Thermostable dUTPase (Pyrococcus fruriosus) was prepared and purified from E.coli strain. dUTPase is specific for dUTP and is critical for the fidelity of DNA replication and repair. dUTPase hydrolyzes dUTP to dUMP and pyrophosphate, simultaneously reducing dUTP levels and providing the dUMP for dTTP biosynthesis.Thermostable dUTPase used for PCR with Pfu DNA polymerase improves the efficiency and longer targets of PCR reaction.
Concentration & Storage Condition- 0.5 ㎍/㎕. Store at -20℃. Storage Buffer- 20 mM Tris-HC1 (pH 8.2), 1 mM DTT, 0.1 mM EDTA, 100 mM KCl, 0.1% Nonidet P40, 0.1% Tween 20 and 50% glycerol Unit Definition- One unit of enzyme catalyzes hadrylazation of 10 nmole of dUTP to dUMP in 1 hour at 85℃. Activity- (A) Measured by its ability to hydrolyze dUTP to dUMP in reaction buffer 20 mM HEPES, pH 7.5, 150 mM KCI, 5 mM MgCl₂, 10 mM dUTP at 85 ℃ for 1 hour. The PPi production was quantified by using the enzymatic determination kit from Sigma.- (B) Enhancing PCR amplication: 50 ㎕ of Pfu PCR reaction system with 1-3 unit of dUTPase to amplify genomic DNA target up to 15-19 kb in length. QC Tests
- Activity, exo and endonuclease activity test, SDS-PAGE purity, performance tests.