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| Full Name | |
| Abbreviated Name-1 | Thermostable dUTPase |
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| Description | GenScript Thermostable dUTPase (pyrococcus fruriosus) maximizes the efficiency of high-fidelity PCR (using proofreading DNA polymerases). It removes contaminating dUTP present in PCR reactions and dNTP solutions. The presence of dUTPase in a proofreading DNA polymerase reaction can prevent dUTP misincorporation by maintaining dUTP levels below their inhibitory concentrations despite the constant generation of the molecule by the spontaneous deamination of dCTP. The incorporation of dUTP into PCR products causes mutations within the amplified product, proofreading polymerases to stall and slows down non-proofreading polymerases such as Taq. The dUTPase increase in PCR product yield, length and fidelity enables further down-stream applications. These effects make dUTPase useful in PCR fidelity and yield-sensitive applications such as cloning and subsequent recombinant protein technology, and gene expression analysis (semi-quantitative RT-PCR techniques and real-time PCR analysis), where small differences in product accumulation can have a significant impact on gene expression analysis. 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. |
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| Source | E. coli |
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| Purity | Greater than 98.0% as determined by:
(a) RP-HPLC analysis
(b) Reducing and non-reducing SDS-PAGE stained Coomassie blue gel analysis |
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| Endotoxin Level | Less than 0.1 ng/µg (IEU/µg) of dUTPase
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| Specific Activity | Measured by its ability to hydrolyze dUTP to dUMP in reaction buffer 20 mM Hepers (A) pH7.5, 150 mM KCI, 5 mM MgCl2, 10 mM dUTP at 85 Centigrade for 1 hour. The PPi production was quantified by using the enzymatic determination kit from Sigma. (B) Enhancing PCR amplication: 50 ul of Pfu PCR reaction system with 1-3 U of dUTPase to amplify genomic DNA target up to 15-19 kb in length. The thermostable dUTPase conforms an activity of 1 MIU/mg.
One unit of enzyme catalyzes hadrylazation of 10 nanomoles of dUTP to dUMP in one hour at 85 Centigrade. |
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| Storage | Lyophilized DsbA although stable at room temperature for three weeks, it's best stored desiccated below -18°C. Upon reconstitution, DsbA may be stored at 4°C for up to one week and below -18°C for longer periods.
For long term storage it is recommended that a carrier protein (0.1% HSA or BSA) be added. Avoid repeated freeze-thaw cycles. Two years when stored at -20°C, 2 weeks at 4°C. |
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| Formulation | dUTPase is supplied in 20 mM Tris-HC1 (pH 8.2), 1 mM DTT, 0.1 mM EDTA, 100 mM KC1, 0.1% Nonidet P40, 0.1% Tween 20 and 50% glycerol at concentration of 10 U/ul of the enzyme |
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| Stability | Lyophilized DsbA although stable at room temperature for 3 weeks, should be stored desiccated below -18°C. Upon reconstitution DsbA should be stored at 4°C between 2-7 days and for future use below -18°C. For long term storage it is recommended to add a carrier protein (0.1% HSA or BSA). Please prevent freeze-thaw cycles. |
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| Applications | Suitable for use in Western Blot. If this protein is to be used for Western Blot analysis, we recommend that the material be diluted in 1X SDS-PAGE sample buffer (1). On a 15-well minigel system, 50 ng of protein per lane should be sufficient when used in a colorimetric Western Blot with D9625 at a dilution of 1:10,000 as the primary antibody and an appropriate alkaline phosphatase conjugated secondary antibody for detection. |
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