マニュアル flexiprep kit...possible causes/solutions 1. centrifugation was too harsh.spin in a...

instructions

FlexiPrep Kitproduct code:

27-9281-01

WarningFor research use only.Not recommended orintended for the diagnosisof disease in humans or animals.Do not use internally orexternally in humans or animals.

Harmful

i 74004103 Ed. AA

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HandlingStorageStore at ambienttemperature.

Page finderComponents of the kit . . . . . . . . . . . . . . . . . . . . . 3

Quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Materials not supplied . . . . . . . . . . . . . . . . . . . . . 3

World Wide Web address. . . . . . . . . . . . . . . . . . . 4

Safety warnings and precautions . . . . . . . . . . . . . 4

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

ProtocolsIntroduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Essential preliminaries . . . . . . . . . . . . . . . . . . . . . 81. Purification of plasmid DNA from 1.5 ml

cultures of E. coli (miniprep procedure). . . . . . 92. Purification of plasmid DNA from 50 ml

cultures of E. coli (midiprep procedure). . . . . 113. Purification of plasmid DNA from 500 ml

cultures of E. coli (maxiprep procedure) . . . . 14

Appendixes1. Bacterial cultures . . . . . . . . . . . . . . . . . . . . . 172. Streamlined protocol for minipreps . . . . . . . . 183. Isolation of cosmid DNA . . . . . . . . . . . . . . . . 20

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . 23

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Licenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Trademarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Components of the kit

The following components are included inthis product:

Solution I 100 mM Tris-HCl (pH 7.5),10 mM EDTA, 400 µg/ml RNase I.

Solution II*† 1 M NaOH, 5.3% (w/v) SDS(30 ml of a 5.3× concentratedsolution). Add 130 ml of distilledwater to solution II and mix byinversion.

Solution III 3 M potassium,5 M acetate solution.

Sephaglas™ FP Sephaglas FP suspended in abuffered solution of guanidine-HCl [7 M Guanidine-HCl,50 mM Tris-HCl (pH 7.5),10 mM EDTA].

Wash buffer* 20 mM Tris-HCl (pH 7.5),2 mM EDTA, 200 mM NaCl.Add 96 ml of absolute ethanolto the wash buffer and mix byinversion. The final concentrationof ethanol is 60%.

*Note: After diluting these components, it is advis-able to indicate on their labels that this step hasbeen completed.

† NaOH is supplied in a concentrated form tocomply with IATA/DOT regulations which limit thevolume of this reagent that can be shipped.

Quality controlFlexiPrep™ Kit is tested forthe ability to isolate apUC18-derived plasmidfrom E. coli. The integrity ofthe plasmid is verified byagarose gel electrophoresisfollowing restriction enzymedigestion and religation.

Materials notsuppliedFor extraction andprufication of DNA:

Reagents

• Absolute ethanol

• Isopropanol

• 70% Ethanol

• TE buffer—10 mMTris-HCl, pH 8.0,1 mM EDTA (autoclave).

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World Wide Web address

http://www.gehealthcare.com/lifesciences

Visit the GE Healthcare home pagefor regularly updated product information.

Safety warnings and precautions

Warning: For research use only. Notrecommended or intended for diagnosisof disease in humans or animals. Do notuse internally or externally in humansor animals.

All chemicals should be considered aspotentially hazardous. Only persons trainedin laboratory techniques and familiar withthe principles of good laboratory practiceshould handle these products. Suitableprotective clothing such as laboratoryoveralls, safety glasses, and gloves shouldbe worn. Care should be taken to avoidcontact with skin or eyes; if contact shouldoccur, wash immediately with water (seeMaterial Safety Data Sheet for specificrecommendations).

Materials notsupplied (continued)

Reagents (continued)

For 100 ml and 500 mlcultures:

• STE—10 mM Tris-HCl(pH 8.0), 100 mM NaCl,1 mM EDTA.

• Lysozyme—Dissolve insolution I at 10 mg/mlprior to use.

