Frequently asked questions

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How do you calculate the annealing temperature for PCR?

The annealing temperature (Ta) chosen for PCR relies directly on length and composition of the primers. Generally, you should use an annealing temperature about 5°C below the Tm of your primers. The optimal annealing temperature (Ta Opt) for a given primer pair on a particular target can be calculated as follows: Ta Opt = 0.3 x (Tm of primer) + 0.7 x (Tm of product) – 14.9; where Tm of primer is the melting temperature of the less stable primer-template pair, and Tm of product is the melting temperature of the PCR product [1].

One consequence of having Ta too low is that one or both primers will anneal to sequences other than the intended target, because internal single-base mismatches or partial annealing may be tolerated. This can lead to nonspecific PCR amplification and will consequently reduce the yield of the desired product. Conversely, when Ta is too high reaction efficiency may be reduced, because the likelihood of primer annealing is reduced significantly. Optimal annealing temperatures give the highest product yield of the correct amplicon.

Reference
  1. Rychlik W, Spencer WJ, Rhoads RE (1990) Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res 18(21):6409–6412.