Site-Directed Mutagenesis (Stratagene protocol)


Posted by jkolibachuk
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Protocol

This is the protocol for site-directed mutagenesis based on the Stratagene kit.

Materials: 
  • Pfu turbo
  • 10X Pfu turbo buffer
  • dNTPs (10mM)
  • Forward and reverse primers (0.1ug/uL, see methods section for design tips)
  • dH2O
  • Dpn1
  • competent cells

 

Methods: 

Primer Design

  • Forward primer should be between 25 and 45 bases in length and contain the desired mutation in the center with correct sequences on both sides; the reverse primer is the reverse complement of this
  • Primers should have a minimum GC content of 40% and terminate in one or more C's or G's
  • Tm should be greater or equal to 78 degrees Celsius and can be calculated as follows:
    • Tm = 81.5 + 0.41(%GC) - 675/(length in bases) - %mismatch

Reaction

Set up as follows:

 

Components Amount
Template DNA (50ng/uL) 1 uL
10X Buffer 5 uL
Forward Primer (0.1 ug/uL) 1 uL
Reverse Primer (0.1 ug/uL) 1 uL
dNTPs (10mM) 1 uL
Pfu turbo 1 uL
dH2O 40 uL

PCR Program

Depending on the length of the plasmid, this program can become very long, so it may be best to run overnight.

  1. 95 degrees for 1 minute
  2. 95 degrees for 50 seconds, 60 degrees for 50 seconds, 68 degrees for 1 minute/kb of plasmid length -- repeat this step 17 times, or 18 cycles total
  3. 68 degrees for 7 minutes
  4. 4 degree hold

Following PCR

Add 1uL of Dpn1 to PCR reaction.  Incubate at 37 degrees Celsius for 1-2 hours to digest parental DNA.  Run 5uL of the digested reaction on a gel and compare to the undigested parental plasmid - there should be some difference in band pattern.  Transform into competent cells.