| Source: |
Purified from E. coli containing a recombinant plasmid encoding the E. coli Mug protein. |
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| Unit Definition: |
One Unit cleaves 1 pmole of a labeled oligonucleotide probe containing 3,N4-ethenocytosine within an oligonucleotide duplex in one hour at 37°C.
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| Assay Conditions: |
1X REC Buffer 6 (20 mM Tris-Cl (pH 8.0), 0.1 mg/ml BSA, 1 mM EDTA, and 1 mM DTT), 4 pmole of labeled 3,N4-ethenocytosine oligonucleotide, annealed to the complement oligonucleotide, and serial dilutions of enzyme in a reaction volume of 20 µl are incubated for 1 hour at 37°C. For analysis, 10 µl of 3X REC Alkali Loading Buffer (cat# 4017-500: 300 mM NaOH, 97% formamide, and 0.2% bromophenol blue) are added, the samples are heated to 95°C for 10 min then fast cooled to 4°C, the cleavage products resolved by 20% denaturing polyacrylamide gel electrophoresis, and percent cleavage quantified. |
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| Specificity: |
E. coli Mug catalyzes the excision of 3,N4-ethenocytosine, a form of DNA damage, in double or single stranded DNA. It also acts to excise uracil in uracil-guanine mismatches.
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| Storage: |
Freeze in working aliquots at -20°C in a manual defrost freezer to avoid repeated freeze-thawings. The enzyme is supplied in 20 mM Tris-Cl (pH 8.0), 2 mM EDTA, 2.5 mM 2-mercaptoethanol, 1 mM PMSF and 50% (v/v) glycerol. |