• RNase A

Equipment

• Centrifuge tubes orbottles—10–500 ml

• Microcentrifuge tubes—1.5 ml

• Microcentrifuge

• Preparative centrifuge

• Tabletop centrifuge

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Introduction

FlexiPrep™ Kit is designed for the rapid extraction and purificationof plasmid DNA from E. coli cultures ranging in volume from 1.5 mlto 500 ml. The procedure can be completed in less than 2 hours(20–45 minutes for minipreps) to yield DNA with a functional purityequivalent to DNA isolated from CsCl gradients.The method employs astandard alkaline cell lysis procedure (1–3), including RNase treatmentand isopropanol precipitation. Plasmid DNA is subsequently purifiedusing Sephaglas FP, a novel glass matrix from GE Healthcare.Sephaglas FP is well suited to DNA purification for two reasons. First,DNA is bound selectively to the glass and eluted in a buffer of low ionicstrength for direct use. Second, the chaotropic salt required for bindingdenatures and removes proteins from the sample (4, 5).

The kit contains sufficient lysis solutions, Sephaglas FP and wash bufferfor the isolation of plasmid DNA from 750 minipreps using1.5 ml cultures, 30 midipreps using 50 ml cultures and 5 maxiprepsusing 500 ml cultures of E. coli. By making appropriate volume substi-tutions for the reagents, the protocols can be scaled to accommodate10 ml, 25 ml and 100 ml cultures as well. Additional procedures forstreamlined minipreps and for isolating cosmid DNA are included inthe appendixes.

The basic protocol for the FlexiPrep Kit involves the following steps:

Step Comments Component

Suspension Bacterial cells are suspended in an Solution Iisotonic solution containing RNase.

Cell lysis Cells are lysed by alkali treatment, Solution IIand chromosomal DNA and proteinsare denatured.

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Neutralization The pH of the lysate is neutralized Solution IIIusing a potassium acetate solution.Proteins, chromosomal DNA andcell debris precipitate and are removedby centrifugation, while supercoiledplasmid DNA remains in solution.

Concentration Plasmid DNA is concentrated forfurther purification steps byisopropanol precipitation.

DNA binding The plasmid DNA pellet is dissolved Sephaglas FPto Sephaglas in the Sephaglas slurry which provides

a guanidine concentration sufficientlyhigh to promote binding of DNAto the glass.

DNA DNA is bound to the Sephaglas.purification

Washing Sephaglas-bound DNA is washed Wash bufferwith ethanolic wash buffer and70% ethanol to removeresidual contaminants.

Drying The Sephaglas is air-dried to removeresidual ethanol which might inhibitsubsequent enzymatic manipulations.

DNA DNA is eluted from the Sephaglas in arecovery buffer of low ionic strength, ready for

use in restriction digestions, labellingreactions and DNA sequencing.

ProtocolsIntroduction

Scaling the protocol

The FlexiPrep Kit protocol can be conveniently adapted to process a widerange of culture volumes, including mini-, midi- and maxipreps. Optimizedprocedures are described for plasmid purification from starting volumesof 1.5 ml, 50 ml and 500 ml cultures. The same protocols can also beused for 10 ml, 25 ml and 100 ml cultures by substituting the appropriatereagent volumes from Table 1 on page 8. Minipreps can be performed in amicrocentrifuge while larger preps require a tabletop or preparative centrifuge.

DNA yield

DNA yield depends on the copy number and size of the plasmid, cell densityof the culture, and efficiency of cell lysis (see Appendix 1). Typically, 2–4 µgof pUC18 DNA can be purified per ml of bacterial culture with greater than80% of the DNA in one or more supercoiled forms.

Streamlining the protocol for minipreps

If a yield of at least 1 µg of plasmid DNA is expected from 1.5 ml cultures,a more rapid, streamlined protocol can be used (see Appendix 2). With thismodification, plasmid DNA equivalent in yield and purity to that obtainedusing the standard miniprep protocol, can be purified in about half the timerequired for the standard protocol.

Protocol for isolating cosmid DNA

High-quality cosmid DNA can be purified using the protocol outlined inAppendix 3. The standard FlexiPrep protocol is modified to use smallervolumes of the various solutions, to handle the sample more gently (tominimize shearing) and to elute at high temperature. Gentle handling isaccomplished by mixing samples either by slowly pipetting up and downor by carefully inverting the tube repeatedly. These modifications givereproducible results and yield purified cosmid DNA suitable for use inrestriction digests and PCR**.

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Table 1. Reagent volume requirements for different culture sizes.

