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H19 Dye for DNA Relaxation and Supercoiling Assays

BHE16300162

This product includes 170 ul of 1500 x H19 dye for high throughput DNA relaxation or DNA supercoiling assays of topoisomerases.

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+1 866.986.9598
Email
[email protected]
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Product Manual Datasheet
Protocols Description Assay Protocol for E. coli gyrase DNA supercoiling assay
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 40 µl including: 24 µl of H2O, 4 µl of 10 x buffer, 4 µl of 10 x relaxed DNA (250 µg/ml relaxed plasmid DNA), 4 µl of 10 x enzyme, 4 µl 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, 25 µg/ml relaxed plasmid DNA, 1 mM ATP and 20 nM E. coli gyrase. 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 1 µl 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 250 µl of the freshly prepared H19 dye with each reaction solution (40 µl). Incubate the mixture at room temperature for 5 min.
(3) Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.
SKU Supplier No. Size Your price Quantity
BHE16300162 DSA1000D 1 $560.35
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BHE16300162 DSA1000D $560.35
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