Steps in the PCR cycle

Temperature

Time

No. of cycles

Initial denaturation

(FastStartTaq DNA Polymerase)

95˚C

10:00 min

1

Denaturation

95˚C

00:10 sec

45

Annealing

63˚C

15:00 min

Extension

72˚C

00:16 sec

For HRM

Second hold

95˚C

1:00 min

1

40˚C

00:01 min

65˚C

00:01 sec

Second temperature increase

95˚C

Fluorescent detection (25 acquisition per 1˚C)

Cooling down

40˚C

00:10 sec