Team:Paris Liliane Bettencourt/Notebook/2011/07/22/

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Team IGEM Paris 2011

Contents

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


Mathias & Laura

Miniprep

-RFP Standard Terminator RBS & T7

-GFP mut3b T7 Terminator RBS & T7

-pT7 I712074

Digestion and gel extraction

-RFP Standard Terminator RBS & T7 by Xba1 and Pst1

-GFP mut3b T7 Terminator RBS & T7 by Xba1 and Pst1

-pT7 I712074 by Spe1 and Pst1

-pVeg + SpoVg and LacI + Term by Spe1 and Pst1

Ligation and transformation

-GFP mut3b T7 Terminator RBS T7 & pT7 => T7 Autoamplifier system + Reporter K606036

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