. 2016 Feb 8;27(6):425–435. doi: 10.1089/hum.2016.011

Table 3.

ASQ thermal cycling

	qPCR with plate reading					PCR + post-PCR plate reading
	Temp (°C)	Time (s)	Reaction			Temp (°C)	Time (s)	Reaction
	95	120	Enzyme activation Denaturation			95	120	Enzyme activation Denaturation
Tier 1	95	15	Denaturation	12×^a	Tier 1	95	15	Denaturation	12×^a
	57.5	18	Annealing			57.5	18	Annealing
	72	60	Extension			72	60	Extension
Tier 2	95	15	Denaturation	25×^a	Tier 2	95	15	Denaturation	25×^a
	62	18	Annealing			62	18	Annealing
	72	60	Extension			72	60	Extension
	45	5	Plate reading			Post-PCR		Plate reading

^{^a}

The number of thermal cycles in tiers 1 and 2 needs to be optimized based on the quality of amplification at each locus and the specificity of the primers for the locus. Twelve times for tier 1 and 25 times for tier 2 are good starting points. The tier 1 cycling may vary between 10 and 14 times and the tier 2 may range from 22 to 35 times.