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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 20 μL of nuclease free water, then cut with E/P and purified from the gel.
- Ligations
| Ligation |
Plate results (lig : neg crtl) 12/12/12
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| 1. E. coli DH5α-T 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 BL-21 KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 25ng |
KAH111/C-His 3:1 No Colonies
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| 3. C-His(E/P)/ 25 ng |
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1 |
2 |
3
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| Insert DNA |
3.12 |
3.12 |
---
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| Vector DNA |
2.94 |
2.94 |
2.94
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| 2x lgn buf (Roche) |
7.06 |
7.06 |
5.0
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| T4 ligase (NEB) |
1.0 |
1.0 |
1.0
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| dH2O |
0 |
0 |
0.88
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14.12 μL |
14.12 μL |
10μl
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PcTF Subcloning in E. coli
