Description
Uracil-DNA Glycosylase 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. This enzyme 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. The 10x UDG Reaction Buffer contains 200 mM Tris-HCI, 10 mM DTT and 10 mM EDTA; pH 8 at 25°.
Storage temperature: –25°C to –15°C Molecular weight: 25,693 Daltons Test Amount tested Specification Purity n/a >95% Specific activity n/a 77,000 U/mg Single-stranded exonuclease 100 U <5% released Double-stranded exonuclease 100 U <1% released Double-stranded endonuclease 100 U No conversion E. coli DNA contamination 100 U <10 copies
The protein is produced by a recombinant E. coli strain carrying the Uracil DNA Glycosylase gene from E. coli K-12. 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.
Usage Instructions 1. Use the included UDG reaction buffer at 1x. This enzyme is active in most molecular biology reaction buffers, so there is no need to exchange buffers. 2. Add 0.5 µL UDG to a reaction with uracil-containing DNA substrate (up to 0.1 µg) in a 20 µL total reaction. 3. Incubate at 37–50°C for 30 minutes. Note: 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 cleaned up the reaction using appropriate methods. Thermolabile UNG (G5030L) is recommended if quick and complete inactivation of UDG at low temperatures is desirable. Note: Protocol can be modified for application-specific usage. Quality Control Specific activity was measured using a twofold serial dilution method. Dilutions of the enzyme were made in 1x reaction buffer and added to 50 µL reactions containing a 3H-dUTP PCR product and 1x UDG Reaction Buffer. Reactions were incubated for 10 minutes at 37°C, placed on ice, and analyzed using a TCA-precipitation method. Protein concentration is determined by OD 280 absorbance. Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the band's mass corresponding to the protein of interest in the diluted sample. Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded exonuclease activity is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded endonuclease activity is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C. E. coli contamination is evaluated using 5 µL replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.
