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:
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.