New England Biolabs, M0372S, Antarctic Thermolabile UDG

Related Categories DNA Repair Enzymes and Structure-specific Endonucleases Applications Loop-Mediated Isothermal Amplification 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 30°C in a total reaction volume of 50 μl in 1X Thermopol II Buffer. Reaction Conditions 1X Standard Taq Reaction Buffer Pack Incubate at 37°C 1X Standard Taq Reaction Buffer Pack 10 mM Tris-HCl 50 mM KCl 1.5 mM MgCl2 (pH 8.3 @ 25°C) Storage Buffer 10 mM Tris-HCl 50 mM NaCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.5 @ 25°C Heat Inactivation 50°C for 5 minutes FAQ Q: What is the activity of UDG in the NEBuffers 1-4? A: Buffers Activity (%) Standard Taq Reaction Buffer 100 Crimson Taq Reaction Buffer 100 LongAmp™ Taq Reaction Buffer 100 OneTaq Stardand Reaction Buffer 100 Isothermal Amplification Buffer 100 Thermopol Reaction Buffer 100 Epimark® Hot Start Taq Reaction Buffer 100 E.coli UDG Reaction Buffer 100 I10x Pfu Turbo® Cx Reaction Buffer 100 Q5 Reaction Buffer 100 Phusion™ HF Buffer 10 5x Green GoTaq® Reaction Buffer 100 NEBuffer 1 25 NEBuffer 2 50 NEBuffer 3 50 NEBuffer 4 100 Q: Will UDG work in T4 DNA ligase buffer? A: Yes. UDG will work well in T4 DNA ligase buffer. Q: What is the molecular weight of the thermolabile UDG? A: The molecular weight of UDG is 24.6 kDa. Q: Is UDG a tagged protein? A: No. UDG is not a tagged protein. Q: I see that the Antarctic Thermolabile UDG works at 25-37°C. Do you have another glycosylase that works at higher temperatures? A: Yes. Afu UDG (NEB# M0279) can release uracil from a uracil-containing DNA at 65°C. Q: Does UDG release uracil from ss and dsDNA? A: Yes. UDG can remove uracils from both ss and dsDNA. Q: Does UDG cut RNA? A: No, UDG should not act on an RNA substrate. UDG 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 is replicated). Q: Can UDG be used to remove dU from a short 21mer oligo? Do the dU residues need to be spaced in any special way to avoid problems with cleavage? A: UDG should work fine on a 21mer. Spacing is not important. Q: Are there any specific recommendations for the use of UDG on single stranded DNA? The materials on the web and data card seem to describe conditions for use with dsDNA. A: UDG acts equally well in ssDNA and dsDNA so the usage recommendations are the same for both types of substrates. Q: Will Antarctic Thermolabile UDG cleave uracil-containing DNA in a form of RNA-DNA duplex? A: Yes. Q: What is the difference between UDG and UNG? A: Both names refer to Uracil-DNA Glycosylase. Q: How much Antarctic Thermolabile UDG I should add in a PCR reaction to prevent carry-over contamination? A: Typically, we add 0.5 ul of Antarctic Thermolabile UDG in a 25 ul of PCR reaction. Q: How do I use Antarctic Thermolabile UDG for carryover prevention in LAMP reactions? A: Carryover contamination prevention requires two parts: incorporation of dUTP by a DNA polymerase during amplicon generation, and excision of those uracils in amplified products and amplicon destruction catalyzed by a UDG. For LAMP, reactions should be run with a ~50% inclusion of dUTP mixed with dTTP (e.g. 1.4 mM dATP, dCTP, dGTP, 0.7 mM dTTP and dUTP) and a Bst DNA polymerase should be used for efficient incorporation of dU without significant inhibition of the reaction. For the subsequent destruction of contaminant products, Antarctic Thermolabile UDG is strongly recommended over the more thermostable E. coli UDG. Include 0.5 μL of Antarctic Thermolabile UDG per 25 μL LAMP reaction, and simply set up and run your LAMP reactions as normal. UDG activity during setup and heating to 65 °C will quickly and efficiently destroy any contaminating products. If more stringent decontamination is required, then 10 minutes at 25 °C can be added to the beginning of the workflow. For simplicity, dUTP and UDG have been included in an updated: WarmStart Colorimetric LAMP 2X Master Mix with UDG and Fluorescent LAMP Kit WarmStart Fluorescent LAMP/RT-LAMP Kit (with UDG).