PCR mixture PCR condition for generic primers

A	B	C
Component	Volume	Final conc.
Template (< 1 µg)	X µL	-
Forward Primer (10 µM)	1 µL	0.4 µM
Reverse Primer (10 µM)	1 µL	0.4 µM
2x Master Mix	12.5 µL	-
ddH20	up to 25 µL	-
Total volume	25 µL	-

PCR condition for generic primers

A	B	C	D
Step	Temp	Sec	Cycle
Initial denaturation	95 ºC	180
Denaturation	95 ºC	30	35 cycles
Annealing	53-58 ºC	30
Extension	72 ºC	60
Final extension	72 ºC	210
Preservation	4 ºC	∞

PCR mixture for barcoded primers

A	B	C
Component	Volume	Final conc.
Template (< 1 µg)	X µL	-
Forward Primer (10 µM)	5 µL	2 µM
Reverse Primer (10 µM)	5 µL	2 µM
2x Master Mix	12.5 µL	-
ddH20	up to 25 µL	-
Total volume	25 µL	-

PCR condition for barcoded primers

A	B	C	D
Step	Temp	Sec	Cycle
Initial denaturation	95 ºC	180
Denaturation	98 ºC	20	35 cycles
Annealing	64 ºC
Extension	72 ºC
Final extension	72 ºC	210
Preservation	Preservation	4 ºC	∞