One Step Gene Disruption/Tagging
Materials
- Appropriate primers
- Appropriate template plasmid (pFA6 series)
- dNTPs
- Taq DNA polymerase and Buffer
Method
- Prepare the following Master Mix:
| Reagent | Volume |
|---|---|
| F1 or F2 Primer | 5 μl |
| R1 Primer | 5 μl |
| 2 mM dNTPs | 50 μl |
| 10X Taq Buffer | 50 μl |
| pFA6 pl.templ. (~250ng) | 5 μl |
| H2O | 382.5 μl |
| Taq | 2.5 μl |
In case of NATMX cassettes add 5% DMSO
- Mix and aliquote 100 μl in each 0.2 ml PCR eppendorf tube.
- Run the following PCR program:
| PCR program | |
|---|---|
| 2' | 95°C |
| 5 cycles | |
| 1' | 94°C |
| 1' | 45°C |
| 4' | 72°C |
| 30 cycles | |
| 1' | 94°C |
| 1' | 52°C |
| 4' | 72°C |
| 10' | 72°C | |