Cycle Step	Temperature	Time	Cycles
Initial denaturation	95	5 min	1
Denaturation	94	10–30 s	30–35
Annealing	∗	15–30 s
Extension	72	30–60 s/kb
Final extension	72	5 min	1

∗ Annealing temperature should be optimized for each pair of primers, based on specific Tm; typically, it should be Tm-5°C.