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P. aeruginosa Gyrase DNA Supercoiling Assay Kit Plus (enzyme included)

BHE16300102

This product includes all the reagents for 100 assays of P. aeruginosa gyrase DNA supercoiling activity. This product includes 600 ul of 10 x Buffer T2, 405 ul of 10 x relaxed DNA, 20 ul of 1500 x H19 dye, 450 ul of 10 x ATP, 3000 ul of 10 x H19 dilution buffer and 50 ul 100 x P. aeruginosa gyrase.

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+1 866.986.9598
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[email protected]
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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 peroxidase-catalyzed 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.
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BHE16300102 DSA100KP 100 assays $738.14
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