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Team:UNICAMP-EMSE Brazil/Notebook/12 September 2011
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Gel 1: Const promo+ RBS+SoxR+T ; SoxS+RBS+GFP+HlyA+T
12 September 2011
Purificate genes
Samples:
- Gel 1:
- Const promo+ RBS+SoxR+T ; SoxS+RBS+GFP+HlyA+T (40ul of digestion and 4ul of buffer)
- Ladder:
- 1kB Plus DNA Ladder (Invitrogen)
- Gel Agarose concentration:
- 1,0%
- Result:
Gel 1: Sox S RBS IL10 (3 samples : A, B ,C)
Check up genes
Samples:
- Gel 2:
- Sox S RBS IL10 (3 samples : A, B ,C) (40ul of digestion and 4ul of buffer)
- Ladder:
- 1kB Plus DNA Ladder (Invitrogen)
- Gel Agarose concentration:
- 1,0%
- Result:
Gel 1: flhDC+ RBS+ GFP+HlyA+T (P1C1, P1C2, P2C1, P2C2); SoxS+RBS+HlyB/D+TolC+T (P1C3) ; flhDC+RBS+IL-12+HlyA+T (P2C1, P2C2)
Checking Electrophoresis
Objective:
- check the inserts sizes (volume of ladder applied 6uL; checking digestion sample:20uL digested sample (E-P, 5uL DNA) +2uL buffer)
Samples:
- Gel 2:
- flhDC+ RBS+ GFP+HlyA+T (P1C1, P1C2, P2C1, P2C2); SoxS+RBS+HlyB/D+TolC+T (P1C3) ; flhDC+RBS+IL-12+HlyA+T (P2C1, P2C2)
- Results:
| Construction | Total size (gene+vector) (bp) | Linear vector size (bp) | Gene size (bp) | Result |
|---|---|---|---|---|
| flhDC+ RBS+ GFP+HlyA+T (P1C1) | 4388 | 3205 | 1183 | Complete digestion, but we dont see the gene (low concentration) |
| flhDC+ RBS+GFP+HlyA+T (P1C2) | 4388 | 3205 | 1183 | Complete digestion, concentration around 3ng/uL |
| flhDC+ RBS+GFP+HlyA+T (P2C1) | 4388 | 3205 | 1183 | Complete digestion, concentration around 3ng/uL |
| flhDC+RBS+ GFP+HlyA+T (P2C2) | 4388 | 3205 | 1183 | Non digested |
| SoxS+ RBS+ HlyB+RBS+HlyD+D+TolC+T (P1C3) | 8257 | 3205 | 5052 | Complete digestion, concentration around 4ng/uL |
| flhDC+RBS+IL-12 +HlyA+T (P2C1) | 5311 | 3205 | 2106 | Complete digestion, concentration around 5ng/uL |
| flhDC+RBS+IL-12 +HlyA+T (P2C2) | 5311 | 3205 | 2106 | Complete digestion, concentration around 8ng/uL |
Ligations:
=> QseB/C (~2100bp) and pSB1C3 (~2000bp) (ratio for ligation 1:1)
14.8 ul - H20
2.2 ul - digested QseB-QseC (~30 ng )
3.3 ul - digested vector (~30 ng)
2 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________
20 ul => TOTAL
=> flhDC (126bp) and pSB1C3 (~2000bp) (ratio for ligation 3:1)
4.7 ul - H20
18 ul - digested flhDC (~90 ng )
3.3 ul - digested vector (~30 ng)
3 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________
30 ul => TOTAL
=> SoxR (~500bp) and pSB1C3 (~2000bp) (ratio for ligation 3:1)
2.4 ul - H20
11.3 ul - digested SoxR (~90 ng )
3.3 ul - digested vector (~30 ng)
2 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________
20 ul => TOTAL
=> IL-10 (~550bp) and pSB1C3 (~2000bp) (ratio for ligation 3:1)
1.7 ul - H20
12 ul - digested IL-10 (~90 ng )
3.3 ul - digested vector (~30 ng)
2 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________
20 ul => TOTAL
=> IL-12 (~1660bp) and pSB1C3 (~2000bp) (ratio for ligation 2:1)
8.7 ul - H20
5 ul - digested IL-12 (~60 ng )
3.3 ul - digested vector (~30 ng)
2 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________
20 ul => TOTAL
=> HlyB (~2200bp) and pSB1C3 (~2000bp) (ratio for ligation 1:1)
11.9 ul - H20
1.8 ul - digested HlyB (~30 ng )
3.3 ul - digested vector (~30 ng)
2 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________
20 ul => TOTAL
=> HlyD (~1500bp) and pSB1C3 (~2000bp) (ratio for ligation 2:1)
8.7 ul - H20
5 ul - digested HlyD (~60 ng )
3.3 ul - digested vector (~30 ng)
2 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________
20 ul => TOTAL
=> TolC (1580bp) and pSB1C3 (~2000bp) (ratio for ligation 2:1)
7.7 ul - H20
15 ul - digested TolC (~60 ng )
3.3 ul - digested vector (~30 ng)
3 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________
30 ul => TOTAL
=> HlyA (~230bp) and pSB1C3 (~2000bp) (ratio for ligation 3:1)
2.7 ul - H20
20 ul - digested HlyA (~90 ng )
3.3 ul - digested vector (~30 ng)
3 ul - 10X Ligation Buffer
1 ul - T4 Ligase
_________________________________
