What is the annealing temperature one should use for a degenerate PCR reaction?
If using degenerate primers and the AT=2 CG=4 C formula for calculating the melting temperature, the annealing temperature is frequently 4-10 C lower even though the primer concentration may be 10 times higher. Tm calculated using the Nearest Neighbor model is lower because of the degeneracy. Suggested annealing temperature is 2 C higher to 4 C lower than the melting temperature for the normal (200 nM) primer concentration. If higher primer concentration is used, melting temperature increases ~1 C for each doubling of the primer concentration.
