| Source: |
Purified from E. coli containing a recombinant plasmid harboring the E. coli Endonuclease V gene. |
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| Unit Definition: |
One unit is the amount of enzyme required to cleave 1 pmoles of a 32P-labeled oligonucleotide probe in one hour at 37°C at a C/C mismatch when hybridized to a complementary oligonucleotide. |
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| Assay Conditions: |
1X RECTM Buffer 2 (20 mM HEPES-NaOH (pH 7.4), 100 mM KCl, 2 mM MnCl2 and 0.1 mg/ml BSA), 4 pmoles of 32P-oligonucleotide probe, 4 pmoles of complementary oligonucleotide (which generate a C/C mismatch when hybridized together) and serial dilutions of enzyme in a 20 µl reaction volume are incubated for 1 hour at 37°C. Cleavage products are resolved by 20% denaturing PAGE. |
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| Specificity: |
Endonuclease V recognizes mismatches in duplex DNA and cleaves the second and third phosphodiester bonds 3’ to the mismatch at 95% and 5% frequency, respectively, in the strand with the mismatch closest to the 5’ end. The opposite strand containing the mismatch closest to its 3’ end is also cleaved, but the extent of cleavage varies depending on the mismatch. Both strands are cleaved if the mismatch is located in the middle of the oligonucleotide duplex and cleavage occurs about equally at the second and third phosphodiester bonds 3’ from the mismatch. Endonuclease V also cleaves DNA duplexes containing inosine, deoxyuridine, AP sites, urea residues, hairpin or unpaired loops, flap, and pseudo-Y structures. |
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| Components: |
Catalog # |
Component |
Size |
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3900-500-02 |
10X REC Buffer 2 |
1 ml |
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|
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| Applications: |
MIDAScanTMAT Kit |
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| Storage: |
Freeze at -80°C in working aliquots to avoid repeated freeze-thaws. The enzyme is supplied in 20 mM Trisc-Cl (pH 7.8), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 50% (v/v) glycerol. |
