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DNA Modifying Enzymes and Cloning >
DNA Repair Proteins >
Uracil-DNA Glycosylase (UDG) |
 | | | | Uracil-DNA Glycosylase (UDG) | | | |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer
Description:
E. coli Uracil-DNA Glycosylase (UDG) catalyses the release of free uracil from uracil-containing DNA. UDG 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 UDG gene from E. coli.
Applications:- Treatment of 0.1 µg of uracil-containing DNA with 1 unit of UDG for 10 minutes at 37°C renders the DNA incapable of being copied by DNA polymerase. The enzyme can be 95% heat killed by incubation at 95°C for 10 minutes. Since UDG remains partially active following heat treatment at 95°C, it is recommended that uracil glycosylase inhibitor be added to prevent degradation of product DNA. Alternatively, reaction products can be immediately extracted with phenol/chloroform.
Reagents Supplied:
UDG Reaction Buffer (10X)
Enzyme Properties
Heat Inactivation:
No
Reaction & Storage Conditions
Reaction Conditions:
1X UDG Reaction Buffer
Incubate at
37°C.
1X UDG Reaction Buffer:
20 mM Tris-HCl
1 mM Dithiothreitol
1 mM EDTA
pH 8.0 @ 25°C
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. 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 Assay Conditions:
1X UDG Reaction Buffer, 1 unit of uracil DNA Glycosylase, 0.2 µg 3H-uracil DNA (104 -105 cpm/µg) for 30 minutes at 37°C in a total reaction volume of 50 µl.
Concentration:
2,000 units/ml
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
0.1 mg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- UDG 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).
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of λ phage DNA
and 50 units of Uracil-DNA Glycosylase (UDG) incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of Uracil-DNA Glycosylase (UDG) with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of Uracil-DNA Glycosylase (UDG) with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
References
- Lindahl, T. et al. (1977) J. Biol. Chem., 252, 3286-3294.
- Wang, Z. et al. (1991) Gene, 99, 31-37.
- Devchand, P.R. et al. (1993) Nucl. Acids Res., 21, 3437-3443.
Reagents Sold Separately
UDG Reaction Buffer
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