- High-fidelity polymerase - Pfu (Stratagene, etc.) or Phusion (NEB)/iProof (Bio-Rad)
- A very small vector back-bone and your desired gene inserted (preferably w/o BsaI or BsmBI cut sites)
- Primers designed for your mutation (see Stemmer et al. on how to do it)
- Restriction enzymes DpnI and BsaI or BsmBI or another similar restriction endonuclease
- Your favorite thermal cycler
Step 1: PCR
- Set up your PCR as follows for 50 uL reaction volume:
- x uL Polymerase Buffer
- 1 uL 10x to 20x diluted template
- 0.5 uL Forward Primer (30 uM) (or appropriately diluted)
- 0.5 uL Reverse Primer (30 uM)
- 1.5 uL [math]MgCl_2[/math] (50 mM)
- 1.5 uL DMSO (optional)
- x uL [math]H_2O[/math]
- Reaction Conditions
- Use the manufacturer specified reaction temperatures and times for optimal performance.
- Touchdown annealing from 60C to 50C with -1.0C steps
- This will be the first 10 cycles. Use your determined extension time.
- Continue with annealing at 50C and extension using determined time.
- Verify your product by standard gel electrophoresis
If your reaction yields are low, some suggest to run more than one and pool the results.
Step 2: Digestion
- Purify the PCR product via standard methods (Qiagen PCR prep or other well established methods)
- Set up the following enzymatic digest for a 50 uL volume
- 43 uL PCR product
- 5 uL 10X Buffer
- 1 uL DpnI (assumed 20,000 U/mL)
- Dpn digests only methylated DNA at its recognition site. This should remove all template DNA.
- React at 37C for 2 hours
- Heat inactivate at 80C for 20 minutes (optional)
- Add 1 uL BsaI (or equivalent)
- React at 50C for 2 hours
- Purify by standard methods (Gel purification, Qiagen PCR prep, etc.)