Component 1.5 ml 10 ml 25 ml 50 ml 100 ml 500 ml

STE wash none none none none 20 ml 100 ml

Lysozyme none none none none 500 µl 1 ml

Solution I 200 µl 2 ml 3 ml 5 ml 10 ml 30 ml

Solution II 200 µl 2 ml 3 ml 5 ml 10 ml 30 ml

Solution III 200 µl 2 ml 3 ml 5 ml 10 ml 30 ml

Isopropanol 420 µl 4.2 ml 6.3 ml 10.5 ml 21 ml 63 ml

Sephaglas FP 150 µl 2 ml 3 ml 5 ml 10 ml 30 ml

Wash buffer 200 µl 2 ml 3 ml 5 ml 10 ml 30 ml

70% Ethanol 300 µl 2 ml 3 ml 5 ml 10 ml 30 ml

TE buffer 50 µl 200 µl 300 µl 500 µl 1 ml 5 ml

Essential preliminaries

Recommended conditions for the culture of E. coli are provided inAppendix 1.

Prior to the first use, the following reagents provided in the kit should bediluted as indicated:

• Add 130 ml of distilled water to solution II and mix by inversion. If neces-sary, warm the solution in a 37 ˚C water bath to assist in dissolving theconcentrate. Store at room temperature.

• Add 96 ml of absolute ethanol to the wash buffer and mix by inversion.Seal the bottle airtight and store at room temperature. If the seal is notairtight, the ethanol will evaporate.

• If 100 ml or 500 ml preparations are planned, prepare STE andlysozyme solutions as described in “Materials not supplied,” page 4.

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➊ Purification of plasmid DNA from 1.5 ml cultures of E. coli–

miniprep procedure

Using the following procedure, 750 minipreps can be prepared withthe kit.

If a yield of at least 1 µg of plasmid DNA is expected from 1.5 mlcultures, a more rapid, streamlined protocol can be used (see Appendix 2).

Cell lysis

1.1 Transfer 1.5 ml of an overnight culture of E. coli to a microcen-trifuge tube and centrifuge at full speed for 30 s to pellet the cells.

1.2 Remove the supernatant by aspiration without disturbing the cellpellet, but leaving it as dry as possible.

1.3 Resuspend the pellet in 200 µl of solution I by vigorous vortexing.

1.4 Add 200 µl of solution II and mix by inverting the tube severaltimes.

Note: The bacterial suspension should clear as cell lysis occurs.

1.5 Add 200 µl of solution III and mix by inverting the tube severaltimes.

1.6 Centrifuge at full speed for 5 min at room temperature.

1.7 Carefully transfer the supernatant to a clean centrifuge tube.

1.8 Add 420 µl (0.7 volume) of ambient temperature isopropanolto the supernatant and vortex to mix. Incubate for 10 min atroom temperature.

1.9 Centrifuge at full speed for 10 min to pellet the plasmid DNA. Re-move the supernatant by aspiration and invert the tube to drain.

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DNA purification

1.10 Suspend the Sephaglas FP slurry by shaking the bottle. Add 150 µl of the suspension to the DNA pellet and vortex gently for 1 min to dissolve the pellet. (No further incubation is required.)

1.11 Centrifuge at full speed for 15 s in a microcentrifuge. Carefullyremove the supernatant without disturbing the Sephaglas pellet.

1.12 Wash the Sephaglas pellet by adding 200 µl of wash buffer andvortexing gently to resuspend it. Centrifuge at full speed for 15 sin a microcentrifuge. Remove the supernatant without disturbingthe pellet.

1.13 Add 300 µl of 70% ethanol to the Sephaglas pellet and vortexgently to resuspend it. Centrifuge at full speed for 15 s in amicrocentrifuge. Remove the supernatant without disturbingthe pellet.

1.14 Vortex or tap the tube to partially disperse the Sephaglas pellet.Allow the pellet to air dry for 10 min.

Note: Do not dry under vacuum. Extensive drying will make it dif-ficult to elute the DNA from the Sephaglas.

1.15 To elute the bound DNA from the Sephaglas, add 50 µl ofTE buffer and vortex to resuspend the pellet. Incubate for 5 minat room temperature, vortexing occasionally to keep theSephaglas in suspension.

