Cloning of shRNA into lentiviral vector pLKO.1
Procedure
Primer annealing
- annealing reaction contains: 1 µl sense primer (stock solution 100 µM), 1 µl antisense primer (stock solution 100 µM), 5 µl NEB buffer 2 and 43 µl ddH2O
- Annealing reaction occurs in thermocycler with the corresponding program:
| 1 | Hot start | 94°C | Pause – addition of enzyme mix |
| 7 repetitions of steps 2-4 with hybridization temperature - 1 ºC per cycle (touch-down) | |||
| 2 | Denauturation | 94°C | 20 seconds |
| 3 | Hybridization | 64-58°C | 20 seconds |
| 4 | Elongation | 72°C | depending on fragment size (1 min/750 bp) |
| 30 repetitions of steps 2-4 with hybridization temperature of 58 ºC | |||
| 5 | Elongation | 72°C | 5-10 minutes |
| 6 | Stop | 8°C | |