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Afu Uracil-DNA Glycosylase (UDG) |
 | | | | Afu 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:
A thermostable homolog of the E. coli Uracil-DNA Glycosylase (UDG) (1,2) from Archaeoglobus fulgidus. Afu UDG catalyzes the release of free uracil from uracil-containing DNA. Afu UDG efficiently hydrolyzes uracil from single-stranded or double-stranded DNA.
Source:
Cloned and overexpressed in E. coli.
Reagents Supplied:
ThermoPol II (Mg-free) Reaction Buffer Pack (10X)
Enzyme Properties
Heat Inactivation:
No
Specific Activity:
96 units/μg
Reaction & Storage Conditions
Reaction Conditions:
1X ThermoPol II (Mg-free) Reaction Buffer
Incubate at
65°C.
1X ThermoPol II (Mg-free) Reaction Buffer:
20 mM Tris-HCl
10 mM KCl
10 mM (NH4)2SO4
0.1 % Triton X-100
pH 8.8 @ 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 65°C.
Unit Assay Conditions:
1X ThermoPol II (Mg-free) Reaction Buffer, 1 unit of Afu Uracil-DNA Glycosylase, 0.2 µg 3H-uracil DNA (104-105 cpm/µg) for 30 minutes at 65°C in a total reaction volume of 50 µl.
Molecular Weight: 22,717 daltons.
Concentration:
2,000 units/ml
Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
0.1 mg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- Afu UDG retains 50% activity in the presence of 150 mM NaCl. Afu UDG retains less than 1% activity after boiling for 30 minutes in standard reaction conditions.
- Under standard reaction conditions, uracil glycosylase inhibitor (UGI) does not inhibit Afu UDG.
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 in ThermoPol Reaction Buffer and 50 units off Afu UDG showed no degradation following overnight incubation at 65°C.
Exonuclease Activity:
Incubation of 50 units for 4 hours at 65°C in 50 μl of ThermoPol Reaction Buffer with 1 μg 3H DNA (105 cpm/μg) released < 0.1% radioactivity.
Endonuclease Assay:
Incubation of 5 units of Afu UDG with 1 μg of ϕX174 RF I DNA for 4 hours at 65°C in 50 μl ThermoPol Reaction Buffer resulted in < 20% conversion to RF II.
References
- Sandigursky, M. and Franklin, W. A. (1999) Current Biology, 9, 531-534.
- Sandigursky, M. and Franklin, W. A. (2000) J. Biol. Chem., 275, 19146-19149.
Reagents Sold Separately
ThermoPol II (Mg-free) Reaction Buffer Pack
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