"
Team:British Columbia/Protocols/Site Directed Mutagenesis
From 2012.igem.org
Site-Directed Mutagenesis with BioRAD iPROOF PCR kit
This protocol closely follows the Stratagene QuikChange Site-Directed Mutagenesis kit, but uses the enzymes and buffers from the BioRAD iPROOF PCR kit.
- 1. Prepare the reaction mixtures:
| dNTP | 4 µL |
| iProof HF Buffer | 10 µL |
| MgCl2 | 1.5 µL |
| Primer, each | ? µL (125 ng) |
| dsDNA template | ? µL (5-50 ng) |
| iProof enzyme | 0.5 µL |
| dH2O | to 50 µL |
- I would suggest trying 5 ng of dsDNA template, as I had the highest mutation success rate at this concentration. This observation, however, has not been confirmed and is more anecdotal
- 2. Thermocycle the reaction according to the standard iPROOF cycling conditions, adjusting the extension time (X:XX) for the expected product length.
| 1. | Hot start | 98ºC | 1:00 |
| 2. | Denaturation | 98ºC | 0:10 |
| 3. | Anneal | 55ºC | 0:30 |
| 4. | Extension | 72ºC | X:XX |
| 5. | GOTO 2 for 12-18 cycles |
The QuikChange protocol suggests the following number of cycles
| Type of mutation | Number of cycles |
| Point mutations | 12 |
| Single amino acid changes | 16 |
| Multiple amino acid deletions or insertions | 18 |
- 3. Check for amplification on agarose gel
- I have had positive transformations even without positive band, so it's worth a shot to transform the product regardless of the outcome.
- 4. Perform DpnI digestion of amplification products to digest the parental DNA (CRITICAL!!!)
- 5. Add 10 U of DpnI to each reaction and incubate at 37 ºC
- 6. Transform 1 µL of PCR product. I did not do any PCR purification before this step
