Step	Temperature (°C)	Time (s)
1	95	240
2	95	30
3	50–55**	30 35x*
4	72	30
5	72	600

^*

Steps 2–4 are repeated 35 times sequentially (2→3→4→2→3→4 etc.)

^**

When using AT-rich (>50%) gRNA sequences, a lower (50°C) annealing temperature could provide better results.