INSA-Lyon/9 October 2010

From 2010.igem.org





October 9th


PCR of :

  1. OmpR234 : on DNA from PHL818, with the same program as the 20/09
  2. pha A : on DNA from PiliI, temperature of hybridization of 53°C
  3. 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 :

  1. L2/1N : with E+P
  2. 2P : with E+X
  3. phasin-intein, phasin : with E+S
  4. 1N and 18A : with S+P
  5. 1N and phasin : X+P

Agarose gel electrophoresis for purification of the digestion : 1h30 at 85V.

  1. L2/1N, phasin-intein, phasin (X+P and E+S) : OK, purification on a silica gel
  2. 1N (S+P and X+P), 18A, 2P : nothing appear on the gel

Ligation, overnight at 15°C :

  1. 18A and OmpR234
  2. OmpR234 in chloramphenicol backbone
  3. Curli in chloramphenicol backbone
  4. phasin+phasin intein in Cm backbone
  5. 18A+1N

There are some clones from the ligation of the 7/10 of 18A/OmpR234 and Phasin/phasin-intein. Each clone has satellites. Liquid cultures of 2 clones of each ligation








June
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
July
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
August
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31
September
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
October
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31