We recommend using the NEB online primer design tool, NEBaseChanger, to provide an optimized annealing temperature for mutagenic primer pairs. For predesigned back-to-back primer sets, a Tm+3 rule can be applied but optimization may be necessary. Note that mismatches in the mutagenic primer are not accounted for using standard Tm calculations and the optimal annealing temperature for Q5 High Fidelity Hot Start DNA Polymerase may differ significantly from that of Taq-based polymerases.

For primer pairs with annealing temperatures close to 72°C, a two-step cycling protocol without a separate annealing step can be used.