡C to lyse the cells and inactivate nucleases.
4. Amplify for 20 to 30 cycles (94¡C for 1 minute, 55¡C for 1 minute, and 72¡C for1Êminute).
5. For the final extension, incubate at 72¡C for 10 minutes. Hold at +4¡C.
6. Visualize by agarose gel electrophoresis.
Long-TermStorage
Once you have identified the correct clone, be sure to purify the colony and make aglycerol stock for long term storage.
¥ Streak the original colony out for single colony on LB plates containing 50ʵg/mlampicillin.
¥ Isolate a single colony and inoculate into 1-2 ml of LB containing 50ʵg/ml ampicillin.
¥ Grow overnight until culture is saturated.
¥ Mix 0.85Êml of culture with 0.15Êml of sterile glycerol and transfer to a cryovial.
¥ Store at -80¡C.
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Map of pCR®2.1-TOPO
pCR¨2.1-TOPOMap
The map below shows the features of pCR¨2.1-TOPO and the sequence surrounding theTOPOª Cloning site. Restriction sites are labeled to indicate the actual cleavage site. Thestart of transcription for T7 polymerase is indicated by the arrow. For the full sequence ofthe vector, you may download it from our Web site or call Technical Service (page 14).
Comments for pCR¨2.1-TOPO 3908 nucleotides
LacZa fragment: bases 1-571M13 reverse priming site: bases 205-221Multiple cloning site: bases 234-357T7 promoter/priming site: bases 364-383M13 Forward (-20) priming site: bases 391-406M13 Forward (-40) priming site: bases 411-426f1 origin: bases 548-962Kanamycin resistance ORF: bases 1296-2090Ampicillin resistance ORF: bases 2108-2968ColE1 origin: bases 3113-3786
CAG GAA ACA GCT ATG ACC ATG ATT ACG CCA AGC TTG GTA CCG AGC TCG GAT CCA CTAGTC CTT TGT CGA TAC TGG TAC TAA TGC GGT TCG AAC CAT GGC TCG AGC CTA GGT GAT
T7 Promoter M13 Forward (-20) Primer M13 Forward (-40) Primer
Not I Xho I Nsi I Xba I Apa I
lacZa
f1
+1
Plac
Ampicillin
Co
lE1
ori
Kanamyc
in
pCR®2.1-TOPO3.9 kb
M13 Reverse PrimerlacZa ATG
Kpn I Sac I Spe IBamH IHind III
EcoR IEcoR IBstX I
BstX IEcoR V
AGA TAT CCA TCA CAC TGG CGG CCG CTC GAG CAT GCA TCT AGA GGG CCC AAT TCG CCC TATTCT ATA GGT AGT GTG ACC GCC GGC GAG CTC GTA CGT AGA TCT CCC GGG TTA AGC GGG ATA
AGT GAG TCG TAT TAC AAT TCA CTG GCC GTC GTT TTA CAA CGT CGT GAC TGG GAA AACTCA CTC AGC ATA ATG TTA AGT GAC CGG CAG CAA AAT GTT GCA GCA CTG ACC CTT TTG
PCR ProductGTA ACG GCC GCC AGT GTG CTG GAA TTC GCC CTT AAG GGC GAA TTC TGCCAT TGC CGG CGG TCA CAC GAC CTT AAG CGG GAA TTC CCG CTT AAG ACG
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Map of pCR®II-TOPO
pCR¨II-TOPOMap
The map below shows the features of pCR¨II-TOPO and the sequence surrounding theTOPOª Cloning site. Restriction sites are labeled to indicate the actual cleavage site. Thestart of transcription for Sp6 and T7 polymerases are indicated by the arrows. For the fullsequence of the vector, you may download it from our Web site or call Technical Service(page 14).
