Team:UNICAMP-EMSE Brazil/Notebook/2 September 2011
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02 August 2011
Checking digestion (E-P) of mini-prep constructs
Objective:
- Checking digestion (E-P) of mini-prep constructs.
Samples:
- Gel 1:
- SoxS+RBS+IL10+HlyA+T C2 (nd)/ SoxS+RBS+IL10+HlyA+T C1/ SoxS+RBS+IL10+HlyA+T C2/ SoxS+RBS+IL10+HlyA+T C3/ SoxS+RBS+IL10+HlyA+T C4/ SoxS+RBS+GFP+HlyA+T C2 (nd)/ SoxS+RBS+GFP+HlyA+T C1/ SoxS+RBS+GFP+HlyA+T C2/ SoxS+RBS+GFP+HlyA+T C3/ SoxS+RBS+GFP+HlyA+T C4/ SoxS+RBS+HlyB+HlyD+TolC+T (nd)/ SoxS+RBS+HlyB+HlyD+TolC+T
- Gel 2:
- flHDC+RBS+HlyB+hlyD+TolC+T C4(nd)/ flHDC+RBS+HlyB+hlyD+TolC+T C2/ flHDC+RBS+HlyB+hlyD+TolC+T C3/ flHDC+RBS+HlyB+hlyD+TolC+T C4/ RBS+GFP+HlyA+T C2 (nd)/ RBS+GFP+HlyA+T C1/ RBS+GFP+HlyA+T C2/ flHDC+RBS+IL12 C5 (nd)/ flHDC+RBS+IL12 C1/ flHDC+RBS+IL12 C2/ flHDC+RBS+IL12 C5.
- Ladder:
- 100-10.000 bp (Fermentas)
- Gel Agarose concentration:
- 1,0%
- Results:
Gene | Total size (gene+vector) | Linear vector size | Gene size | Result |
---|---|---|---|---|
SoxS+RBS+IL10+HlyA+T C1 | 4270 bp | 3205 bp | 1065 bp | Did not digest |
SoxS+RBS+IL10+HlyA+T C2 | 4270 bp | 3205 bp | 1065 bp | Did not digest |
SoxS+RBS+IL10+HlyA+T C3 | 4270 bp | 3205 bp | 1065 bp | Did not digest |
SoxS+RBS+IL10+HlyA+T C4 | 4270 bp | 3205 bp | 1065 bp | Did not digest |
SoxS+RBS+GFP+HlyA+T C1 | 4365 bp | 3205 bp | 1160 bp | OK |
SoxS+RBS+GFP+HlyA+T C2 | 4365 bp | 3205 bp | 1160 bp | Smaller bands |
SoxS+RBS+GFP+HlyA+T C3 | 4365 bp | 3205 bp | 1160 bp | Smaller bands |
SoxS+RBS+GFP+HlyA+T C4 | 4365 bp | 3205 bp | 1160 bp | OK |
SoxS+RBS+HlyB+HlyD+TolC + T | 8257 bp | 2800 bp | 5457 bp | No bands |
fLHDC+RBS+HlyB+HlyD+TolC + T C2 | 8280 bp | 2800 bp | 5480 bp | OK |
fLHDC+RBS+HlyB+HlyD+TolC + T C3 | 8280 bp | 2800 bp | 5480 bp | OK |
fLHDC+RBS+HlyB+HlyD+TolC + T C4 | 8280 bp | 2800 bp | 5480 bp | OK |
RBS+GFP+HlyA+T C1 | 4262 bp | 3205 bp | 1057 bp | Did not digest |
RBS+GFP+HlyA+T C2 | 4262 bp | 3205 bp | 1057 bp | Did not digest |
fLHDC+RBS+IL12 C1 | 4584 bp | 2800 bp | 1784 bp | OK |
fLHDC+RBS+IL12 C1 | 4584 bp | 2800 bp | 1784 bp | Diluted (?) |
fLHDC+RBS+IL12 C1 | 4584 bp | 2800 bp | 1784 bp | No bands |
- Checking digestion again:
- SoxS+RBS+IL10+HlyA+T and RBS+GFP+HlyA+T
- Real digestion for ligation:
- fLHDC+RBS+IL12 C1 with HlyA+T
- Ready Biobricks:
- fLHDC+RBS+HlyB+HlyD+TolC + T / SoxS+RBS+GFP+HlyA+T / SoxS+RBS+HlyB+HlyD+TolC + T.
- Ready to test:
- Device that produces and secrete IL10 ! :)
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