Team:UNICAMP-EMSE Brazil/Notebook/31 August 2011
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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.!
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:
- 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:
- Ligation of samples purified from the gel
- Transformation and platting
- Inoculation of fLHDC+RBS+GFP C6 from permanent culture and new colonies of fLHDC+RBS+ IL12 from the plate (C8, C9,C10,C11).