1.16 Centrifuge at full speed for 1 min in a microcentrifuge. Transferthe supernatant to a clean microcentrifuge tube. If any Sephaglasis observed in the eluted sample, centrifuge the eluant and care-fully transfer the supernatant to a clean tube, taking care to avoidthe Sephaglas pellet at the bottom of the tube.

Note: Do not store the eluted DNA in the presence of Sephaglas.

1.17 Depending on the application requirements, the DNA may be further concentrated by ethanol precipitation.

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➋ Purification of plasmid DNA from 50 ml cultures of E. coli–

midiprep procedure

Using the following procedure, 30 midipreps can be prepared using50 ml cultures. By making appropriate volume substitutions for thereagents (Table I, page 8), the protocol can be used to prepare plas-mid DNA from 10 ml and 25 ml cultures as well.

Cell lysis

2.1 Transfer 50 ml of an overnight culture of E. coli to a 50 ml tubeand centrifuge at 7 700 × g (~ 8 000 rpm in a Beckman JA-20rotor) for 10 min to pellet the cells.

Note: The appropriate centrifugation speed can be calculatedusing the following formula: RCF = (1.12)(r)(rpm/1 000)2, whereRCF = relative centrifugal force; r = radius in mm measured fromcenter of spindle to bottom of rotor bucket; and rpm = revolutionsper minute. For a force of 7 700 × g, where r = 108 mm, theappropriate speed would be 8 000 rpm.

2.2 Carefully decant the supernatant without disturbing the cellpellet, but leaving it as dry as possible.

2.3 Resuspend the pellet in 5 ml of solution I by vigorous vortexing.

2.4 Add 5 ml of solution II and mix by inverting the tube severaltimes. Incubate at room temperature for 5 min.


2.5 Add 5 ml of solution III and mix by inverting the tube severaltimes. Incubate on ice for 10 min.

2.6 Centrifuge at 12 000 × g (~ 10 000 rpm in a Beckman JA-20rotor) for 15 min at room temperature.

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2.7 Carefully decant the supernatant into a clean centrifuge tube.(The denatured chromosomal DNA will stick to the side of thetube as the supernatant is removed.)

2.8 Add 10.5 ml (0.7 volume) of ambient temperature isopropanolto the supernatant and vortex to mix. Incubate for 10 min atroom temperature.

2.9 Centrifuge at 12 000 × g for 20 min to pellet the plasmid DNA.Decant the supernatant and invert the tube to drain.

DNA purification

2.10 Suspend the Sephaglas FP slurry by shaking the bottle. Add 5 ml of the suspension to the DNA pellet and vortex gently for 1 min todissolve the pellet.

2.11 Incubate at room temperature for 10 min. Gently vortex thesample every 2 min to keep the Sephaglas in suspension.

2.12 Centrifuge at 1 000 × g for 2 min in a tabletop centrifuge.Carefully remove the supernatant without disturbing theSephaglas pellet.

2.13 Wash the Sephaglas pellet by adding 5 ml of wash buffer andvortexing gently to resuspend it. Centrifuge at 1 000 × g for 2 minin a tabletop centrifuge. Remove the supernatant without disturb-ing the pellet.

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2.14 Add 5 ml of 70% ethanol to the Sephaglas pellet and vortex gen-tly to resuspend it. Centrifuge at 1 000 × g for 2 min in a tabletopcentrifuge. Remove the supernatant without disturbing the pellet.

2.15 Following the ethanol rinse, centrifuge without a cap at 1 000 × gfor 1 min. Carefully remove any residual ethanol with a pipette.

2.16 Tap the tube to partially disperse the Sephaglas pellet. Allow thepellet to air-dry for 20–30 min at room temperature.

Note: Do not dry under vacuum. Extensive drying will make it difficult to elute the DNA from the Sephaglas.

2.17 To elute the bound DNA from the Sephaglas, add 500 µl of TE buffer and vortex to resuspend the pellet. Incubate for 10 minat room temperature, vortexing occasionally to keep theSephaglas in suspension.

2.18 Centrifuge at 1 000 × g for 5 min in a tabletop centrifuge.Transfer the supernatant to a clean microcentrifuge tube. If anySephaglas is observed in the eluted sample, centrifuge the eluant and carefully transfer the supernatant to a clean tube, taking care to avoid the Sephaglas pellet at the bottom of the tube.