Comments for pCR¨II-TOPO 3950 nucleotides
LacZa gene: bases 1-588M13 Reverse priming site: bases 205-221Sp6 promoter: bases 239-256Multiple Cloning Site: bases 269-399T7 promoter: bases 406-425M13 (-20) Forward priming site: bases 433-448M13 (-40) Forward priming site: bases 453-468f1 origin: bases 590-1004Kanamycin resistance ORF: bases 1338-2132Ampicillin resistance ORF: bases 2150-3010ColE1 origin: bases 3155-3828
lacZ
f1
+1Plac
Ampicillin
Co
lE1
ori
Kanamyc
in
pCR®II-TOPO3.9 kb
M13 Reverse Primer Sp6 Promoter
T7 Promoter M13 (-20) Forward Primer M13(-40) Forward Primer
Nsi I Hind III Kpn I Sac I Spe IBamH I
BstX I Not I Xho I Nsi I Xba I Apa I
BstX I EcoR I EcoR I EcoR V
lacZa ATG
CAG GAA ACA GCT ATG ACC ATG ATT ACG CCA AGC TAT TTA GGT GAC ACT ATA GAAGTC CTT TGT CGA TAC TGG TAC TAA TGC GGT TCG ATA AAT CCA CTG TGA TAT CTT
TAC TCA AGC TAT GCA TCA AGC TTG GTA CCG AGC TCG GAT CCA CTA GTA ACG GCCATG AGT TCG ATA CGT AGT TCG AAC CAT GGC TCG AGC CTA GGT GAT CAT TGC CGG
CCA TCA CAC TGG CGG CCG CTC GAG CAT GCA TCT AGA GGG CCC AAT TCG CCC TATGGT AGT GTG ACC GCC GGC GAG CTC GTA CGT AGA TCT CCC GGG TTA AGC GGG ATA
PCR ProductGCC AGT GTG CTG GAA TTC GCC CTT AAG GGC GAA TTC TGC AGA TATCGG TCA CAC GAC CTT AAG CGG GAA TTC CCG CTT AAG ACG TCT ATA
AGT GAG TCG TAT TAC AAT TCA CTG GCC GTC GTT TTA CAA CGT CGT GAC TGG GAA AACTCA CTC AGC ATA ATG TTA AGT GAC CGG CAG CAA AAT GTT GCA GCA CTG ACC CTT TTG
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TOPO™ TA Cloning® Control Reactions
Introduction We recommend performing the following control TOPOª Cloning reactions the first timeyou use the kit to help you evaluate your results. Performing the control reactionsinvolves producing a control PCR product using the reagents included in the kit and usingit directly in a TOPOª Cloning reaction.
Producing ControlPCR Product
1. To produce the 750 bp control PCR product, set up the following 50ʵl PCR:
Control DNA Template (100Êng) 1ʵl
10X PCR Buffer 5ʵl
50 mM dNTPs 0.5ʵl
Amplification Primer #1 (0.1ʵg/µl) 1ʵl
Amplification Primer #2 (0.1ʵg/µl) 1ʵl
Sterile Water 40.5ʵl
Taq Polymerase (1Êunit/µl) 1ʵl
Total Volume 50ʵl
2. Overlay with 70 µl (1 drop) of mineral oil.
3. Amplify using the following cycling parameters:
Step Time Temperature Cycles
Initial Denaturation 2 minute 94¡C 1X
Denaturation 1 minute 94¡C
Annealing 1 minute 55¡C 25X
Extension 1 minute 72¡C
Final Extension 7 minutes 72¡C 1X
4. Remove 10 µl from the reaction and analyze by agarose gel electrophoresis. Adiscrete 750Êbp band should be visible. Proceed to the Control TOPOª CloningReactions, next page.
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TOPO™ TA Cloning® Control Reactions, continued
Control TOPOª
Cloning ReactionsUsing the control PCR product produced above and the TOPOª TA Cloning¨ vector, setup two 5ʵl TOPOª Cloning reactions as described below.
1. Set up control TOPOª Cloning reactions:
Reagent "Vector Only" "Vector + PCR Insert"
Control PCR Product -- 1ʵl
Sterile Water 4ʵl 3ʵl
TOPOª TA Cloning¨
vector1ʵl 1ʵl
2. Incubate at 25¡C (room temperature) for 5 minutes and place on ice. Do not incu-bate longer than 5 minutes.
3. Transform 2ʵl of each reaction into separate vials of TOP10 One Shotª cells(pageÊ4).
4. Spread 10-50ʵl of each transformation mix onto LB plates containing 50ʵg/mlkanamycin (or ampicillin) and X-Gal (and IPTG, if using TOP10F« cells) (seepageÊ13). Be sure to plate two different volumes to ensure well-spaced colonies. Forplating small volumes, add 20ʵl of SOC to allow even spreading.
5. Incubate overnight at 37¡C.
Analysis of Results Hundreds of colonies from the vector + PCR insert reaction should be produced. Greaterthan ninety-five percent of these colonies will be white and 90% (or more) of these willcontain the 750Êbp insert when analyzed by EcoRÊI digestion and agarose gelelectrophoresis.
Relatively few colonies will be produced in the vector-only reaction and most of thesewill be dark blue. You may observe a few white colonies. This results from removal ofthe 3« deoxythymidine overhangs creating a blunt-end vector. Ligation (re-joining) of theblunt ends will result in disruption of the LacZa reading frame leading to the productionof white colonies.
