Cycle step	Temperature	Time	Cycles
Initial denaturation	95	5 min	1
Denaturation	94	15 s	30–35
Annealing	∗	20 s
Extension	72	15 s
Final Extension	72	5 min	1

∗ Annealing temperature should be optimized for each primer set based on the primer Tm; typically, it should be Tm-5°C.