Team:Paris Liliane Bettencourt/Notebook/2011/08/30/

From 2011.igem.org

Revision as of 09:10, 1 September 2011

Cyrille

S24 + YFP-TetR:

• Prepare the 9 miniprep
• Replicate into glycerol
• PCR colony was done

YFP-TetR PstI-- 5re;oving the qdditionnal PstI site

• 5 Miniprep prepared

Competent cell preparation of MH1

Axel

extraction of ComS from 5 different colonies

same protocol as those from the 17th of august:

We are going to extract the ComS gene from the PY79 strain. Oligo has been designed for this purpose.

• Preparing the tube

Prepare 5 sterile tube with 20 µL of H2O for each colony Pick the colony using a pipetman woth a sterile tube. The pipetor is set at 3 µL for mixing

• Dissolving the primers

Resuspend the primers adding 10µL of water by nmol of primer. Final concentration at 100 mM. Add 1µL in 2 ml of water to get the 50 µM

• PCR mastermix

60    µL HF phusion buffer
6      µL dNTPs
15    µL of colony mix
6      µL Primer fwd at (50µM)
6      µL Primer rev at (50µM)
157 µL of H2O 

Aliquot 49 µL in 5 PCR tubes Add 1 µL of phusion in each tube.

• PCR temperature

1 - 98°C - 15 min
2 - 98°C - 30 sec
3 - 70°C - gradient +-10  - 1 min
4 - 55°C  - 1 min
5 - 72°C - 1 min
6 - Repeat 2-5 39 times
7 - 7°C forever

result of the gel: (biobricked ComS is about 160bp) ladder 100bp

ladder - col1 - col2 - col3 - col4 - col5 - control no col