==== Mutagenesis ====


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 //Dpn//I : add 6 µl tampon 4 Biolab’s + 4 µl //Dpn//I 2h 37°C.


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