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Team:Aix-Marseille/Notebook W3110-sdaBC-cheA
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Revision as of 02:21, 12 October 2014
Notebook
Week 10 : 09/01/2014 ➡ 08/07/2014
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PCR cheA-kan-cheA using the iGEM PCR protocol with Q5 polymerase. The temperature of annealing is 60°C, the elongation time is 1’30”.
Mix:
- 1µL pKD4 vector
- 2,5µL primer 3
- 2,5µL primer 4
- 2,5µL dNTP
- 10µL Q5 buffer
- qsp 50µL H2O
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Clean-up of the previous PCR. Elution in 45µL of water.
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Electroporation of W3110 ∆sdaBC electrocompetent cells from Aimeric (clone 43)
40µL cells + 10µL PCR
40µL cells + 0,5µL pKT25 (negative control)
==> Spread bacteria on LB-Kan
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Many clones have grown on the negative control but no clone has grown for the cheA mutant. So cells are competent but they don’t express the recombinase.
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Streak out W3110 ∆sdaBC 43 and 63 from Aimeric on LB and LB-Kan
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The bacteria are sensitive to kanamicyn.
Week 11 : 09/08/2014 ➡ 08/14/2014
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Repreparation of W3110 ∆sdaBC 43 electrocompetent cells (see Lambda Red mutant construction protocol) and restart from this step (day 1).
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The clones obtained were KanR and ApS ==> OK
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The clones are pooled 3 by 3 to be tested by PCR with primers 52 and 53.
The pools 1 and 2 seemed to contain mutants (1786bp).
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Restart PCR with primers 52 and 53 on the isolated clones from pools 1 and 2.
The clones 1a, 2a and 2b seemed good, they are kept at -80°C. So the W3110 ∆sdaBC ∆cheA is OK.
