Site Directed Mutagenesis Guidelines

Gregory Broussard

Revision: 4/19/12

(Please use, change and disseminate freely!)

• Design primers according to the Quickchange SDM protocol.
• Isolate plasmid DNA from a dam+ E. coli strain.
• Run the SDM PCR:
  • Use 125 ng of each primer
  • Use ~25 ng of plasmid template DNA
  • Use 1 μl (2.5 U/μl) of Pfu Turbo DNA Polymerase
    • 95 °C            30 sec.
    • ——————
    • 95 °C            30 sec.
    • 55 °C            1 min.                        12-18 cycles
    • 68 °C            1 min./kb
    • ——————
    • 4 °C            indef.

• Point mutations            12 cycles
• Single amino acid changes            16 cycles
• Multiple amino acid deletions or insertions             18 cycles
• Add 1 μl DpnI (10U/ul) directly to each PCR rxn. Mix in a 1.5 ml tube and incubate for 1 hr. at 37 °C.  (This will digest the parental DNA; sometimes overnight digestion gives better results.)
• Transform 1 μl of each rxn into XL1-Blue or NEB5α cells and plate with selection.  Incubate at 37 °C, overnight.

Notes:

1. Do a control for DpnI activity:
   1. Do control PCR w/o primers
   2. Digest w/ DpnI, as with the true SDM rxn.
   3. Transform and plate.  There should be less colonies on the control (0-2 cfu is ideal).
   4. Use 25-50 ng of template DNA in the PCR.
   5. Math for primer conc.:

(125 ng/(330 X number of bp)) X 1000 = number of pmole primer for 125 ng

primer stocks are 10 μM = 10 pmole/μl

So, for example add 0.9 μl of 10 μM primer for 9 pmoles