TransformationControl
pUC18 plasmid is included to check the transformation efficiency of the One Shotª
competent cells. Transform with 10Êpg per 50ʵl of cells using the protocol on page 4. Forplating instructions, please refer to the table below:
Cells Volume to Plate Transformation Efficiency
TOP10 10ʵl + 20ʵl SOC 1 x 109 cfu/µg DNA
TOP10F« 50ʵl 1 x 108 cfu/µg DNA
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TOPO™ TA Cloning® Control Reactions, continued
Factors AffectingCloning Efficiency
Please note that lower cloning efficiencies will result from the following variables. Mostof these are easily correctable, but if you are cloning large inserts, you may not obtain theexpected 95% (or more) cloning efficiency.
Variable Solution
Incubations longer than 5 minutes Be sure to incubate for only 5 minutes.Incubations longer than 5 minutes willdecrease transformation and cloningefficiencies.
pH>9 Check the pH of the PCR amplificationreaction and adjust with 1ÊM Tris-HCl, pH 8.
Incomplete extension during PCR Be sure to include a final extension step of 7to 30 minutes during PCR. Longer PCRproducts will need a longer extension time.
Cloning large inserts (>1Êkb) Increase amount of insert. Or gel purify asdescribed on page 11.
Excess (or overly dilute) PCR product Reduce (or concentrate) the amount of PCRproduct.
Cloning blunt-ended fragments Add 3« A-overhangs by incubating with Taqpolymerase (page 12) or use the Zero Bluntª
PCR Cloning Kit (Catalog no. K2700-20).
PCR cloning artifacts ("false positives") TOPOª Cloning is very efficient for smallfragments (< 100Êbp) present in certain PCRreactions. Gel-purify your PCR product(page 11).
PCR product does not contain sufficient3« A-overhangs even though you usedTaq polymerase
Increase the final extension time to ensure all3« ends are adenylated.
Taq polymerase is less efficient at adding anontemplate 3« A next to another A. Taq ismost efficient at adding a nontemplate 3« Anext to a C. You may have to redesign yourprimers so that they contain a 5« G instead ofa 5« T (Brownstein et al., 1996).
11
Appendix
Purifying PCR Products
Introduction Smearing, multiple banding, primer-dimer artifacts, or large PCR products (>1Êkb) maynecessitate gel purification. If you intend to purify your PCR product, be extremelycareful to remove all sources of nuclease contamination. There are many protocols toisolate DNA fragments or remove oligonucleotides. Please refer to Current Protocols inMolecular Biology, Unit 2.6 (Ausubel et al., 1994) for the most common protocols. Twosimple protocols are provided below that work for most people.
Using theS.N.A.P.ªMiniPrep Kit
The S.N.A.P.ª MiniPrep Kit (Catalog no. K1900-01) allows you to rapidly purify PCRproducts from regular agarose gels. You will need to prepare 6ÊM sodium iodide in sterilewater before starting. A small amount (spatula tip-full) of sodium sulfite may be added tothe NaI solution to prevent oxidation.
1. Electrophorese amplification reaction on a 1 to 5% regular TAE agarose gel.
2. Cut out the gel slice containing the PCR product and melt it at 65¡C in 2 volumes of6ÊM NaI.
3. Add 1.5 volumes Binding Buffer (provided in the S.N.A.P.ª MiniPrep Kit).
4. Load solution (no more than 1Êml at a time) from Step 3 onto a S.N.A.P.ª column.Centrifuge 1 minute at full speed in a microcentrifuge and discard the supernatant.
5. If you have solution remaining from Step 3, repeat Step 4.
6. Add 900 µl of the Final Wash Buffer (provided in the S.N.A.P.ª MiniPrep Kit).
7. Centrifuge 1 minute at full speed in a microcentrifuge and discard the supernatant.
8. Repeat Step 7.
9. Elute the purified PCR product in 40ʵl of TE or sterile water. Use 4ʵl for theTOPOª Cloning reaction and proceed as described on page 4.
Low-Melt AgaroseMethod
This is probably the easiest method for purification and TOPOª Cloning of PCRproducts. Please note that gel purification will result in a dilution of your PCR product.
1. Electrophorese as much as possible of your PCR reaction on a low-melt agarose gel(0.8 to 1.2%) in TAE buffer.
2. Visualize the band of interest and excise the band.
3. Place the gel slice in a microcentrifuge tube and incubate the tube at 65¡C until thegel slice melts.
4. Place the tube at 37¡C to keep the agarose melted.
5. In a fresh tube, mix together 4ʵl of the melted agarose containing your PCRproduct and 1ʵl of the TOPOª TA Cloning¨ vector.
6. Incubate at 37¡C for 5 to 10 minutes.
7. Transform 2 to 4ʵl directly into TOP10 One Shotª cells using the method onpageÊ4.
NOTE
Please note that cloning efficiency may decrease with purification of the PCR product.You may wish to optimize your PCR to produce a single band.
