Site-directed mutagenesis

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arsonkamal
arsonkamal's picture
Site-directed mutagenesis

 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?