Site-directed-Mutagenesis

1 PCR:

Add
100ng of template DNA
5 ÎŒl 10 X buffer
125ng of forward primer
125ng of reverse primer
1ÎŒL of dNTP mix
3ÎŒL of quick solution reagent
2.5U of Pfu Turbo DNA polymerase.
Water to 50ÎŒL

1 min at 95ºC
16 cycles of 30s at 95ºC, 1 min at 55ºC and 11 min at 68ºC.

2. Digest with Dpn I enzyme for three hours.

3. Transform chemical competent bacteria, grow at 37 oC or 30 oC.

4. Make miniprep DNA

5. Sequence to confirm correct mutagenesis.