| Source: |
Recombinant E. coli photolyase is purified from an E. coli strain harboring the photolyase phr gene. |
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| Unit Definition: |
One unit is the amount of enzyme required to repair 100 ng of a supercoiled plasmid irradiated with 50 mJ of 254 nm UV light in 1 hour at 37°C during photoreactivation by 365 nm blue light at the rate of 2 mW/cm. The repair is measured as a loss in conversion of the supercoiled plasmid to relaxed, open circle form following treatment with the CPD-specific endonuclease T4-PDG. |
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| Assay Conditions: |
Reactions of 20 µl contain 250 ng of supercoiled plasmid irradiated with 50 mJ of 254 nm UV light, 1X REC™ Buffer 14 (20 mM Tris-Cl (pH 7.8), 1 mM EDTA, 1 mM DTT, 50 mM NaCl), and photolyase. Reactions are set up in yellow light to minimize activation of the photolyase. The enzyme is photoactivated by irradiation with 365 nm light at a fluence of 2 mW/cm and the reaction is allowed to proceed for 1 hour at 37°C. 5 units of T4-PDG are added and the tubes are incubated for 1 hour at 37°C. Supercoiled and relaxed forms of the plasmid are resolved by 1% agarose gel electrophoresis and detected by ethidium bromide staining. |
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| Applications: |
Studies involving UV-induced DNA damage
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| Storage: |
Freeze in working aliquots at -80°C to avoid repeated freeze-thawing. The enzyme is supplied in a buffer containing 20 mM Tris-Cl (pH 7.8), 1 mM EDTA, 50% glycerol, 50 mM NaCl, and 1 mM DTT. |
