Gel documentation equipment

Bands
were visualized
by ethidium staining for 10 min, destained for 10 min in water, analyzed
by gel documentation equipment (Syngene, Cambridge, U.K.) and quantitated
using Syngene Gene Tools software. Raw gel data (fluorescent band
volumes) were collected from Syngene, Gene Tools gel analysis software
was calculated as a % of the 100% control (fully supercoiled DNA band)
and converted to % inhibition. The raw gel data were analyzed using
SigmaPlot Version 13 (2015). The global curve fit nonlinear regression
tool was used to calculate IC₅₀ data using the following
equation: Exponential Decay, Single, 2 Parameter f = a* exp(−b*x)

One unit (the amount of enzyme required to fully supercoil the substrate) of M. tuberculosis gyrase was incubated with 0.5 μg of relaxed pBR322 DNA in a 30-μL reaction mixture at 37°C for 30 min under the following conditions: 50 mM HEPES, KOH (pH 7.9), 6 mM magnesium acetate (MgOAc), 4 mM dithiothreitol (DTT), 1 mM ATP, 100 mM potassium glutamate, 2 mM spermidine, and 0.05 mg/mL BSA. Each reaction was stopped by the addition of 30 μL chloroform–iso-amyl alcohol (24:1) and 20 μL stop dye (40% sucrose, 100 mM Tris-HCl [pH 7.5], 10 mM EDTA, 0.5 μg/mL bromophenol blue) before being loaded on a 1.0% TAE (0.04 mM Tris-acetate, 0.002 mM EDTA) gel run at 80 V for 3 h. Bands were visualized by ethidium staining for 10 min, destained for 10 min in water, analyzed by gel documentation equipment (Syngene, Cambridge, UK), and quantitated using Syngene GeneTools software. Raw gel data (fluorescent band volumes) collected from Syngene GeneTools gel analysis software were calculated as a percentage of the 100% control (the fully supercoiled DNA band) and converted to percent inhibition. The raw gel data were analyzed using SigmaPlot version 13 (2015). The global curve fit nonlinear regression tool was used to calculate IC₅₀ data using the following equation: exponential decay, single, two-parameter f = a × exp(−b × x).