Topoisomerase Type I Nuclear Extraction/Assay Kit Product Description
This Kit is designed to allow a researcher to prepare a nuclear extract enriched for type I topoisomerase (topo I) activity and intact 100 kDa polypeptide. The appropriate assay buffer specific for topo I is included. This kit is suitable for most animal cell lines growing in vitro but is optimized for human and rodent species. This Kit also allows the investigator to quantify topo I activity in any given cell line and provides the end user a source of enzyme for preliminary drug screening or HTS.
1. 10x TEMP Buffer. (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 4 mM MgCl2, 0.5 mM PMSF). The kit includes 10 mL (in a 15 mL conical tube).
2. 10x TEP Buffer. (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 mM PMSF). A microfuge tube with 1 mL is provided.
3. 1 M NaCl for extraction (0.5 mL in microfuge tube).
4. pHOT1 DNA (25 ug total), supercoiled substrate provided at a concentration of 0.25 ug/ml. DNA concentration may vary in some cases; therefore verify DNA concentrations by referring to the tube label.
5. Marker DNA, relaxed pHOT1; 0.05 ug/ul in 1x gel loading buffer.
6. 10x Topo I Reaction Buffer (10x TGS; 300 ul).
7. 10x gel loading buffer (300 ul).
8. 10x Proteinase K (500 ul).
9. 10% SDS (300 ul).
The kit may be shipped at ambient temperature or on ice (dry ice or wet ice) and should be stored at -20° C upon receipt. Avoid frequent freeze/thaw cycles which may cause DNA nicking. The kit may be store for up to 1 month at 4° C.