Team:UNICAMP-EMSE Brazil/Notebook/9 September 2011
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09 September 2011
Digestion and Electrophoresis
- Digestion recipe:
- DNA – 7ul
- Buffer O – 2ul
- EcoRI – 0,5ul
- PstI – 0,5ul
- Water – 10ul
Samples:
- Gel 1:
- SoxS+RBS+GFP+HlyA+T nd/ SoxS+RBS+GFP+HlyA+T E-P/ Const. Promoter+ RBS+SoxR+ T nd/ Const. Promoter+ RBS+SoxR+ T E-P/ SoxS+RBS+HlyB+HlyD+TolC + T nd/ SoxS+RBS+HlyB+HlyD+TolC + T E-P
- Ladder:
- 100-10.000 bp (Fermentas)
- Gel Agarose concentration:
- 1,0%
- Results:
Construction | Total size (gene+vector) (bp) | Linear vector size (bp) | Gene size (bp) | Result |
---|---|---|---|---|
1) SoxS+RBS+GFP+HlyA+T | 4365 | 3205 | 1160 | ok |
2) Const. Promoter+ RBS+SoxR+ T | 2732 | 2079 | 653 | ok |
3) SoxS+RBS+HlyB+HlyD+TolC + T | 8257 | 2800 | 5457 | ?? |
Samples:
- Gel 2:
- SoxS+RBS+GFP+HlyA+T E-X/ / Const. Promoter+ RBS+SoxR+ T E-S
Check up electrophoresis
Objective:
- Checking digestion
Samples:
- Gel 3:
- Sox S + RBS + IL10 ; flHDC + RBS +IL12+ HlyA + T (digest by (E-P))
- Ladder:
- 100-10.000 bp (Fermentas)
- Gel Agarose concentration:
- 1,0%
- Results:
Construction | Total size (gene+vector) (bp) | Linear vector size (bp) | Gene size (bp) | Result |
---|---|---|---|---|
Sox S + RBS + IL10 | 3543 | 2800 | 743 | No Gene size band |
Sox S + RBS + IL10 | 3543 | 2800 | 743 | No Gene size band |
Sox S + RBS + IL10 | 3543 | 2800 | 743 | No Gene size band; and partial digestion |
flHDC +RBS +IL12+ HlyA +T | 5311 | 3205 | 2106 | Ok |
flHDC +RBS +IL12+ HlyA +T | 5311 | 3205 | 2106 | Ok |