Cycle process	Temperature	Time	Cycles
Nick translation	72 °C	20 min	1
Initial denaturation	95 °C	5 min	1
Denaturation	95 °C	15 s	2–10a
Annealing	62 °C	15 s
Extension	70 °C	1 min
Final extension	70 °C	5 min	1
Hold	4 °C	1	1

^a

Adjust the number of cycles based on starting amount of DNA as follows:

• 100 ng to 1 μg: 6–8 cycles
• 1–2 μg: 4–6 cycles
• 2–5 μg: 2–3 cycles