( urthermore extensive denaturation time...)Optimizing DNA Amplification Protocols using the Eppendorf Mastercycler

urthermore, extensive denaturation times result in a substantial loss of polymerase activity (see below).

Annealing

During the annealing phase, the primers are rapidly hybridized. This operation is completed within a few seconds. Therefore, annealing times of 1020 seconds are usually fully adequate (4).

Very long annealing times normally do not improve yield, but rather produce an increase in spurious priming and, thus, greater amounts of nonspecific PCR products.

Extension

During primer extension (at approx. 72C), the insertion rate of commonly used DNA polymerases is at least 50 nucleotides per second (7). It is therefore possible to keep extension times shorter 15 seconds for PCR products less than 400 bp long. In addition, nucleotides are already inserted during the annealing phase, in particular, if it is possible to carry out annealing at relatively high temperatures. At 55C, for example, Taq polymerase has an insertion rate of approx. 24 nucleotides per second (7). Of course, very long PCR products demand correspondingly longer extension times. In these cases, it may be advisable to provide for longer extension times in each PCR cycle (delay function) in order to compensate for increasing viscosity in the sample.

Magnesium ion concentration

A very simple but significant strategy for optimization is titration of the magnesium ion concentration (4). In addition to Mg2+ ions bound by the template DNA, the nucleotides (dNTPs) and the primers, Taq DNA polymerase also requires free Mg2+ ions. Their concentration has
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