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Team:Paris Liliane Bettencourt/Notebook/2011/07/22/
From 2011.igem.org
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Welcome on the Paris Bettencourt wiki. This is a very basic layout for the time being for as long as we don't properly master the wiki language.
We are fifteen parisian students coming from many different disciplines who came together to participate to the iGEM competition. You can find out more about our project in the Project section. We are also dedicated to Safety. |
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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
