Quinolone-resistant E. coli Gyrase Mutant D87G Assay kit Plus-100

Mfr.No.	D87G100KE
Description	Quinolone molecules such as ciprofloxacin are commonly used antibiotics that target bacterial DNA topoisomerases. Mutations S83W and D87G in the gyrA subunit of E.coli gyrase are proven causing quinolone resistance. Solving the drug resistance problem requires discovery of new molecules that inhibit the mutant topoisomerases. The Quinolone-resistant E. coli Gyrase Mutant DNA Supercoiling Assay Kits are designed to measure the inhibitory activities of molecules against mutant bacterial gyrases. The assays can be used to test new quinolone compounds in drug discovery. The Quinolone-resistant E. coli Gyrase Mutant D87G Assay Kit Plus-100 (Catalog number D87G100KE) includes all the reagents for 100 assays of E. coli gyrase mutant D87G DNA supercoiling activity. It includes 600 µl of 10 x Buffer T2, 405 µl of 10 x relaxed DNA, 20 µl of 1500 x H19 dye, 450 µl of 10 x ATP, 3000 µl of 10 x H19 dilution buffer and 50 µl 100 x E.coli gyrase mutant D87G.
Drug target assays	Antibacterial drug targets

Product Manual	Datasheet
Protocols Description	Assay Protocol The following assay protocol is based on a 96-well assay plate format using a standard black 96-well plate such as Greiner 655076. 1. Reaction: The total volume of each reaction mixture is 40µl including: 24µl of H2O, 4µl of 10 x buffer, 4µl of 10 x relaxed DNA (250µg/ml relaxed plasmid DNA), 4µl of 10 x enzyme, 4µl of 10 mM ATP. Incubate the reaction mixture at 37°C for 60 min. Note: The final concentrations are 20 mM Tris-HCl, pH 8, 35 mM NH4OAc, 4.6 % glycerol, 1 mM DTT, 0.005% Brij35, 8 mM MgCl2, 25µg/ml relaxed plasmid DNA, 1 mM ATP and 50 nM topoisomerase II. The 10 x enzyme is prepared by dilution of the 100x enzyme in 1x assay buffer. Magnesium is essential for the reaction and assay. EDTA should be avoided. 2. Assay (1) Make 1 x H19 Dilution Buffer by dilution of the 10 x H19 Dilution Buffer 10-fold with water. Freshly prepare the H19 dye by dilution of 1µl the 1500 x H19 dye stock solution with 1.5 ml of 1 x H19 Dilution Buffer (1500 x dilution). Mix the solution evenly by inverting the tube a few times. (2) Mix 250µl of the freshly prepared H19 dye with each reaction solution (40µl). Incubate the mixture at room temperature for 5 min. (3) Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.

Have you used this product?

SKU	Supplier No.	Size	Your price	Quantity
BHE16300103	D87G100KE	100 assays	$983.06		Add to Cart