Optimization of a PCR Reaction

Second, DNA polymerases need magnesium ions for activity a correct Mg2+ concentration is crucial for successful amplification as well as correctness in sequence of the amplified product. This fact can be used as a tool for directed evolution to introduce sequence errors deliberately during amplification (error-prone PCR see Chapter 11, Section 11.3) by adding geometrically similar Mn2+ to the PCR buffer already containing Mg2+. In most cases, the concentration of the buffer provided in a commercial PCR kit is optimized for the corresponding polymerase, so adjustment of Mg2+ concentration should rarely have to be considered. 

A short list of possible PCR kits and useful websites is given in Table 4.3. 

For large PCR products or difficult templates (secondary structure or high GC content), high-fidelity (low error rate) polymerases such as the Advantage PCR (Clontech) or Failsafe PCR (Epicentre) are highly recommended. 

Advantage PCR Clontech http / /www.hemo.mrc.ac.uk/GenomeWeb/nuc-nrimer html 

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