Team:Paris Liliane Bettencourt/Notebook/2011/08/24/

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Cyrille

TetR-YFP

The Gel extraction was carried on and the tubes pooled. the final concentration is around 10ng/µL

2 times 500ng of the plasmid containing the YFP-tetR wasdigested in E and X 3 times 160ng of the PCR product was digested in E and S

The opened plasmid was PCR purified to eliminate the small fragment. An inactivation of EcoRI enzyme was carried on on the PCR tube at 80°C during 5 min.

In the PCR product, the insert account for 1/3 of the final mass in ng. The digested fragment is 144 bp. The original vector is 3427.

Mvector = 50 ng Cvector = Cinsert = 10 ng/µL

3 ratio will be attempted:

1:1 : Minsert = 6,30 ng -> 0,63 µL (ligation in 20 µL) 1:6: Minsert = 38 ng -> 3,8 µL (ligation in 20 µL) 1:50 Minsert = 315 ng -> 32 µL (ligation in 50 µL)

Then tranformation by eat chock, one hour of growth, and then plating in Cm plates.

Quick change to eliminate PstI inside TetR-YFP

Quick change mutagenesis using the stratagene protocol.

""Dilution""

For te two primers Mother solution 100 µM

  -> 1 µL in 20µL -> daughter solution 5µM

125 ng = 8,25 pmol 5 µM = 5.0 pmol/µL

For the Template DNA

Mother concentration: 180 ng/µL

1µL of DNA in 35µL of water

Final concentration: 5µg/µL

""PCR mastermix""

For control tube:

5 µL of phusion HF buffer 1,65 µL of primer 1 solution 1,65 µL of primer 2 solution 10µL of template DNA at 5µg/µL 30,7 H2O

1µL of phusion

For QCM site:

5 µL of phusion HF buffer 1,65 µL of primer 1 solution 1,65 µL of primer 2 solution 1 µL of dNTPs 1 - 5 - 10 µL of template DNA at 5µg/µL 38,7 - 34,7 - 29,7 µL H2O

1µL of phusion

Primer sequence GTG GTC GGG GTA GCG GGC GAA GCA TTG GAG GCC GTA GCC GAA GGT GGT CA

Tm primer = 72°C

The gel was runned and the result are shown on this gel

ladder - 5ng - 25ng - 50ng

Camille

Meeting on the morning. Then on the afternoon I worked on the PCR product controlled yesterday.