| Source: |
Purified from E. coli containing a recombinant plasmid harboring the E. coli ung gene. |
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| Unit Definition: |
The amount of enzyme required to cleave a uracil-containing oligonucleotide within an oligonucleotide duplex at the rate of 60 pmole/min. at 37°C. |
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| Assay Conditions: |
1 X RECTM Buffer 6 (20 mM Tris-Cl (pH 8.0), 1mM EDTA, 1 mM DTT, 0.1 mg/ml BSA), 4 pmoles of uracil oligonucleotide (Cat# 3852-100-01) labeled with 32P, 2 pmoles of oligo complement B (Cat# 3849-100-02), and Uracil-N-Glycosylase in a 20 µl reaction volume. The tubes are incubated for 1 hour at 37°C and then treated with 0.1 M NaOH for 15 minutes at 95°C. For analysis, 10µl of 3X REC Alkali Loading Buffer (Cat# 4017-500: 300 mM NaOH, 97% formamide, 0.2% bromophenol blue) are added, the samples are heated to 95°C for 10 minutes then fast cooled to 4°C, and cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis. The bands are cut out and radioactivity counted to quantify cleavage products. |
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| Specificity: |
Uracil-N-Glycosylase releases uracil, 5-hydroxy-2’-deoxyuridine, 5,6-dihydroxyuracil, 5-fluorouracil from both double or single stranded DNA. |
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| Applications: |
CometAssay®/FLARETM
Supercoiled DNA relaxation assay
Alkaline elution assay
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| Storage: |
Freeze at -80°C in working aliquots to avoid repeated freeze-thawing. The enzyme is supplied in 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 50% glycerol. |
