| 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 | |
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Enzymes and Buffers
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Gels
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| METHOD | |
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| 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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