This Kit is designed to allow a researcher to prepare a nuclear extract enriched for type II topoisomerase (topo II) activity. The appropriate assay buffer specific for topo II 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 II activity in any given cell line. This Kit also provides the end user a source of enzyme for preliminary drug screening for topo II active agents.
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. kDNA (20 ug total) substrate at the concentration specified on the tube provided.
5. Decatenated kDNA marker (25 ul) in gel loading buffer.
6. Linearized kDNA marker (25 ul) in gel loading buffer
7. 10x gel loading buffer (300 ul).
8. 10x Proteinase K (500 ul).
9. 10% SDS (300 ul).
10. 10x Topo II Incomplete Assay Buffer A (250 ul).
11. 10x ATP Buffer B (250 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.