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From 2010.igem.org

Week-16

Start to apply high fidelity PCR due the construct are getting larger and the regular Taq polymerase may not perform as well as the Phusion Polymerase at this length. Based on the company´s manual, we calculated the

	Temperatur	Time	Cycle
Initial denaturation	98	30 s	1
Denaturation	98	10	30
Annealing	55.3	10s	30
Extension	72	1 min 45 s	30
Final extension	72	5 min	1
	4	forever

The purpose of this was to get more reliable band when doing a PCR and running the gel.

<COL WIDTH=134> <COL WIDTH=99>

Mastermix	Volume (µL)
H20	10.8
5X phusion HF Buffer	4
dNTPs	2
VF2	1
VR	1
DNA-template	1
Phusion Polymerase	0.2