4.8.2. Annealing temperature
The annealing temperature for the assay should be optimized experimentally, with a temperature gradient, which can be generated by most modern thermocyclers in a single run. Set the annealing temperature at 1-2oC below the highest temperature that still generates a signal/band, to make sure the assay is both specific and robust. For primers with a Tm of 56oC, the optimized assay annealing temperature is usually around 58oC and the maximum annealing temperature still generating a (weak) signal around 60oC.