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From 2012.igem.org

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Contents 1 PCR with Phusion High-Fidelity DNA Polymerase 1.1 Tube preparation 1.2 Cycling instructions 2 Préparation of LB medium and LB Agar:

PCR with Phusion High-Fidelity DNA Polymerase

Tube preparation

Put items in this order:

Component	50µl reaction	Comments
H2O	32
5x Phusion HF Buffer	10
10mM dNTPs	1
Primer FW	2	Primers have to be at 10µM
Primer RV	2	Primers have to be at 10µM
Template DNA	1
DMSO (optional)	1,5	recommended for GC-rich amplicons < 20kb
Phusion DNA polymerase	0,5

Cycling instructions

Préparation of LB medium and LB Agar:

=> LB Agar : -18,5g LB Agar -300ml H2O

=> LB medium : -6g LB broth -300ml de H2O

Autoclaved at 250°C

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Cycle step	Temperature	Time	Cycles
Initial denaturation	98°C	4min	1
Denaturation	98°C	20s	30
Annealing	Lower Tm of primers	30s
Extension	72°C	30S/kb
Final extension	72°C	10min	1
	4°C	hold