Novagene kod hot start dna polymerase

Random mutagenesis was done via error-prone PCR. YPQ1 coding sequence fragments were amplified from plasmids expressing pSSH4::YPQ1^X-GFP-URA3 (where x = S14D, L70D, or M73D) using Taq polymerase from TaKaRa Bio (R001). Primers used were pSSH4-forward (5′-GAA​CGC​TTG​TTT​GTT​CTC​GTC​AC-3′) and GFP-reverse (5′-GTA​CAT​AAC​CTT​CGG​GCA​TGG​CAC-3′). To enhance the error rate, concentrations of dNTPs were modified as follows: 0.4 mM dCTP, 0.4 mM dTTP, 0.08 mM dATP, and 0.08 mM dGTP. Additionally, 0.2 mM MnCl₂ was added to the PCR mixture.
Site-directed mutagenesis was performed with Novagene KOD Hot-Start DNA polymerase (71086; MilliporeSigma).

Site-directed mutagenesis was performed on PFA390 (pRS305-pTEF1::Ypq1-3C-GFP(S65T)-8xHis) and PFA391 (pRS306-pTEF1::Ypq1-3C-GFP(S65T)-8xHis) with Novagene KOD Hot-Start DNA polymerase (71086; MilliporeSigma). Mutant plasmids were then integrated into yeast as described above.

Site-directed mutagenesis was performed on PFA390 (pRS305-pTEF1::Ypq1-3C-GFP(S65T)-8xHis) and PFA391 (pRS306-pTEF1::Ypq1-3C-GFP(S65T)-8xHis) with Novagene KOD Hot-Start DNA polymerase (71086; Millipore Sigma). Mutant plasmids were then integrated into yeast as described above.