Team:Paris Liliane Bettencourt/Notebook/2011/08/30/
From 2011.igem.org
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Contents |
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
Camille
From the overnight culture I did minipreps, and obtained :
Strain | [DNA] in ng/µL | ratio |
S38+pHyperspank 1 | 483.1 | 1.76 |
S38+pHyperspank 2 | 281.9 | 1.67 |
I sent them for sequencing.