Uracil-DNA Glycosylase
Source:
E. coli strain containing an overproducing clone of E. coli Uracil-DNA Glycosylase.
Description:
Uracil-DNA Glycosylase excises uracil from dU-containing DNA by cleaving the N-glycosidic bond between the uracil base and the sugar backbone. This cleavage generates alkali sensitive apyrimidinic sites that are blocked from replication by DNA polymerase or prevented from becoming a hybridization site.
Double and single-stranded dU-containing DNA are substrates for Uracil-DNA Glycosylase whereas RNA and normal dT-containing DNA are not(1).
Uracil-DNA Glycosylase can also be used to increase the cloning efficiency of PCR products having dU-containing primers incorporated into them(2) as well as increasing the efficiency of site-directed mutagenesis(3).
Properties:
Molecular Weight: 31 kDa
Optimum pH: 8.0
Optimum Temperature: 37°C
Inactivation: 95°C for 10 min. Note: Enzyme activity is partially restored at temperatures
lower than 55°C.
Purity:
Tested for contaminating single and double-stranded exonucleases and endonucleases.
Storage Buffer:
10mM Tris-HCl (pH 7.5), 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.05% Tween-20, and 50% glycerol.
Assay Conditions:
Activity is measured by release of [3H]-uracil in a 50 μl reaction containing 0.2 μg DNA (104-105 cpm/μg) in 30 minutes at 37°C.
Unit Definition:
One unit is the amount of enzyme that catalyzes the release of 60 pmol of uracil per minute from double-stranded, uracil-containing DNA.
Concentration:
2 units/μl
Uracil-DNA Glycosylase Reaction Buffer (1 ml included, PN 71961):
200mM Tris-HCl (pH 8), 10mM DTT, 10mM EDTA.
References:
- LINDAHL, T., LJUNGQUIST, S., SIEGERT, W., NYBERG, B. AND SPERENS, B. (1977) J. Biol. Chem. 252, 3286-3294.
- NISSON, P. E., RASHTCHIAN, A. AND WATKINS, P. C. (1991) PCR Methods Appl. 1, 120-123.
- KUNKEL, T. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492.
- VAUGHAN, P. AND MCCARTHY, T. V. (1998) Nucl. Acids Res. 26, 810-815.
- DEUCHAND, P. R., MCGHEE, J. D. AND van de SANDE, J. H. (1993) Nucl. Acids Res. 21, 3437-3443.
Applications:
- Study of DNA repair and mutation detection(4).
- Increase cloning efficiency of PCR products.
- Increase the efficiency of site-directed mutagenesis.
- Study of protein-DNA interactions(5).
Storage:
Shipped on dry ice. Store at -20°C.