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Human Topoisomerase I Assay Kit
(Catalog # K002)


This assay kit contains reagents for routine detection of type I topoisomerase activity. DNA marker is included to allow clear detection of the enzyme. The assay is specific for topoisomerase I. This assay has been shown to work with crude extracts. Human topoisomerase I, active (Cat# P005) is strongly recommended to be used along with this kit.


  • Studying the effects of supercoiling on transcription in vitro.
  • Studying chromatin reconstitution in vitro.
  • Determining the degree of supercoiling of naturally occurring DNA.
  • Detecting mutant plasmids that differ in length by only one basepair.
  • Increasing restriction endonuclease digestion of resistant DNA substrates by unwinding the DNA coils to expose restriction sites.
  • Anticancer drug screening.

Quality Control Tests:

  1. A test for nuclease contamination is carried out by assaying for the formation of linear KDNA and linear plasmid DNA. Incubation with KDNA or supercoiled pGEM 5Z DNA (4 hrs. at 37oC in the presence of 10 mM MgCl2) is performed. Linear DNA or breakdown products are not generated under these conditions.
  2. A check for cross contamination with Topo II is negative. There must be no decatenation of KDNA in topo II reaction conditions.

Kit Contents:

  • Supercoiled plasmid substrate DNA control (cat# D001, 100ul pLAE1 DNA, 0.2 ug/ul, good for 100 standard assays).
  • Relaxed plasmid DNA marker (cat# D002, 100 ul pre-relaxed pLAE1 DNA by Topo I at 0.04 ug/ul in gel loading buffer, good for 20 standard assays).
  • 5X Topo I reaction buffer (cat# B001, 500 ul, good for 100 standard assays).
  • Topo I Stop buffer/gel loading dye (cat# B002, 500 ul, good for 100 standard assays).

This kit is shipped at ambient temperature and should be stored at 4oC.

Assay Conditions:

  • Relaxation assays are recommended to carry out in a final volume of 10 ul with 5 unit of Topo I, active (Cat# P005) in topo I reaction buffer (40 mM Tris-Cl, pH 7.5, 100 mM NaCl or KCl, 10mM MgCl2, 0.5 mM EDTA, 30 ug/ml BSA).
  • Standard Assays: Supercoiled plasmid DNA (Cat# D001) is used at 0.2 ug/reaction. Reactions are terminated with 5 ul (in 10 ul reaction volume) of stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol). Reaction products can then be analyzed on a 0.8% native agarose gel.
  • Load 5 ul (0.2 ug) Relaxed plasmid DNA marker (Cat# D002) per lane as control.

Legal consideration:

Wang, J.C. (1996) Annu. Rev. Biochem. 65: 635-692.

A unit of topo I incubated with 0.2 ug pGEM 5Z DNA in 30 min at 37oC.