Rao:EI-PCR

You'll Need

 * 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
If your reaction yields are low, some suggest to run more than one and pool the results.
 * 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 $$MgCl_2$$ (50 mM)
 * 1.5 uL DMSO (optional)
 * x uL $$H_2O$$
 * 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

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.)

Step 3: Ligation and transformation

 * Proceed with standard ligation and transformation protocols