New England Biolabs, M1282S, WarmStart® Afu Uracil-DNA Glycosylase (UDG)

Related Categories DNA Repair Enzymes and Structure-specific Endonucleases Specification Unit Definition One unit is defined as the amount of enzyme required to release 60 pmol per minute of a fluorescently labeled 47 mer single-stranded DNA oligonucleotide containing a single uracil base in 30 minutes at 65°C in a total reaction volume of 50 μl in 1X Thermopol II Buffer. Reaction Conditions 1X ThermoPol® II (Mg-free) Reaction Buffer Incubate at 65°C 1X ThermoPol® II (Mg-free) Reaction Buffer 20 mM Tris-HCl 10 mM (NH4)2SO4 10 mM KCl 0.1% Triton® X-100 (pH 8.8 @ 25°C) Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 0.1 mg/ml Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C Heat Inactivation No Molecular Weight Theoretical: 22717 daltons FAQ Q: What is the activity of WarmStart Afu Uracil-DNA Glycosylase (UDG) in NEBuffers? A: WarmStart Afu Uracil-DNA Glycosylase (UDG) and Afu Uracil-DNA Glycosylase (UDG) exhibit very little activity in NEBuffers r1.1, r2.1, r3.1 and rCutSmart. Q: Will WarmStart Afu Uracil-DNA Glycosylase (UDG) work in Thermopol® Buffer? A: WarmStart Afu Uracil-DNA Glycosylase (UDG) is active in Thermopol buffer. Q: What is the molecular weight of WarmStart Afu Uracil-DNA Glycosylase (UDG)? A: The molecular weight of the Afu Uracil-DNA Glycosylase (UDG) in WarmStart Afu Uracil-DNA Glycosylase (UDG) is 22.7 kDa. Q: Is WarmStart Afu Uracil-DNA Glycosylase (UDG) a tagged protein? A: No. WarmStart Afu Uracil-DNA Glycosylase (UDG) is not a tagged protein. Q: Does the Uracil Glycosylase inhibitor (UGI) inhibit WarmStart Afu Uracil-DNA Glycosylase (UDG)? A: No. Under standard conditions UGI does not inhibit the WarmStart Afu Uracil-DNA Glycosylase (UDG). Q: Does WarmStart Afu Uracil-DNA Glycosylase (UDG) release uracil from ss and dsDNA? A: WarmStart Afu Uracil-DNA Glycosylase (UDG) acts equally well in ssDNA and dsDNA, so the usage recommendations are the same for both types of substrates. Q: Does WarmStart Afu Uracil-DNA Glycosylase (UDG) cut RNA? A: No, WarmStart Afu Uracil-DNA Glycosylase (UDG) should not act on an RNA substrate. UDG enzymes evolved to repair dU residues in DNA that can be created by deamination of dC (hence avoiding the mutation of a C:G base pair to T:A that would occur when the U-strand was replicated). Q: Will WarmStart Afu Uracil-DNA Glycosylase (UDG) work in Fpg buffer? A: No. WarmStart Afu Uracil-DNA Glycosylase (UDG) does not work in Fpg buffer. Q: What does WarmStart mean? A: WarmStart® is the term we use to describe a mesophylic enzyme that is inactive at room temperature, and activated when the reaction is warmed above approximately 40°C. The WarmStart technology can be found in the following products: Bst 2.0 WarmStart DNA Polymerase (NEB #M0538), WarmStart RTx Reverse Transcriptase (NEB #M0380), Luna one-step RT-qPCR products (www.lunaqpcr.com), WarmStart Nt.BstNBI (NEB #R0725) and WarmStart Afu Uracil-DNA Glycosylase (UDG) (NEB #M1282S). For more information on our use of aptamers to control enzyme activity, refer to this article. Q: How can WarmStart Afu Uracil-DNA Glycosylase (UDG) be used for carryover prevention? A: WarmStart Afu Uracil-DNA Glycosylase (UDG) is inactive at lower temperatures and does not interfere with SDA Amplification below 42°C. In contrast, the Afu Uracil-DNA Glycosylase (UDG) is active <42°C and completely inhibits SDA amplification at these temperatures. Additionally, WarmStart Afu Uracil-DNA Glycosylase (UDG) is fully active at 65°C and can excise incorporated uracils from the SDA product once the temperature is raised to 65°C. Carryover contamination prevention by WarmStart Afu Uracil-DNA Glycosylase (UDG) requires two parts: incorporation of dUTP at lower temperatures (<42°C) by a DNA polymerase during amplicon generation, and post-amplification excision of those uracils in amplified products and amplicon destruction catalyzed by a WarmStart Afu Uracil-DNA Glycosylase (UDG) at higher temperatures (~65°C). Q: What is Strand Displacement Amplification (SDA)? A: Strand displacement amplification is an isothermal in vitro DNA amplification method that relies on a nicking enzyme to nick one strand of double-stranded DNA and a DNA polymerase with exonuclease deficiency to extend the 3´-end at the nick and displace the downstream DNA strand (GT Walker, 1992). This method can be applied to rapidly detect a target nucleic acid with high sensitivity in a point-of-care setting and has become a popular molecular diagnosis method. We show a typical SDA reaction with WarmStart Nt.BstNBI (NEB #R0725) and Bst 2.0 WarmStart® DNA polymerase (NEB #M0538) to detect hBRCA1 target in HeLa genomic DNA in WarmStart Nt.BstNBI. To learn more and to view our strand displacement amplification and nicking enzyme amplification reaction product offerings, please visit here.