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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