Step	Temperature	Time	Cycle number
Phosphorylation	37 °C	45 min	1
Denaturation	95 °C	2 min, 30 s	1
Cool down 1a	−0.3 °C/s until 50 °C	-	1
Holda	50 °C	10 s	1
Cool down 2a	−0.3 °C/s until 22 °C	-	1
Finish	On ice	-	1

^a

If you do not use a PCR machine for −0.3 °C/s cooling down, you can leave the product at room temperature CRT, 20-25 °C) for 30 min for cooling down, then put the tube on ice before the next step.