What annealing temperature should be used in the cycling conditions?
Set the annealing temperature approximately 3°C higher than the lowest Tm primer for oligos greater than 20 nucleotides. For oligos shorter than 20 nucleotides, set the annealing temperature equal to the Tm of the lowest Tm primer. When the Tm of the primer pairs is ≥ 72°C, use two-step cycling conditions where the annealing and extension steps are combined. If DMSO is needed in the reaction, a reduction of the annealing temperature is often necessary.