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DNA gyrase supercoiling inhibition assay

SS Shayna Sandhaus
PC Prem P. Chapagain
YT Yuk-Ching Tse-Dinh
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E. coli DNA gyrase was obtained from New England BioLabs. Two units of the enzyme were added to a reaction buffer provided by the manufacturer (35 mM Tris-HCl, pH 8.0, 4 mM MgCl2, 24 mM KCl, 2 mM DTT, 1.75 mM ATP, 5 mM spermidine, 0.1 mg/mL BSA, and 6.5% glycerol). 0.5 µL of the compounds dissolved in DMSO or the solvent alone were added to the enzyme mixture. 300 ng of relaxed covalently closed plasmid DNA was then added for a final volume of 20 µL. The reactions were incubated for 30 minutes at 37 °C before termination by the addition of 4 µL of the SDS stop buffer. The samples were then loaded into a 1% agarose gel and run at 25 V overnight39. The experiments were replicated twice.

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