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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

  • 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]\displaystyle{ MgCl_2 }[/math] (50 mM)
    • 1.5 uL DMSO (optional)
    • x uL [math]\displaystyle{ 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.)

Step 3: Ligation and transformation