Uracil DNA Glycosylase (UNG)

Catalyzes the hydrolysis of N-glycosylic bond between the uracil and sugar, leaving an apyrimidinic site in uracil-containing single or double-stranded DNA. It shows no activity on RNA (1).\n\nUnit Definition:\n1unit of Uracil DNA Glycosylase catalyses the release of 1nanomole of uracil from uacil containing DNA template in 60 min at 37 degrees .\n\nStorage Buffer:\nSupplied as a liquid in 30mM Tris-HCl, pH 7.5, 150mM sodium chloride, 1mM EDTA, 1mM DTT, 0.05% Tween 20, 50% glycerol.\n\n10X Reaction Buffer U1900-05A):\nSupplied as a liquid in 200mM Tris-HCl, pH 8.2, 10mM EDTA, 100mM sodium chloride.\n\nApplications:\nSuitable for GMPD detection, Site-directed mutagenesis (2), as a Probe for protein-DNA interaction (3), Rapid, efficient cloning of PCR products (4) anddH2Oelimination of carry over contamination in PCR.\nNote:\nThe abasic sites formed in DNA by uracil-DNA glycosylase may be cleaved by heat under alkaline conditions. Any of the five USBio buffers can be used instead of 10X uracil-DNA glycosylase reaction buffer. Uracil-DNA glycosylase should be inactivated by heating at 95 degrees C for 10 minutes. Enzyme activity is partially restored at temperatures lower than 55 degrees C. Put PCR products on ice after PCR and load directly on a gel.\n\nInhibitors: Ugi protein from the Bacillus subtilis phage PBS2, protein p56 from the Bacillus subtilis phage phi29 (7).\nUGI is active in the presence or absence of divalent cations.\n\nQuality Control:\nEndodeoxyribonuclease Assay:\nNo detectable conversion of covalently closed circular DNA to nicked DNA was observed after incubation of 10u of Uracil DNA Glycosylase with 1ug of pUC19 DNA in 50ul of reaction buffer (33mM Tris- acetate (pH7.9 at 37C), 10mM Mg-acetate, 66mM K-actetate) for 4 hours at 37 degrees C.\n\nLabeled Oligonucleotide Assay:\nNo detectable degradation of single- stranded and double-stranded labeled oligonucleotide was observed after incubation with 2u of Uracil DNA Glycosylase, for 4 hours at 37 degrees C.\n\nRibonuclease Assay:\n(same/less than)0.5% of the total radioactivity was released into trichloroacetic acid-soluble fraction after incubation of 10 units of Uracil DNA Glycosylase with 1ug of [3H]-RNA in 50ul of reaction buffer (33mM Tris- acetate (pH7.9 at 37C), 10mM Mg-acetate, 66mM K-actetate) for 4 hour at 37 degrees C.\n\nStorage and Stability:\nAliquot to avoid repeated freezing and thawing. Store at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

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