Validating a multi-locus metabarcoding approach for characterizing mixed-pollen samples

Table 3 PCR1 and PCR2 program specifications

Program	Step	Temperature ( °C)	Time	Number of cycles
1	Initial denaturation	94	10 min	1×
	Denaturation	94	30 s	40×
	Primer annealing	54	40 s
	Extension	72	1 min
	Final extension	72	10 min	1×
2	Initial denaturation	94	10 min	1×
	Denaturation	94	30 s	40×
	Primer annealing	56	40 s
	Extension	72	1 min
	Final extension	72	10 min	1×

Bolded values are changes in annealing temperature between programs. PCR3 followed the program specifications indicated above, with an annealing temperature of 60 °C

ISSN: 1746-4811