Table 2.

PCR thermocycling conditions

PCR TYPE	STEP	CONDITIONS	Tc °C
Conventional PCR	Initial denaturation	98°C for 30 s
	Thermocycling: 35 cycles	98°C for 10 s Ta for 20 s 72°C for 10 s	N/A
	Melting Curve	Ramping 0.2°C/s, 65–98°C
fast-COLD-PCR	Initial denaturation	98°C for 30 s
	Stage 1 cycling: 5 cycles	98°C for 10 s Ta for 20 s, fluorescent reading 72°C for 10 s
	Stage 2 cycling: 20–35 cyclesa	Tc for 10 s Ta for 20 s, fluorescent reading 72°C for 10 s	87 bp, Tc = 85.5°C 115 bp, Tc = 85.4°C
	Melting curve	Ramping 0.2°C/s, 65–98°C
full-COLD-PCR	Initial denaturation	98°C for 30 s
	Stage 1 cycling: 5 cycles	98°C for 10 s Ta for 20 s, fluorescent reading 72°C for 10 s
	Stage 2 cycling: 20–35 cyclesa	98°C for 10 s 70°C for 30 s Tc for 10 s Ta for 20 s, fluorescent reading 72°C for 10 s	87 bp, Tc = 85.5°C 115 bp, Tc = 85.4°C
	Melting Curve	Ramping 0.2°C/s, 65–98°C
ice-COLD-PCR	Initial denaturation	98°C for 30 s
	Stage 1 cycling: 5 cycles	98°C for 10 s Ta for 20 s, fluorescent reading 72°C for 10 s
	Stage 2 cycling: 20-35 cyclesa	98°C for 10 s 70°C for 30 s Tc for 10 s Ta for 20 s, fluorescent reading 72°C for 10 s	87 bp, Tc = 84.1°C 115 bp, Tc = 83.4°C
	Melting Curve	Ramping 0.2°C/s, 65–98°C

T_a, annealing temperature; 60 and 55°C for the 87- and the 115-bp amplicons, respectively; T_c, Critical denaturation temperature used in COLD-PCR reactions.

^a20 and 35 cycles were performed respectively for the 87-bp and the 115-bp amplicons.