Description:

E. coli Uracil DNA Glycosylase (UNG) catalyses the release of free uracil from uracil-containing DNA. UNG efficiently hydrolyzes uracil from single-stranded or double-stranded DNA, but not from oligomers (6 or fewer bases).

Source:

An E. coli strain that carries the UNG gene from E. coli.

Applications:

Glycosylase mediated single nucleotide polymorphism detection (GMPD)

Site-directed mutagenesis

As a probe for protein-DNA interaction studies

Rapid and efficient cloning of PCR products

Elimination carry-over contamination in PCR

Unit Definition:

One unit is defined as the amount of enzyme that catalyzes the release of 60 pmol of uracil per minute from double-stranded, uracil-containing DNA in a total reaction volume of 50 ¦Ìl in 30 minutes at 37^oC in 1X Uracil DNA Glycosylase Reaction Buffer with 1 unit of uracil DNA Glycosylase and 0.2 µg [³H]-uracil DNA (10⁴-10⁵ cpm/µg).

Quality Control

Activity, SDS-PAGE (purity), 16-Hour Incubation, Exonuclease and Endonuclease Activity.

Storage:

UNG in 10 mM Tris-HCl (pH 7.4 at 25^oC), 50 mM KCl, 1 mM Dithiothreitol, 0.1 mM EDTA, 0.1 mg/ml BSA, 50% Glycerol should be stored at -20^oC.

10X UNG Reaction Buffer:

200mM Tris-HCl (pH 8.0 at 25^oC), 10mM Dithiothreitol, 10mM EDTA.

Reaction Conditions:

1X UNG Reaction Buffer, incubate at 37^oC.

Inhibition and Inactivation

Inactivated by heating at 95^oC for 10min. Enzyme activity is partially restored at temperatures lower than 55^oC.

Usage notes:

UNG is active over a broad pH range with an optimum at pH 8.0, does not require divalent cation, and is inhibited by high ionic strength (> 200 mM).

The abasic sites formed in DNA by UNG may be cleaved by heat, alkali-treatment or endonucleases that cleave specifically at abasic sites.