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INSA-Lyon/20 September 2010
From 2010.igem.org
September 20th
Digestion with E+P of :
- 1F/24A (3 clones)
- 4O/10L
- Curli/22B (2 clones)
- I0500 just with E (clone from the 13/08 and the 17/08)
Verification of the weight of the part and the ligation on an agarose gel electrophoris :
- I0500, 4O/10L : the part are not correct, we will ask for I0500, 4O and 10L again
- One clone of Curli/22B and all the 1F/24A seams OK
Start of 6 liquid cultur of PHL1273 with 2 condition of cultur :
- waterbath at 31°C, shaking speed 100 rpm
- waterbath at 32°C, shaking speed 100 rpm
Lyophilisated primers ompR234 Forward and Reverse are diluted in sterile water (at 100µM): Forward primer: 450µl added, and Reverse primer: 560µl added.
3 PCR: vial 1 corresponded to negative control with 10µl sterile water, vial 2 corresponded to genomic DNA 1/5 diluted and vial 3 corresponded to genomic DNA 1/10 diluted.
MIX composition for one PCR:
- 5µl 10X buffer with MgCl2
- QSP H2O
- 5µl dNTP (PCR Dig Labeling Mix, Roche)
- 10 or 5 µl DNA
- 1µl of each primer
- 0,75µl Taq Pol (Euromedex)
NB: Hybridation temperature is 56°C (program OMPR). PCR products and diluted primers are always stored at -20°C.
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