Site-Directed Mutagenesis of H gamma DC in pQE.1 (MSKC)
Adapted from Stratagene Quikchange


1. Design complementary primers covering your mutation
2. Dilute primers to 125 ug/mL in TE
3. Prepare 3 reactions on ice containing the following:

  10x Buffer dNTP Primer 1 Primer 2 DNA ddH2O
Sample1 5 µL 1 µL 1 µL 1 µL 5 µL 36 µL
Sample 2 5 µL 1 µL 1 µL 1 µL 2 µL 39 µL
Sample 3 5 µL 1 µL 1 µL 1 µL 1 µL 40 µL

4. After everything else is added, add 0.7 mL of Pfu Turbo Polymerase
5. Mix reactions thoroughly
6. Add 1 drop of mineral oil on top of samples and spin briefly
7. Cycle using the following parameters on the old thermocycler
(Program “Liss”)

1X step 1 95°C for 1 minute
12X steps 2-4 95°C for 30 seconds
  55°C for 1 minute
  68°C for 15 minutes
1X step 5 68°C for 10 minutes
  4°C hold


8. After PCR add 1 mL of Dpn1 and incubate for 1 hour at 37°C
9. Run 10 µL of product on a 1% agarose gel and stain with EtBr
(A band should be present at ~4 kb)
10. Transform 1 µL of PCR product into XL-1 Blue supercompetent cells
11. Plate 300 µL of transformation mixture and grow overnight at 37°C
12. Miniprep DNA and send to MGH for sequencing