Team:Paris Liliane Bettencourt/Notebook/2011/08/17/

From 2011.igem.org

Team IGEM Paris 2011

Contents

Cyrille

PCR colony on ComS

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

Kevin

Send to sequencing

3 of each plasmid was sent to sequencing GATC.