Heat-labile Uracil-DNA Glycosylase (C09-0912-102)

Product description:Thermosensitive UDG enzyme can release uracil and produce alkali sensitive pyrimidine free sites (AP-DNA) by catalytic hydrolysis of uracil glycoside bonds (base excision) of DNA strands containing U-DNA sites. This enzyme can act on single and double stranded DNA containing uracil. The activity of heat sensitive UDG enzyme to double stranded DNA is lower than that to single stranded DNA. The enzyme is active to small U-DNA oligonucleotides and dUMP, but inactive to RNA or normal uracil free DNA. As the thermal UDG enzyme has no requirement for metal ions, it is active in the presence of Mg2+or EDTA. However, glycerol, Mg2+and high ionic strength can reduce the activity of this enzyme. The thermostable UDG enzyme from BMTU 3346 can be inactivated faster (at 95 ℃ for 2 min) than the corresponding enzyme from E. coli (at 95 ℃ for 10 min).Product content:Heat-labile Uracil-DNA Glycosylase （1U/ μ l）Source: BMTU 3346 strain with heat sensitive UDG enzyme gene was cloned.Activity definition: one unit is defined as complete degradation within 60 min at 37 ℃ μ G The amount of heat sensitive UDG enzyme required for purified single stranded uracil DNA (phage M13, E. coli CJ236dut ung).Enzyme storage solution:20mM Tris HCl, pH 8.0 (4 ℃), 0.1mM EDTA, 100mM KCl, 1mM DTT, 50% glycerin, 0.5% Nonidet, 0.5% Tween 20.In the buffer without stabilizer (such as PCR buffer: 10mM Tris HCl, pH8.3, 50mM KCl, 1.5mM MgCl2), the enzyme can be rapidly inactivated at elevated temperature.Application:1) DNA was cleaved at sites where deoxyuridine residues had been incorporated. Through alkali treatment, high temperature or endonuclease (such as T4 endonuclease) specifically cleaves pyrimidine free sites, which can catalyze the hydrolysis of AP-DNA.2) Uracil containing DNA (U-DNA) can be prepared in vitro. Thermosensitive UDG enzymes can be used to achieve site specific, strand specific or general cleavage, depending on how U-DNA is prepared.3) The enzyme can be used to improve the efficiency of directed mutagenesis and obtain highly labeled oligonucleotide probes.4) Thermosensitive UDG enzyme can be used together with dUTP to eliminate PCR contamination of DNA synthesis reaction. In order to make the PCR product easy to degrade, dTTP must be replaced by dUTP in the PCR reaction mixture. Before PCR, the PCR reaction mixture must be pretreated with heat sensitive UD enzyme to degrade the DNA containing uracil. Natural DNA does not contain uracil, so the sample will not be degraded.usage method:Reaction system (50 μ l) 1) Incubate the product at 15 ℃ - 25 ℃ for 10min.2) Heat at 95 ℃ for 2min to inactivate heat sensitive UDG enzyme.3) Perform PCR.4) Short term preservation of PCR products can be stored at 2 ℃ - 8 ℃. For long-term storage, please place the PCR products in - 15 ℃ to - 25 ℃ for frozen storage.Thermal deactivation: heating at 95 ℃ for 2min. (If used for RT-PCR, heat at 55 ℃ for 10min for thermal deactivation.). Specification: 1U/μl Related Documents: https://ald-pub-files.oss-cn-shanghai.aliyuncs.com/aladdinsci/pdp/sds/1/H292766-SCI_3dc97601c161248da6d4ab7a795cda10.pdf