Site directed mutagenesis
Procedure
- Prepare the PCR reaction mix according to the hot start procedure. Include one negative control sample without Pfu polymerase (incubate sample at the lowest annealing temperature of your gradient)
- Perform gradient PCR using the following thermal cycling conditions:
| Step | Temperature °C | Time | No. of cycles |
|---|---|---|---|
| Initial denauturation | 94 | 1-3 min | 1 |
| Hot Start / add enzyme | 94 | pause | 1 |
| Denauturation | 94 | 20 sec | 20 |
| Annealing | 51, 55, 59, 63 | 20 sec | |
| Extension | 72 | 1 min/500 bp | |
| Final extension | 72 | 10 min | 1 |
- DpnI digestion: prior to DpnI digestion, separate 15 μl PCR product of negative control (will serve as positive control). Add 2 μl DpnI to every PCR sample. Incubate for 2 hours at 37 °C
- Inactivation of DpnI: incubate the digested PCR products at 70 °C for 10 min
- Perform transformation of E. coli cells with the PCR products: 10 μl PCR products (digested or undigested) for 90 μl E. coli competent cells
- Check positive/negative controls. If negative control shows no or low background, check out the mutagenesis samples: starting with the highest gradient temperature, pick every colony and streak out on fresh agar plate (cake style). Next day, isolate the plasmid from the first 12 - 24 clones plate using Birnboim- Dooley protocol
- Restriction analysis: digest the plasmid with the newly incorporated restriction enzyme. Check the size of fragments on the electrophoresis gel compared to the original (wildtype) version.
- Sequence one positive clone and save in strain collection, if correct