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 | </font>
|
| 1. E. coli BL-21 KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng | KAH111/C-His 3:1 No Colonies
|
| 2. E. coli DH5α-T KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 26ng | KAH111/C-His 3:1 No Colonies
|
| 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
|
| 4. E. coli DH5α-T KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng | KAH112/C-His 3:1 No Colonies
|
| 5. C-His(E/P)/ 26 ng (Control Plate) |
|
| | 1 | 2 | 3 |
|
| Insert DNA | 3.12 | 3.12 | --- |
|
| Vector DNA | 2.94 | 2.94 | 2.94 |
|
| 2x lgn buf (Roche) | 7.06 | 7.06 | 5.0 |
|
| T4 ligase (NEB) | 1.0 | 1.0 | 1.0 |
|
| dH2O | 0 | 0 | 0.88 |
|
| | 14.12 μL | 14.12 μL | 10μl |
|
|
PcTF Subcloning in E. coli
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