TopI-NucEx Kit


The TopI-NucEx 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. The TopI-NucEx Kit also allows the investigator to quantify topoisomerase I activity in any given cell line and provides the end user a source of enzyme for preliminary drug screening or high throughput screening (HTS).

Note that this kit contains everything needed to assay topo I in a crude extract (including plasmid DNA, buffers and markers). The kit is suitable for drug screening experiments. Additional materials for the assay should be ordered to replenish the kit, including DNA substrate and marker DNA, as well as control drugs, such as Camptothecin or etoposide if you are performing drug screening experiments.

TopI-NucEx Kit (100 Reaction Set)

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.  pHOT-1 DNA (25 μg total), supercoiled substrate provided at a concentration of 0.25 μg/ml.  DNA concentration may vary in some cases; therefore verify DNA concentrations by referring to the tube label.

5.  Marker DNA, relaxed pHOT-1; 0.05 μg/ul in 1x gel loading buffer.

6.  10x Topo I Reaction Buffer (10x TGS; 300 μl).

7.  10x gel loading buffer (300 μl).

8.  10x Proteinase K (500 μl).

9.  10% SDS (300 μl).

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.


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