DNA Gyrase Assay Kit is designed to allow quick and specific detection of DNA gyrase. This kit facilitates the purification and characterization of type II topoisomerase enzymes (DNA Gyrase) and contains all reagents necessary for routine assays of type II enzymes that either have or do not have the ability to supercoil.

The Gyrase Assay is Based Upon a Two Step Process:
1. Decatentation of kinetoplast DNA (kDNA)
2. Supercoiling of the resulting decatenated monomer kDNA species

1. kDNA (20 ug total) substrate at the concentration specified on the tube provided. Typically one should use 0.1 to 0.2 ug per reaction.
2. Decatenated kDNA marker (25 ul) in gel loading buffer. Run 2ul of decatenated DNA marker per gel.
3. Linearized kDNA marker (25 ul) in gel loading buffer; run 2 ul of linear marker per gel.
4. 5x Gyrase Assay Buffer (600 ul). 1x buffer contains 35 mM Tris-HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2, 1 mM ATP, 2 mM Dithiothreitol, 1.8 mM Spermidine, 6.5% glycerol, 100 ug/ml BSA.
5. 5x Stop Buffer/gel loading dye (600 ul): 5x buffer is 5% Sarkosyl, 0.125% bromophenol blue, 25% glycerol.

This kit may be stored at 4°C; however, for long term storage (>1 month) we recommend placing it at -20°C. Try to avoid repeated freezing and thawing of the kDNA since this will enhance degradation of the networks.