Uracil DNA Glycosylase (10000 U)

From QIAGEN, this recombinant Uracil DNA Glycosylase (UDG) catalyzes the hydrolysis of the N-glycosylic bond between uracil and the sugar, leaving an abasic site in uracil-containing single- or double-stranded DNA. The enzyme shows no measurable activity on short oligonucleotides (<6 bases) or RNA substrates.

The protein is produced by a recombinant E. coli strain carrying the Uracil DNA Glycosylase gene from E. coli K-12 and is supplied in 10 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 7.5 at 25°C. A 10x UDG Reaction Buffer (200 mM Tris-HCl, 10 mM DTT and 10 mM EDTA; pH 8 at 25°C) is included. The enzyme is active in most molecular biology reaction buffers, so there is no need to exchange buffers before use. A typical setup adds 0.5 µL UDG to a 20 µL reaction with uracil-containing DNA substrate (up to 0.1 µg), followed by incubation at 37–50°C for 30 minutes. Heating at 95°C for 10 minutes only partially inactivates the enzyme; if full inactivation is needed, a uracil glycosylase inhibitor should be added or the reaction cleaned up using appropriate methods.

One unit is defined as the amount of enzyme that catalyzes the release of 1.8 nmol of uracil in 30 minutes from double-stranded, tritiated, uracil-containing DNA at 37°C in 1x UDG Reaction Buffer. Quality specifications include >95% purity, a specific activity of 77,000 U/mg, single-stranded exonuclease activity <5% released (100 U tested), double-stranded exonuclease activity <1% released (100 U tested), double-stranded endonuclease activity showing no conversion (100 U tested), and E. coli DNA contamination <10 copies (100 U tested). UDG also functions as the first step in the Uracil Cleavage System workflow, where it generates the abasic site that Endonuclease VIII subsequently cleaves 3ʹ and 5ʹ to liberate the deoxyribose sugar.