PCR产物纯化方法 Purification of PCR Products in Preparation for Cloning

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Purification of PCR Products in Preparation for Cloning

Joseph Sambrook
Peter Maccallum Cancer Institute and The University of Melbourne, Australia
David W. Russell
University of Texas Southwestern Medical Center, Dallas
Excerpted From Molecular Cloning: A Laboratory Manual Third Edition

ABSTRACT

The residual enzymatic activity of thermostable DNA polymerases that survive the rigors of PCR can compromise subsequent enzymatic reactions. This protocol describes how to use proteinase K to destroy thermostable enzymes and to purify amplified DNA in preparation for cloning.

MATERIALS

Reagents and Solutions

Ammonium acetate (10 M)
Amplified DNA from polymerase chain reactions
Chloroform
Ethanol
Phenol:chloroform (1:1, v/v)
TE (pH 8.0)

Enzymes and Buffers

Proteinase K (20 mg/ml)
Genomic grade proteinase K that has been shown to be free of DNase and RNase activity.

Gels

EthanolAgarose gel containing 0.5 µg/ml ethidium bromide

METHOD

Pool up to eight PCRs (400 µl) containing 1 µg of the desired amplification product. (See Troubleshooting.)

If mineral oil was used to prevent evaporation during PCR, centrifuge the pooled samples briefly and transfer the lower (aqueous) phase to a fresh microcentrifuge tube.
Add 0.2 volume of 5x proteinase K buffer and proteinase K to a final concentration of 50 µg/ml. Incubate the mixture for 60 minutes at 37°C.
Inactivate the proteinase K by heating to 75°C for 20 minutes.
Extract the reaction mixture once with phenol:chloroform and once with chloroform.
Add 0.2 volume of 10 M ammonium acetate and 2.5 volumes of ethanol. Mix the solution well and store it for 30 minutes at 4°C.
Recover the DNA by centrifugation at maximum speed for 5 minutes at 4°C in a microcentrifuge. Discard the supernatant and then wash the pellet with 70% ethanol. Centrifuge again, remove the supernatant, and then allow the DNA to dry.

The DNA may be further purified by chromatography or by gel electrophoresis. This step is recommended when primers have been used to add restriction sites to the ends of the amplified DNA. Unused primers and primer-dimers should be removed before digesting the DNA with the appropriate restriction enzymes (please see Removal of Oligonucleotides and Excess dNTPs from Amplified DNA by Ultrafiltration).
Dissolve the pellet in TE (pH 8.0). Assume that the recovery of amplified DNA is 50-80% and dissolve the DNA in TE (pH 8.0) at an estimated concentration of 25 µg/ml (25 ng/µl).
Analyze approx. 25 ng of the purified DNA by agarose-ethidium bromide gel electrophoresis, using markers of an appropriate size. Check that the amplified band fluoresces with the intensity expected of approx. 25 ng of DNA.

TROUBLESHOOTING

Nonspecific amplification products are present at significant levels (e.g., are detectable by gel electrophoresis).

Step 1

Purify the desired product by electrophoresis through low-melting-temperature agarose before proceeding (please see Recovery of DNA from Low-melting-temperature Agarose Gels: Organic Extraction).

REFERENCES

Crowe J.S., Cooper H.J., Smith M.A., Sims M.J., Parker D., Gewert D. 1991. Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion. Nucleic Acids Res. 19: 184-184.[Free Full Text]

Wybranietz W.A. and Lauer U. 1998. Distinct combination of purification methods dramatically improves cohesive-end subcloning of PCR products. BioTechniques 24: 578-580.[Medline]

Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these material, please consult:
Molecular Cloning: A Laboratory Manual Third Edition , by Joseph Sambrook and David W. Russell, © 2001 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. . 8.25-8.26.

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