2.19 Depending on the application requirements, the DNA may befurther concentrated by ethanol precipitation.

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➌ Purification of plasmid DNA from 500 ml cultures of E. coli–

maxiprep procedure

With the following procedure, five maxipreps can be prepared using500 ml cultures. By making the appropriate volume substitutions forthe reagents (Table I, page 8), the protocol can be used to prepareplasmid DNA (15 preps) from 100 ml cultures as well.

Cell lysis

3.1 Transfer 500 ml of an overnight culture of E. coli to an appropri-ate tube(s) or bottle(s) and centrifuge at 7 700 × g (~ 7 000 rpmin a Beckman JA-10 rotor) for 10 min to pellet the cells.

3.2 Carefully decant the supernatant without disturbing the cellpellet, but leaving it as dry as possible.

3.3 Resuspend the pellet in 100 ml of ice cold STE buffer(“Materials not supplied,” page 4) by pipetting. Centrifuge asabove and drain the pellet.

3.4 Resuspend the pellet in 30 ml of solution I by pipetting or vortex-ing. Add 1 ml of freshly made lysozyme (Appendix 2) and mix.Place on ice for 5 min.

3.5 Add 30 ml of solution II and mix by inverting the tube severaltimes. Incubate at room temperature for 5 min.


3.6 Add 30 ml of solution III and mix by inverting the tube severaltimes. Incubate on ice for 10 min.

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3.7 Centrifuge at 12 000 × g (~ 8 000 rpm in a Beckman JA-10rotor or ~ 10 000 rpm in a JA-20 rotor) for 15 min at roomtemperature.

3.8 Carefully decant the supernatant into a clean centrifuge tube.(The denatured chromosomal DNA will stick to the side of thetube as the supernatant is removed.)

3.9 Add 63 ml (0.7 volume) of ambient temperature isopropanolto the supernatant and mix well. Incubate for 10 min atroom temperature.

3.9 Centrifuge at 12 000 × g for 20 min to pellet the plasmid DNA.Decant the supernatant and invert the tube to drain.

DNA purification

3.10 Suspend the Sephaglas FP slurry by shaking the bottle. Add30 ml of the suspension to the DNA pellet and vortex gently for 1 min to dissolve the pellet.

3.11 Incubate at room temperature for 10 min. Gently vortex thesample every 2 min to keep the Sephaglas in suspension.

3.12 Centrifuge at 1 000 × g for 2 min in a tabletop centrifuge.Carefully remove the supernatant without disturbing theSephaglas pellet.

3.13 Wash the Sephaglas pellet by adding 30 ml of wash buffer andvortex gently to resuspend it. Centrifuge at 1 000 × g for 2 min ina tabletop centrifuge. Remove the supernatant without disturbingthe pellet.

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3.14 Add 30 ml of 70% ethanol to the Sephaglas pellet and vortexgently to resuspend it. Centrifuge at 1 000 × g for 2 min in atabletop centrifuge. Remove the supernatant without disturbingthe pellet.

3.15 Following the ethanol rinse, centrifuge without a cap at 1 000 × gfor 1 min. Carefully remove any residual ethanol with a pipette.

3.16 Tap the tube to partially disperse the Sephaglas pellet. Allow the pellet to air-dry for 20–30 min at room temperature.

Note: Do not dry under vacuum. Extensive drying will make itdifficult to elute the DNA from the Sephaglas.

3.17 To elute the bound DNA from the Sephaglas, add 5 ml ofTE buffer and vortex to resuspend the pellet. Incubate for 10 minat room temperature, vortexing occasionally to keep theSephaglas in suspension.

3.18 Centrifuge at 1 000 × g for 5 min in a tabletop centrifuge.Transfer the supernatant to a clean centrifuge tube. If anySephaglas is observed in the eluted sample, centrifuge the eluantand carefully transfer the supernatant to a clean tube, taking careto avoid the Sephaglas pellet at the bottom of the tube.


3.19 Depending on the application requirements, the DNA may befurther concentrated by ethanol precipitation.

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Appendix 1:Bacterial cultures

• For 1.5 ml to 100 ml cultures, use a single colony from an agar platecontaining the appropriate antibiotics to inoculate growth medium.For 500 ml cultures, it is advisable to use 3–5 ml of an overnight cul-ture as the starting inoculum.

