| Source: |
Purified from E. coli containing a recombinant plasmid harboring the E. coli MutY gene. |
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| Unit Definition: |
One unit is the amount of enzyme required to cleave 1 pmole of an oligonucleotide duplex containing an A/G mismatch in 1 hour at 37∞C. Only the strand with the A is cleaved.
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| Assay Conditions and Analysis: |
8 pmoles of each oligo in an A/G mismatch oligonucleotide set with A oligo end-labeled with γ-32P, 1X REC Buffer 4 (10 mM HEPES-KOH (Ph 7.4), incubated for 1 hour at 37°C. For analysis, 10µl of 3X REC Alkaline Loading Buffer (0.3 M NaOH, 97% formamide, and 0.2% bromophenol blue) are added, the samples are heated at 95°C for 15 min then fast cooled to 4°C, and the cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis. The bands are cut out and radioactivity counted to quantify the cleavage products.
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| Applications: |
Mismatch cleavage assays
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| Storage: |
Freeze in working aliquots at -20∞C in a manual defrost freezer to avoid repeated freeze-thawing. Enzyme is supplied in 10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 1 mM EDTA, 0.1 mg/ml BSA, and 50%(v/v) glycerol. |
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| Specificity: |
MutY DNA glycosylase recognizes A/G and A/8-oxo-dG mismatches in duplex DNA and cleaves the strand containing the A. The opposite strand is not cleaved. |
