4.6. DNA supercoiling assay

The E. coli DNA supercoiling assay was performed using a protocol described previously,38 with slight modification. In brief, for each fragment, a 20 μL reaction was prepared by mixing 35 mM Tris HCl pH 7.5, 24 mM KCl, 4 mM MgCl₂, 2 mM dithiothreitol, 1 mM ATP, 1.8 mM spermidine, 0.1 mg mL^–1 BSA, 6.5% glycerol, 125 ng relaxed pHTO-1 plasmid (TopoGEN), and 1 mM fragment (dissolved at 50 mM in 100% DMSO). The reaction was initiated by adding 25 nM of E. coli DNA gyrase, and then incubated at 30 °C for 30 min. The reaction was stopped by adding 5 μL of 5× stop buffer (5% sarkosyl, 0.125% bromophenol blue, and 25% glycerol). The samples were loaded onto a standard 1% agarose gel, and ran at an electric field of 5 V cm^–1 for 90 min. The gel was stained with 3× 4S Red Plus nucleic acid stain (BBI Life Sciences), and visualized under UV light. Reactions containing 1 mM novobiocin (dissolved at 50 mM in 100% DMSO) or 2% DMSO were used as the positive or blank control, respectively.