1) Design the mutagenesis primers (fwd and rev) with 15-18 bp each side of the mutated codon

Ex : Carm1 P409A

   Primer fwd TGGCTATCCACAGCCGCAACAGAGCCCCTGAC
   Primer rev GTCAGGGGCTCTGTTGCGGCTGTGGATAGCCA     
   

2) PCR :

• 1 µl plasmid DNA (200ng/µl)
• 5 µl 10x pfu buffer
• 5 µl dNTP 2mM
• 5 µl DMSO
• 0.5 µl primer fwd 100µM
• 0.5 µl primer rev 100µM
• 1 µl pfu polymerase
• qsp H2O 50 µl

Cycles : 20x(94°C 30’’, 55°C 30’’, 68°C 20’)

3) Digest DpnI : add 6 µl tampon 4 Biolab’s + 4 µl DpnI 2h 37°C.

4) Transform DH5a with 10 µl. Spread on LB+AB plate.