Pfu ultra 2 hs fusion dna polymerase

Site directed mutagenesis was performed on pCMV-Myc clones in order to substitute lysine by alanine at position 14 using the QuickChange Site-Directed mutagenesis Kit (Stratagene) with Pfu Ultra II HS fusion DNA polymerase (Agilent). Mutated DNA was sequenced to confirm the desired changes.

Plasmids pG4-1 and pG4-1-v1 (5 ηg each) served as templates for PCR amplification (in duplicate, 25 μl per reaction mixture) using 1X buffers supplied by vendors and DNA primers M13F (5’-TGTAAAACGACGGCCAGT-3’) and M13R (5’-CAGGAAACAGCTATGAC-3’) at 0.5 mM each. For high fidelity PCR, deoxynucleoside triphosphates were included at 0.25 mM each, Mg²⁺ at 2 mM, and Pfu Ultra II HS Fusion DNA polymerase (Agilent) at 1/50 reaction volume. For Taq-based PCR amplification, deoxynucleoside triphosphates were included at 0.5 mM each, Mg²⁺ at 2.5 mM, and GoTaq DNA polymerase (Promega) at 1/200 reaction volume (0.625 units). PCR products were purified using the QIAquick PCR Purification Kit (Qiagen).