APPENDIX 3K Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
Published Online: 1 AUG 2006
Copyright © 2006 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Cytometry
How to Cite
Kramer, M. F. and Coen, D. M. 2006. Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization. Current Protocols in Cytometry. 37:3K:A.3K.1–A.3K.15.
- Published Online: 1 AUG 2006
- Published Print: JUL 2006
This unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield. The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. Once assembled, the mixture is cycled many times (usually 30) through temperatures that permit denaturation, annealing, and synthesis to exponentially amplify a product of specific size and sequence. The PCR products are then displayed on an appropriate gel and examined for yield and specificity. Recommended optimization conditions are included.