• During growth, cultures should be well aerated. The volume ofculture medium should represent only about one-fifth the totalvolume of the container used for growth.

Recipes are given below for common media that have been shown togive consistent yields of plasmid DNA from the FlexiPrep Kit:

• LB medium: Dissolve 10 g of Bacto-tryptone, 5 g of Bacto-yeastextract and 10 g of NaCl in 900 ml of deionized water. Adjust thepH to 7.0 by the addition of 1 M NaOH, and adjust the volume to1 liter with deionized water. Sterilize by autoclaving.

• 2× YT medium: Dissolve 16 g of Bacto-tryptone, 10 g of Bacto-yeastextract and 5 g of NaCl in 1 liter of distilled water. Mixand autoclave.

• TB medium: Dissolve 12 g of Bacto-tryptone, 24 g of Bacto-yeastextract and 4 ml of glycerol in 900 ml of deionized water. Sterilize byautoclaving. Allow the medium to cool to < 60 ˚C, then add 100 mlof a sterile solution of 0.17 M KH2PO4, 0.72 M K2HPO4. (Preparethe phosphate solution by dissolving 2.31 g of KH2PO4 and 12.54 gof K2HPO4 in 90 ml of deionized water. After the salts have dis-solved, adjust the volume to 100 ml with deionized water and steril-ize by autoclaving).

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Appendix 2:Streamlined protocol for minipreps

When a yield of at least 1 µg of plasmid DNA is expected from 1.5 mlcultures, the streamlined protocol gives plasmid DNA of yield and puri-ty equivalent to that obtained using the standard miniprep protocol.

Cell lysis

1. Transfer 1.5 ml of an overnight culture of E. coli to a micro-centrifuge tube and centrifuge at full speed for 30 s to pelletthe cells.

2. Remove the supernatant by aspiration without disturbing the cellpellet, but leaving it as dry as possible.

3. Resuspend the pellet in 200 µl of solution I by vigorous vortexing.

4. Add 200 µl of solution II and mix by inverting the tube severaltimes.


5. Add 200 µl of solution III and mix by inverting the tube severaltimes.

6. Centrifuge at full speed for 5 min at room temperature.

7. Carefully transfer the supernatant to a clean centrifuge tube.

8. Add 420 µl (0.7 volume) of ambient temperature isopropanol tothe supernatant and vortex to mix. Incubate for 1 minute at room temperature.

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9. Centrifuge at full speed for 5 min to pellet the plasmid DNA.Remove the supernatant by aspiration and invert the tube to drain.

DNA purification

10.Suspend the Sephaglas FP slurry by shaking the bottle. Add 150 µlof the suspension to the DNA pellet and vortex gently for 1 min todissolve the pellet. (No further incubation is required.)

11.Centrifuge at full speed for 15 s in a microcentrifuge. Carefullyremove the supernatant without disturbing the Sephaglas pellet.

12.Wash the Sephaglas pellet by adding 200 µl of wash buffer andvortex gently to resuspend it. Centrifuge at full speed for 15 sin a microcentrifuge. Remove the supernatant without disturbingthe pellet.

13.Add 300 µl of 70% ethanol to the Sephaglas pellet and vortex gentlyto resuspend it. Centrifuge at full speed for 15 s in a micro-centrifuge. Remove the supernatant without disturbing the pellet.

14.Vortex or tap the tube to partially disperse the Sephaglas pellet.

15.Centrifuge at full speed for 15 s in a microcentrifuge. Carefullyremove any residual liquid without disturbing the pellet.


16.To elute the bound DNA from the Sephaglas, add 50 µl of TE bufferand vortex to resuspend the pellet. Incubate for 5 min at room tem-perature, vortexing occasionally to keep the Sephaglas in suspension.

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17.Centrifuge at full speed for 1 min in a microcentrifuge. Transfer thesupernatant to a clean microcentrifuge tube. If any Sephaglas isobserved in the eluted sample, centrifuge the eluant and carefullytransfer the supernatant to a clean tube, taking care to avoid theSephaglas pellet at the bottom of the tube.


