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 any 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) - 25; 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.

One consequence of having too low a Ta is that one or both primers will anneal to sequences other than the intended target, as 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, too high a Ta may reduce reaction efficiency, as the likelihood of primer annealing is reduced significantly.  Optimal annealing temperatures will result in the highest product yield with the correct amplicon.

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  • Ta