I am doing DNMT1 site-directed Mutagenesis (using PCR) using Thermos Scientific Phusion Flash High-Fidelity PCR master Mix as my DNA polymerase to mutate my desired side. After PCR and gel extraction it transforms into E.coli, and I send the liquid culture for sequencing.
But I never get my mutant site. My primer designs are all okay. Do I have a problem on my primer ?
Irene C Schneider
Do you know if the Phusion Polymerase performs strand displacment? This is the clou with the classical site directed Mutagenesis polymerases as they don´t displace the mutagenesis primers and introduce therefor the changes. I have VERY good experience in Mutagenesis with the KOD Hot Start Polymerase (Merck).
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