Calculating the optimal PCR annealing temperature

MacVector has several tools to help with primer design and testing. The Analyze | Primer Design/Test (Pairs) function uses the popular Primer3 algorithm to find suitable pairs of primers to amplify specified segments of DNA. You can also enter pairs of pre-designed primers and test their suitability for use in PCR. In both cases, the Tm of each primer is reported, along with the optimal annealing temperature (Ta).

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The optimal annealing temperature (degrees C) is calculated as follows (from W. Rychlik, W.J. Spencer, and R. E. Rhoads, Nucl.Acids.Res. 18:6409–6412(1990));

(Lowest Primer Tm x 0.3) + (Product Tm x 0.7) - 14.9

This means that you can get an optimal annealing temperature for a PCR experiment that is significantly different from the optimal annealing temperature for an individual primer (e.g. in a sequencing experiment) because of the large influence of the product in the calculation.

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