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

From 2011.igem.org

(Difference between revisions)
(→Cyrille)
(→Cyrille)
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Competent cell preparation of MH1
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
 +
[[File:ComS extraction.jpg|thumb|center|ladder - 5ng - 25ng - 50ng]]

Revision as of 09:06, 1 September 2011

Cyrille

S24 + YFP-TetR:

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

CP3008 PCRColony.jpg

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 - 5ng - 25ng - 50ng
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