Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.ip66

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Optimization and Troubleshooting in PCR

Kenneth H. Roux

Adapted from PCR Primer: A Laboratory Manual, 2nd edition (eds. Dieffenbach and Dveksler). CSHL Press, Cold Spring Harbor, NY, USA, 2003.


INTRODUCTION

The use of polymerase chain reaction (PCR) to generate large amounts of a desired product can be a double-edged sword. Failure to amplify under optimum conditions can lead to the generation of multiple undefined and unwanted products, even to the exclusion of the desired product. At the other extreme, no product may be produced. A typical response at this point is to vary one or more of the many parameters that are known to contribute to primer-template fidelity and primer extension. High on the list of optimization variables are Mg++ concentrations, buffer pH, and cycling conditions. With regard to the last, the annealing temperature is most important. The situation is further complicated by the fact that some of the variables are quite interdependent. For example, because dNTPs directly chelate a proportional number of Mg++ ions, an increase in the concentration of dNTPs decreases the concentration of free Mg++ available to influence polymerase function. This article discusses various optimization strategies, including touchdown PCR and hot-start PCR.


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