18.Depending on the application requirements, the DNA may befurther concentrated by ethanol precipitation.

Appendix 3:Isolation of cosmid DNA

Using the following procedure, cosmid DNA can be isolated from 10 mlof an overnight culture.

Cell lysis

1. Transfer 10 ml of an overnight culture of E. coli to a 15 ml cen-trifuge tube. Centrifuge cells at 7 700 × g (~ 8 000 rpm in aBeckman JA-20 rotor with adapters for 15 ml tubes) for 10 min.

Note: The appropriate centrifugation speed can be calculated usingthe following formula: RCF = (1.12)(r)(rpm/1 000)2, where RCF =relative centrifugal force; r = radius in mm measured from center ofspindle to bottom of rotor bucket; and rpm = revolutions perminute. For a force of 7 700 × g, where r = 108 mm, the appropri-ate speed would be 8 000 rpm.

2. Carefully decant the supernatant without disturbing the cell pellet,but leaving it as dry as possible.

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3. Add 200 µl of solution I and fully resuspend the pellet by gentlypipetting the solution up and down. Transfer the cell suspension toa 1.5 ml microcentrifuge tube.

4. Add 400 µl of solution II and mix by gently inverting the tube sever-al times. Incubate at room temperature for 5 min.


5. Add 400 µl solution III and mix by gently inverting the tube severaltimes. Incubate on ice for 10 min.

6. Centrifuge at full speed in a microcentrifuge for 10 min at roomtemperature.

7. Carefully transfer the supernatant to a fresh microcentrifuge tube.

8. Add 700 µl of ambient temperature isopropanol to the supernatantand mix by gently inverting several times. Incubate for 10 min atroom temperature.

9. Centrifuge at full speed in a microcentrifuge for 20 min at roomtemperature to pellet the plasmid DNA. Decant the supernatant andinvert the tube to drain.

DNA purification

10.Suspend the Sephaglas FP slurry by shaking the bottle and add 250 µl to the DNA pellet. Resuspend the DNA pellet by gentlypipetting up and down. Incubate at 37 ˚C for 2 min.

11.Centrifuge at full speed in a microcentrifuge for 2 min at roomtemperature. Carefully remove and discard the supernatant withoutdisturbing the Sephaglas pellet.

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12.Resuspend the Sephaglas pellet in 300 µl of wash buffer by gentlypipetting up and down. Centrifuge at full speed in a microcentrifugefor 2 minutes at room temperature. Carefully remove and discardthe supernatant without disturbing the Sephaglas pellet.

13.Resuspend the Sephaglas pellet in 300 µl of 70% ethanol by gentlypipetting up and down. Centrifuge at full speed in a microcentrifugeat room temperature for 2 min. Carefully remove and discard thesupernatant without disturbing the Sephaglas pellet.

14.Tap the tube to partially disperse the Sephaglas pellet. Allow thepellet to air dry for 10 min at room temperature.


15.To elute the bound DNA from the Sephaglas pellet, add 50 µl of TE buffer and gently pipette up and down to resuspend the pellet.Incubate for 5 min at 72 ˚C.

16.Centrifuge at full speed in a microcentrifuge for 2 min at room tem-perature. Transfer the supernatant to a clean microcentrifuge tube.

17.Add another 50 µl of TE buffer, then resuspend the pellet andincubate at 72 ˚C as before to perform a second elution from theSephaglas.

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18.Centrifuge at full speed in a microcentrifuge for 2 min at roomtemperature. Transfer the supernatant to the microcentrifuge tubecontaining the first elution. If any Sephaglas is observed in theeluted sample, centrifuge the eluant and carefully transfer the super-natant to a clean tube, taking care to avoid the Sephaglas pellet atthe bottom of the tube.


19.Add 1 µl of 1 µg/ml RNase A to the pooled supernatants.

20.Depending on the application requirements, the DNA may befurther concentrated by ethanol precipitation.

Troubleshooting

Problem: The Sephaglas is difficult to resuspend (miniprep procedure).

Possible causes/solutions

1. Centrifugation was too harsh. Spin in a microcentrifuge at full speedfor 5–10 s rather than for 15 s. Be sure that the pellet is not looseand will not be disturbed during removal of the supernatant.

