Team:UNICAMP-EMSE Brazil/Notebook/31 August 2011

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May
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
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


Electrophoresis Gel 1, 31/08/2011.
Gel 1: HlyA+Terminator (E-X)/ HlyA+Terminator (E-X)/ HlyA+Terminator (E-X)/ HlyA+Terminator (E-X)/ SoxS+RBS+GFP (E-S)/ SoxS+RBS+GFP (E-S)/ RBS+HlyD+TolC+Terminator (E-X)/ RBS+HlyD+TolC+Terminator (E-X)/ RBS+HlyD+TolC+Terminator (E-X)/ RBS+HlyD+TolC+Terminator (E-X)/ SoxS+RBS+IL 10 (E-S)/ SoxS+RBS+IL 10 (E-S)/ flHDC+RBS+HlyB (E-S)/ flHDC+RBS+HlyB (E-S)/flHDC+RBS+IL 12 (E-S)/ flHDC+RBS+GFP (E-S)/

31 August 2011

Digestion, purification and ligation!!!

Objective:

  • Digestion of constructs to purify and ligate them.

Samples:

  • Gel 1:
  • HlyA+Terminator (E-X)/ HlyA+Terminator (E-X)/ HlyA+Terminator (E-X)/ HlyA+Terminator (E-X)/ SoxS+RBS+GFP (E-S)/ SoxS+RBS+GFP (E-S)/ RBS+HlyD+TolC+Terminator (E-X)/ RBS+HlyD+TolC+Terminator (E-X)/ RBS+HlyD+TolC+Terminator (E-X)/ RBS+HlyD+TolC+Terminator (E-X)/ SoxS+RBS+IL 10 (E-S)/ SoxS+RBS+IL 10 (E-S)/ flHDC+RBS+HlyB (E-S)/ flHDC+RBS+HlyB (E-S)/flHDC+RBS+IL 12 (E-S)/ flHDC+RBS+GFP (E-S)/
  • Ladder:
  • 100-10.000 bp (Fermentas)
  • Gel Agarose concentration:
  • 1,0%


  • Results:

Construction Total size (gene+vector) Linear vector size Gene size Result
HlyA+Terminator 3527 3205 No cut Ok
HlyA+Terminator 3527 3205 No cut Ok
HlyA+Terminator 3527 3205 No cut Ok
HlyA+Terminator 3527 3205 No cut Ok
SoxS+RBS+GFP 3535 2800 838 Ok
SoxS+RBS+GFP 3535 2800 838 Ok
RBS+HlyD+TolC+Terminator 6376 3205 No cut Run more
RBS+HlyD+TolC+Terminator 6376 3205 No cut Run more
RBS+HlyD+TolC+Terminator 6376 3205 No cut Run more
RBS+HlyD+TolC+Terminator 6376 3205 No cut Run more
SoxS+RBS+IL 10 3543 2800 743 Ok
SoxS+RBS+IL 10 3543 2800 743 Ok
flHDC+RBS+HlyB 5109 2800 2309 Run more
flHDC+RBS+HlyB 5109 2800 2309 Run more
flHDC+RBS+IL 12 4584 2800 1784 Where is the vector?
flHDC+RBS+GFP 3661 2800 861 No digestion


OBS: flHDC+RBS+IL 12 and flHDC+RBS+GFP did not work! Inoculate again from a permanent culture.!





Electrophoresis Gel 2, 31/08/2011.
Gel 2: HlyA+Terminator (E-X)/ HlyA+Terminator (E-X)/ HlyA+Terminator (E-X)/ HlyA+Terminator (E-X)/ SoxS+RBS+GFP (E-S)/ SoxS+RBS+GFP (E-S)/ RBS+HlyD+TolC+Terminator (E-X)/ RBS+HlyD+TolC+Terminator (E-X)/ RBS+HlyD+TolC+Terminator (E-X)/ RBS+HlyD+TolC+Terminator (E-X)/ SoxS+RBS+IL 10 (E-S)/ SoxS+RBS+IL 10 (E-S)/ flHDC+RBS+HlyB (E-S)/ flHDC+RBS+HlyB (E-S)/flHDC+RBS+IL 12 (E-S)/ flHDC+RBS+GFP (E-S)/

Same electrophoresys but after a longer period run:

  • Ladder:
  • 100-10.000 bp (Fermentas)
  • Gel Agarose concentration:
  • 1,0%

Samples:

  • Gel 2: Same of Gel1!
  • HlyA+Terminator (E-X)/ HlyA+Terminator (E-X)/ HlyA+Terminator (E-X)/ HlyA+Terminator (E-X)/ SoxS+RBS+GFP (E-S)/ SoxS+RBS+GFP (E-S)/ RBS+HlyD+TolC+Terminator (E-X)/ RBS+HlyD+TolC+Terminator (E-X)/ RBS+HlyD+TolC+Terminator (E-X)/ RBS+HlyD+TolC+Terminator (E-X)/ SoxS+RBS+IL 10 (E-S)/ SoxS+RBS+IL 10 (E-S)/ flHDC+RBS+HlyB (E-S)/ flHDC+RBS+HlyB (E-S)/flHDC+RBS+IL 12 (E-S)/ flHDC+RBS+GFP (E-S)/

Results:

  • RBS+HlyD+TolC+Terminator has two bands, but the smaller one can be the closed vector while the larger ones can be the linearized vector. We cut the larger ones.


Quantification of digestion:

Objective:

Electrophoresis Gel 3, 31/08/2011.
Gel 3: HlyA+T (E-X) 1/ HlyA+T (E-X) 2/ HlyA+T (E-X) 3/ HlyA+T (E-X) 4/ SoxS+RBS+IL10 (E-S) 1/ SoxS+RBS+IL10 (E-S) 2/ RBS+HlyD+TolC+T (E-X) 1/ RBS+HlyD+TolC+T (E-X) 2/ RBS+HlyD+TolC+T (E-X) 3/ RBS+HlyD+TolC+T (E-X) 4/ / flHDC+RBS+HlyB (E-S) 1/ flHDC+RBS+HlyB (E-S) 2/ SoxS+RBS+GFP (E-S) 1/ SoxS+RBS+GFP (E-S) 1
  • Quantification of samples purified from the gel
  • Ladder:
  • 100-10.000 bp (Fermentas)
  • Gel Agarose concentration:
  • 1,0%
  • Gel 3:
  • HlyA+T (E-X) 1/ HlyA+T (E-X) 2/ HlyA+T (E-X) 3/ HlyA+T (E-X) 4/ SoxS+RBS+IL10 (E-S) 1/ SoxS+RBS+IL10 (E-S) 2/ RBS+HlyD+TolC+T (E-X) 1/ RBS+HlyD+TolC+T (E-X) 2/ RBS+HlyD+TolC+T (E-X) 3/ RBS+HlyD+TolC+T (E-X) 4/ / flHDC+RBS+HlyB (E-S) 1/ flHDC+RBS+HlyB (E-S) 2/ SoxS+RBS+GFP (E-S) 1/ SoxS+RBS+GFP (E-S) 1



  • Other tasks made today:
  1. Ligation of samples purified from the gel
  2. Transformation and platting
  3. Inoculation of fLHDC+RBS+GFP C6 from permanent culture and new colonies of fLHDC+RBS+ IL12 from the plate (C8, C9,C10,C11).





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