Team:Evry/Protocols

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Contents

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

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

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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