Problem: The Sephaglas does not pellet completely during centrifuga-tion (midi- and maxipreps).


1. Insufficient centrifugation. Be sure that the centrifuge is set at anrpm that corresponds to 1 000 × g. Centrifugation times can beincreased from 5 min to fully pellet the Sephaglas.

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Problem: The yield of DNA is low.


1. The yield of plasmid from the culture is low. Typical yields for ahigh-copy-number plasmid such as pUC18 are 2–4 µg/ml of bacterialculture. If yields are substantially lower, either the growth conditionsare not optimal or the copy number of the plasmid is low. The yieldmay be increased by:

– Growing the cells in a flask with at least five volumes of air spaceand incubating at 37 ˚C overnight with shaking at 250 rpm.

– Growing the cells in a richer medium than LB (e.g. TB).

– Amplifying the plasmid by treating the bacterial culture withchloramphenicol or spectinomycin: when the A600 of the culturereaches 1.0, add chloramphenicol to 170 µg/ml or spectinomycinto 100 µg/ml, then continue incubation for 12–16 hours.

2. DNA is eluted in the wash buffer. This may happen if the washbuffer was not diluted with the correct volume of absolute ethanolor if the wash buffer was stored for long periods at room tempera-ture, in which case the ethanol may have evaporated fromthe solution.

– If the integrity of the wash buffer is questionable, use freshly pre-pared 70% ethanol in place of the wash buffer.

3. DNA is irreversibly bound to the Sephaglas. Do not overdry theSephaglas and do not dry under vacuum.

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Problem: Upon agarose gel analysis, the DNA looks smeared.


1. The DNA was stored with Sephaglas. Do not store the DNA in thepresence of Sephaglas—this can shear the DNA.

Problem: DNA does not cut to completion with restriction enzymes.


1. The DNA may be denatured. Denatured DNA can be seen on anagarose gel as a band that migrates more quickly than thesupercoiled band. Denaturation can be avoided by shortening theincubation time in solution II. The incubation with solution IIshould last only until the cell suspension clears.

2. The DNA may contain residual salt. Ethanol precipitate the elutedDNA and wash with 70% ethanol.

Problem: DNA is too dilute.


1. The eluted DNA can be ethanol precipitated and resuspended in asmaller volume. Do not use less TE buffer for elution to increase theconcentration—this may reduce recoveries.

If problems persist, please contact GE Healthcare TechnicalService for assistance.

● p25

References1. Birnboim, H.C. and Doly, J., Nucl. Acids Res. 7, 1513 (1979).

2. Ish-Horowicz, D. and Burke, J. F., Nucl. Acids Res. 9, 2989 (1981).

3. Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A laboratorymanual, Cold Spring Harbor Laboratory, 2nd Ed., pp 1.25-1.28 (1989).

4. Vogelstein, B. and Gillespie, D., Proc. Natl. Acad. Sci. USA 76, 615 (1979).

5. Marko, M. A., Chipperfield, R. and Birnboim, H. C., Anal. Biochem. 121, 382(1982).

● p26

All goods and services are sold subject to the terms and conditions of sale of the company within theGeneral Electric Company group which supplies them. A copy of these terms and conditions isavailable on request.

** The Polymerase Chain Reaction (PCR) is covered by patents owned by Roche Molecular Systems andF Hoffmann-La Roche Ltd. A license to use the PCR process for certain research and development activi-ties accompanies the purchase of certain reagents from licensed suppliers such as GE Healthcareand affiliates when used in conjunction with an authorized thermal cycler.

Printed in the United States.

● p27

● p28

GE Healthcare Bio-Sciences Ltd

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tel 44 0870 606 1921 fax 44 01494 544350

GE Healthcare Bio-Sciences AB

SE-751 84 Uppsala, Sweden

tel 46 (0) 18 612 00 00 fax 46 (0) 18 612 12 00

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tel 1-800-526-3593 fax 1-800-329-3593 or 877-295-8102

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tel 49 761 4519-0 fax 49 761 4519 159

TrademarksGE Healthcareare trademarks ofGeneral Electric Company.

FlexiPrep and Sephaglasare trademarks ofGE Healthcare Bio-Sciences Ltd.

マニュアル flexiprep kit...possible causes/solutions 1. centrifugation was too harsh.spin in a...

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