How do I determine the annealing temperature from the melting temperature (Tm) of the primers?
Determining the PCR annealing temperature depends on the method used to calculate Tm. When using more accurate and improved nearest-neighbor thermodynamic parameters (as employed in the SantaLucia equations used in the RefSplice primer design tool), the annealing temperature should be set at the lowest Tm value of the primers minus two degrees. If using the Tm values provided to you by an oligonucleotide supplier, consult them for suggested annealing temperatures.
