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 | </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 | 4 | 5
|
| Insert DNA | 2.34 | 2.34 | 2.05 | 2.05 | ---
|
| Vector DNA | 1.92 | 1.92 | 1.92 | 1.92 | 1.92
|
| 2x lgn buf (Roche) | 5.26 | 5.26 | 5.0 | 5.0 | 5.0
|
| T4 ligase (NEB) | 1.0 | 1.0 | 1.0 | 1.0 | 1.0
|
| dH2O | 0 | 0 | 0.03 | 0.03 | 2.08
|
| | 10.52 μL | 10.52 μL | 10μl | 10μL | 10μL
|
|
PcTF Subcloning in E. coli
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