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Home / Assay Kits

S. aureus Gyrase DNA Supercoiling Assay Kit Plus (enzyme included)

SKU : BHE16300106

This product includes160 ul of 10 x Buffer T2, 84 ul of 10 x relaxed DNA, 6 ul of 1500 x H19 dye, 90 ul of 10 x ATP, 10 ul 7.5 uM S. aureus gyrase, 180 ul of 2 M potassium glutamate and 550 ul of 10 x H19 dilution buffer. It is for 20 assays in a 96-well plate format.

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Mfr.No.	DSA020KSE
Description	DNA topoisomerases such as bacterial topoisomerase II (gyrase) convert relaxed circular DNA into supercoiled DNA (DNA supercoiling reaction). The DNA Topoisomerase II (Gyrase) Assay Kit is based on the principle that the supercoiled DNA and relaxed DNA yield different fluorescent intensity when interact with fluorescence dye H19. The relaxed DNA suppresses the fluorescent intensity much more than the supercoiled DNA in the presence of magnesium. Therefore, when the relaxed DNA is converted into its supercoiled form, the fluorescent signal increases. The change of fluorescence intensity is used to measure the supercoiling reaction of gyrases and high throughput screen of gyrase inhibitors. The S. aureus DNA Topoisomerase II (Gyrase) Assay Kit Plus (Catalog No. DSA020KSE) includes160 µl of 10 x Buffer T2, 84 µl of 10 x relaxed DNA, 6 µl of 1500 x H19 dye, 90 µl of 10 x ATP, 10 µl 7.5 uM S. aureus gyrase, 180 µl of 2 M potassium glutamate and 550 µl of 10 x H19 dilution buffer. It is for 20 assays in a 96-well plate format.
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 (Greiner 655076). For 384-well plate assays using a standard black 384-well plate (Matrix 4318), please reduce the reagent volumes proportionally. 1. Reaction: The total volume of each reaction mixture is 40µl including: 19.6µl of H2O, 4µl of 10 x buffer, 8µl of 2 M potassium glutamate, 4µl of 10 x relaxed DNA (250µg/ml), 0.4µl of 7.5µM S. aureus gyrase, 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, 400 mM potassium glutamate, 25µg/ml relaxed plasmid DNA, 1 mM ATP and 75 nM topoisomerase II. A negative control reaction can be the reaction mixture without addition of ATP. 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. 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.

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Combination SKU	Supplier No.	Size	Your price	Quantity
BHE16300106-1	DSA020KSE	20 assays	$327.89		Add to Cart
BHE16300106-2	DSA100KSE	100 assays	$894.19		Add to Cart

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