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 PcTF Subcloning in E. coli
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01/12/2013
Ligation of KAH111, KAH112 and C-His vector in DH5α-T and BL21
- The 4 μL of C-His Vector from stock is diluted with 16 μL of nuclease free water, then cut with E/P and purified from the gel.
- Ligations
| Ligation |
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| 1. E. coli BL-21 KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng |
KAH111/C-His 3:1 No Colonies
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| 2. E. coli DH5α-T KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 26ng |
KAH111/C-His 3:1 No Colonies
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| 3. E. coli BL-21 KAH112(E/P)/size, 22.8 ng + C-His(E/P)/3600, 26ng |
KAH112/C-His 3:1 No Colonies
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| 4. E. coli DH5α-T KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng |
KAH112/C-His 3:1 No Colonies
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| 5. C-His(E/P)/ 26 ng (Control Plate) |
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- Calculations are for the ng of insert we need to get a 3:1 ratio of insert molecules to 50 ng vector molecules
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1 |
2 |
3 |
4 |
5
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| Insert DNA |
2.34 |
2.34 |
2.05 |
2.05 |
---
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| Vector DNA |
1.92 |
1.92 |
1.92 |
1.92 |
1.92
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| 2x lgn buf (Roche) |
5.26 |
5.26 |
5.0 |
5.0 |
5.0
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| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
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| dH2O |
0 |
0 |
0.03 |
0.03 |
2.08
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10.52 μL |
10.52 μL |
10μl |
10μL |
10μL
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- The incubation time for the ligation process was 30min at room temperature.
- Fast transformation, 30 min on ice.
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PcTF Subcloning in E. coli
