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INSA-Lyon/9 October 2010
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Revision as of 20:15, 25 October 2010
October 9th
PCR of :
- OmpR234 : on DNA from PHL818, with the same program as the 20/09
- pha A : on DNA from PiliI, temperature of hybridization of 53°C
- pha B : on DNA from PiliI, temperature of hybridization of 64°C
Resuspension of pha C (we receive the synthesized part yesterday) in 50µL of sterile water
DNA extraction of L2/1N, 1N, 18A, phasin, phasin-intein, 2P, curli and 14K
Digestion on 25µL of DNA :
- L2/1N : with E+P
- 2P : with E+X
- phasin-intein, phasin : with E+S
- 1N and 18A : with S+P
- 1N and phasin : X+P
Agarose gel electrophoresis for purification of the digestion : 1h30 at 85V.
- L2/1N, phasin-intein, phasin (X+P and E+S) : OK, purification on a silica gel
- 1N (S+P and X+P), 18A, 2P : nothing appear on the gel
Ligation, overnight at 15°C :
- 18A and OmpR234
- OmpR234 in chloremphenicol backbone
- Curli in chloremphenicol backbone
- phasin+phasin intein in Cm backbone
- 18A+1N
There is some clones from the ligation of the 7/10 of 18A/OmpR234 and Phasin/phasin-intein. Each clone have satalites.Liquid culture of 2 clones of each ligation
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