I am doing site-directed mutagenesis for DNMT1 protein. I used PCR master mix as my DNA polymerase. After PCR, I used DPN1 enzyme to digest my paternal plasmid (dpnl: a restriction enzyme which cleaves only methylated DNA, while mutant one will be left) . After that I did a transformation into TOPO10 E.coli. I picked up the colony and sent for sequencing, but after the sequencing, I didn't get my desired mutant site, instead, the mutant is on the next site. My desired mutant is on Thr320-C, but I get it on 960 position. Is this a problem of polymerase?
Moreover, which polymerase is good for site-directed mutagenesis?
Hi,
I too suggest you to move on to a high fidelity Polymerase like Pfu-Turbo from Stratagene. Also check for amplification of your plasmid on gel prior to transformation. Normally you will find enhanced fluorescence on agarose gel.
Another suggestion is to follow the protocol from Stratagene, since I had very good results using their protocol.
