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Team:Paris Liliane Bettencourt/Notebook/2011/08/17/
From 2011.igem.org
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.
