Uracil-DNA Glycosylase (UNG), heat-labile

Product Information

Product description: Uracil-DNA Glycosylase (UNG), heat-labile is a recombinant protein in E.coli, from marine bacterium. The enzyme hydrolyzes uracil-glycosidic bonds at U-DNA in single- and double-stranded DNA excising uracil and creating alkali sensitive abasic sites in the DNA. The enzyme is inactive on RNA and native, uracil-free DNA. Since Uracil-DNA Glycosylase (UNG), heat-labile has no metal ion requirements, it is fully active in the presence of EDTA.

Uracil-DNA Glycosylase (UNG), heat-labile can be used with dUTP to eliminate PCR "carry over" contaminations from previous DNA synthesis reactions. To make PCR products suspectible to degradation, dTTP has to be substituted by dUTP in the PCR reaction mix. Subsequent PCR reaction mixes must be pretreated with Uracil-DNA Glycosylase (UNG), heat-labile prior to PCR to degrade uracil-containing DNA. Native DNA does not contain uracil so that the sample is not degraded by this procedure.

Source: Recombinant E.coli

Concentration: 1U/μL

Unit Definition: One unit is defined as the amount of Uracil-DNA Glycosylase (UNG), required to completely degrade 1μg of purified single-stranded uracil-containing DNA at 37℃ in 60min.

Storage Buffer: 20mM Tris-HCl (pH 8.0), 0.1mM EDTA, 100mM KCl, 1mM DTT, 0.5%(v/v) Tween-20, 0.5%(v/v) NP-40, 50%(v/v) glycerol.

Key points of operation

1. Uracil-DNA Glycosylase from E.coli has to be inactivated for 10min at 95℃, but Uracil-DNA Glycosylase (UNG), heat-labile is inactivated for 2min at 50℃.

2. In buffers lacking stabilizers (as PCR buffer: 10mM Tris-HCl, PH8.0, 50mM KCl, 2.5mM MgCl₂) the enzyme is rapidly inactivated at elevated temperatures.

3. It is suggested that adding 1U Uracil-DNA Glycosylase (UNG), heat-labile to 50μL PCR System.

4. Uracil-DNA Glycosylase (UNG), heat-labile is fully active at 15-25℃.

Security Information

Storage Conditions: -20℃

Quality Assurance: Free of Endonuclease, Exonuclease, Nickase and RNase activities.

PCR carry-over prevention: 5ng dUTP-containing PCR products as template to amplify 200bp target DNA. Before the amplification reaction, add 1 unit of Uracil-DNA Glycosylase (UNG), heat-labile to PCR mixes, after 10min at 25℃, no reamplification is possible.

Physical Purity: ＞95% by SDS-PAGE.

Reference

1. H. Sobek, M. Schmidt, B. Frey, K. kaluza. FEBS Letters. 388, 1-4(1996).