| Applications |
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| Study of UV damage to DNA and its repair, including DNA damage in single cells. Detection of differential UV damage repair of transcribed sequences. Detection of UV mutational hotspots. |
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| T4 Endonuclease V functions as part of a base excision repair pathway to recognize and remove |
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| cyclobutane pyrimidine dimers. The enzyme binds to UV-irradiated DNA and processively |
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| scans the DNA until a pyrimidine dimer is encountered. T4 Endonuclease V then cleaves |
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| the glycosyl bond of the 5'-pyrimidine of the dimer and the 3' phosphodiester bond, resulting |
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| in breakage of the DNA strand.1,2 Strand breakage is evident during subsequent electrophoresis |
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| as a change in conformation (i.e., from covalent closed circular to relaxed circular molecules), or as the appearance of unique DNA fragments. The enzyme is a small protein that does not require divalent cations or other cofactors.1 |
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| Catalog # | T0200 |
| Source | gene denV in E. coli |
| Activity | 20u/ul |
| Unit Definition | One unit will change 1ug of UV-irradiated plasmid DNA from covalently closed circles (form I) to nicked closed circles (form II) in 30 minutes at 37°C. |
| Activity Assay | The activity assay is performed in a 50ul reaction containing 50mM Tris-HCl pH 7.5) and 5mM using plasmid DNA irradiated for 1 minute at 254nm. |
| Contaminating Activity Assays | T4 Endonuclease V is free of detectable exonuclease, non-T4 Endonuclease V-specific endonuclease and RNase activities as judged by electrophoresis after incubation of 1mg of various DNA substrates with 500-1000U of enzyme at 37°C for 16 hours. |
| Storage Buffer | 50 mM Tris-HCl, pH 7.5, 100mM sodium chloride, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% glycerol. |
| Working Solution | Dilute the enzyme in 0.01M EDTA with 0.1mg/ml nuclease-free BSA, and buffer of choice (0.04M sodium phosphate or equivalant). The presence of EDTA is critical. |
| Quality Control | T4 Endonuclease V is free of detectable nonspecific exo-and endonuclease activities and RNase activity. |
| Storage and Stability | Stable for one year at -20ºC. |
| Concentration | 20 U/uL |
| Form | Supplied as a frozen solution in 0.01M phosphate buffer, pH 6.5 containing 0.01M EDTA, 0.001M DTT and 20% glycerol. |
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| Important Note | This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. |
| Alternate names | gene denV in E. coli |
