rS. Solfataricus Dpo-4 with Buffer and Cations

Dpo4 is a thermophilic Y-family DNA polymerase capable of performing translesion synthesis and allows for coupling to PCR for amplification of damaged DNA. Dpo4 has a molecular weight of 42 kDa and is 95% pure by SDS-PAGE. Translesion synthesis is enhanced by the presence of Mn2+ in the reaction. One unit of enzyme incorporates 1 nmole of dNTP onto the 3’ end of annealed primer in 30 minutes at 60°C.

1X REC Buffer 15 [10 mM HEPES-NaOH (pH 7.4), 1 mM DTT, 50 mM NaCl, 100 μg/ml BSA, 0.1% Triton X-100], 5 mM MgCl2, 4 pmoles of labeled annealed primer, and serial dilutions of enzyme in a 20 μl reaction volume are incubated for 30 minutes at 60°C. Reaction products are resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent primer extended.