Kod xtreme hot start dna polymerase kit

FLAG-tagged WT and constitutively active (G23V) RALB constructs were provided by Prof. Dan Theodorescu (Denver University, Colorado)^{21 (link)}. Site-directed mutagenesis to generate the other RALB mutants (S28N, C203S) was performed using the KOD Xtreme™ Hot Start DNA Polymerase kit (Merck Millipore). DNA transfection was carried out using XtremeGene-HP reagent (Roche).

Site-directed mutagenesis was carried out using the KOD Xtreme™ Hot Start DNA Polymerase kit (Merck). Briefly, a PCR reaction (100 µL scale) incorporating 50 µL 2x buffer, 20 µL of 2 mM dNTPs, 6 µL each of 5 µM forward and reverse primers (containing the desired mutations), 16 µL of 10 ng/µL template DNA and 2 µL KOD polymerase were set up, according to the manufacturer’s instructions. Fifty microliters of the completed PCR reaction mixture was then digested with 2.5 µL Dpn1 and 5.8 µL CutSmart® Buffer (New England Biolabs) for 3 h at 37 °C to degrade methylated template DNA. CaCL₂ competent DH5α bacteria were then transformed with the digested PCR mixture and plated onto agar with the appropriate selection marker. Subsequent colonies were then expanded, and clonal DNA was isolated using the QIAprep Spin Miniprep Kit (QIAGEN). Mutations in DNA were then verified by DNA sequencing (GATC BIOTECH).