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DNA Topoisomerase II (Gyrase) Assay Kit (enzyme not included)

BHE16300096

This product includes the reaction buffer (T2 Buffer), relaxed plasmid DNA and fluorescence dye H19 for 20 assays of DNA supercoiling reactions a 96-well plate assay format or 40 assays in a 384-well assay format.

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Reference 1. Asha M.K. et al, In vitro anti-Helicobacter pylori activity of a flavonoid rich extract of Glycyrrhiza glabra and its probable mechanisms of action, Journal of Ethnopharmacology, Vol.145(2), pp.581-586 (2013).
2. Mora-Pale M et al, Antimicrobial mechanism of resveratrol-trans-dihydrodimer produced from peroxidasecatalyzed oxidation of resveratrol. Biotechnol Bioeng.Vol 112, pp2417–2428 (2015). 
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: 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 20 nM topoisomerase II. The 10 x enzyme is prepared by dilution of the 100x enzyme in 1x assay buffer. 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. 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.
Combination SKU Supplier No. Specifications Size Your price Quantity
BHE16300096-1 DSA020K enzyme not included 20 assays $250.74
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BHE16300096-2 DSA020KE enzyme included 20 assays $302.17
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BHE16300096-3 DSA100KE enzyme included 100 assays $775.05
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