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From 2011.igem.org

Cyrille

Prearation of the pHM3 plasmid. This plasmid from H. Pfutzer contains 2 EcoRI excision site. One will be removed by quick change mutagenesis.

42 base oligo couple has been ordered from eurogentec. The protocol is the one [http://labs.fhcrc.org/hahn/Methods/mol_bio_meth/quick_change.htmlhere] using the phusion enzyme.

The two primers pHM3-fwd, pHM3-rev where resuspended at 1 microM. The template was mesured with the TECAN and is at 448 ng/microL

The reaction mix contains:

• Buffer HF 5x : 10 microL
• dNTP (2mM) : 5 microL
• Phusion  : 1 microL
• Primer fwd  : 2 microL
• Primer rev  : 3 microL
• Template  : 0,7 microL

QSP H2O 50 MicroL

Cycles:

• 98 °C 2 min

• 98°C 10s
• 63°C 1 min
• 72°C 12 min
• Repeat 20 times

• 72°C 10 min
• 4°C forever

The PCR was done overnight and then freeze until monday