How do you calculate the annealing temperature for PCR?

Jul 29, 2013, 16:46 PM

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.

  • PCR/qPCR
  • anneal
  • Ta