12
Addition of 3´ A-Overhangs Post-Amplification
Introduction Direct cloning of DNA amplified by Vent¨ or Pfu polymerases into TOPOª TACloning¨ vectors is often difficult because of very low cloning efficiencies. This isbecause proofreading polymerases do not have the terminal transferase activity whichadds the 3«ÊA-overhangs necessary for TA Cloning¨. Invitrogen has developed a simplemethod to clone these blunt-ended fragments.
Before Starting You will need the following items:
¥ Taq polymerase
¥ A heat block equilibrated to 72¡C
¥ Phenol-chloroform
¥ 3ÊM sodium acetate
¥ 100% ethanol
¥ 80% ethanol
¥ TE buffer
Procedure This is just one method for adding 3« adenines. Other protocols may be suitable.
1. After amplification with Vent¨ or Pfu polymerase, place vials on ice and add 0.7-1Êunit of Taq polymerase per tube. Mix well. It is not necessary to change thebuffer.
2. Incubate at 72¡C for 8-10 minutes (do not cycle).
3. Extract immediately with an equal volume of phenol-chloroform. Extraction withphenol-chloroform removes all the polymerases. Note: If you have more than oneband, you may wish to gel-purify your fragment after the phenol-chloroformextraction.
4. Precipitate the DNA by adding 1/10 volume of 3ÊM sodium acetate and 2X volumeof 100% ethanol.
5. Centrifuge at maximum speed for 5 minutes at room temperature to pellet the DNA.
6. Remove the ethanol, rinse the pellet with 80% ethanol, and allow to air dry.
7. Resuspend the pellet in TE buffer to the starting volume of the DNA amplificationreaction. The DNA amplification product is now ready for ligation into the TOPOª
TA Cloning¨ vector.
NOTE
You may also gel-purify your PCR product after amplification with Vent¨ or Pfu. Afterpurification, add Taq polymerase buffer, dATP, and 0.5Êunit of Taq polymerase andincubate 10-15 minutes at 72¡C and use as is in the TOPOª Cloning reaction.
13
Recipes
LB (Luria-Bertani)Medium and Plates
Composition:
1.0% Tryptone0.5% Yeast Extract1.0% NaClpHÊ7.0
1. For 1 liter, dissolve 10Êg tryptone, 5Êg yeast extract, and 10Êg NaCl in 950Êmldeionized water.
2. Adjust the pH of the solution to 7.0 with NaOH and bring the volume up to 1 liter.
3. Autoclave on liquid cycle for 20 minutes at 15Êpsi. Allow solution to cool to 55¡Cand add antibiotic if needed.
4. Store at room temperature or at +4¡C.
LB agar plates
1. Prepare LB medium as above, but add 15Êg/L agar before autoclaving.
2. Autoclave on liquid cycle for 20 minutes at 15Êpsi.
3. After autoclaving, cool to ~55¡C, add antibiotic (50ʵg/ml of either ampicillin orkanamycin), and pour into 10Êcm plates.
4. Let harden, then invert and store at +4¡C, in the dark.
X-Gal StockSolution
1. To make a 40Êmg/ml stock solution, dissolve 400Êmg X-Gal in 10Êmldimethylformamide.
2. Protect from light by storing in a brown bottle at -20¡C.
3. To add to previously made agar plates, warm the plate to 37¡C. Pipette 40ʵl of the40Êmg/ml stock solution onto the plate, spread evenly, and let dry 15 minutes.Protect plates from light. Note: If you need to add IPTG, see below.
IPTG StockSolution
1. Prepare a 100ÊmM stock solution by dissolving 238Êmg of IPTG in 10Êml deionizedwater.
2. Filter-sterilize and store in 1Êml aliquots at -20¡C.
3. Add 40ʵl of the IPTG stock solution onto the center of the plate and spread evenlywith a sterile spreader.
4. Allow the solution to diffuse into the plate by incubating at 37¡C for 20-30 minutes.Plates are now ready for use.
14
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16
References
References Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.,and, K. (1994) Current Protocols in Molecular Biology. (New York: GreenePublishing Associates and Wiley-Interscience).
Brownstein, M. J., Carpten, J. D., and Smith, J. R. (1996). Modulation of Non-TemplatedNucleotide Addition by Taq DNA Polymerase: Primer Modifications that FacilitateGenotyping. BioTechniques 20, 1004-1010.
Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. S. (1990) PCR Protocols: AGuide to Methods and Applications. (San Diego, CA: Academic Press).
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A LaboratoryManual, Second Edition (Plainview, New York: Cold Spring Harbor LaboratoryPress).
Shuman, S. (1994). Novel Approach to Molecular Cloning and Polynucleotide SynthesisUsing Vaccinia DNA Topoisomerase. J. Biol. Chem. 269, 32678-32684.
©1997 Invitrogen Corporation. Reproduction forbidden without permission.