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E. coli DNA Gyrase DNA Supercoiling Assay Kits

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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