4.2. Topoisomerase I Relaxation Activity Assay

To assay the relaxation activity of topoisomerase I, each protein was serially diluted as indicated and incubated with 180 ng of supercoiled pBAD/Thio plasmid DNA in 20 μL reaction (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1 mg/mL gelatin, 6 mM MgCl₂). After incubation at 37 °C for 30 min, the reaction was stopped by addition of 4 μL stop solution (50 mM Ethylenediaminetetraacetic acid, 50% glycerol, 0.5% (v/v) bromophenol blue). The plasmid DNA was electrophoresed in 1% agarose gel with TAE buffer (40 mM Tris-acetate, pH 8.3, 2 mM EDTA) and visualized with ultraviolet light.