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96-Well E. coli Topo IV DNA Decatenation Assay Kit Plus (enzyme included)

BHE16300109

This product includes 600 ul of 10 x assay buffer, 500 ul of 10 x concatenated DNA, 110 ul of 10 mM ATP, 8 ul of 1000 x E. coli topo IV, 600 ul of 0.4 M EDTA, 260 ul of 20 x fluorescence dye, 2 ml of 10 x rinse buffer, one V-bottom plate, a TDD filter plate and one black 96-well plates for 96 assays of DNA decatenation reactions.

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Product Manual Datasheet
Protocols Description Assay Protocol
1. Reagent preparation and filtration unit
Dilute the 10 mM ATP with water 5-fold to make 2 mM ATP.
Dilute the 20 x fluorescence dye 20-fold to make 1x fluorescence dye.
Dilute the 10 x Rinse buffer 10-fold with water to make 1 x Rinse buffer.
Assembly the filtration unit by connecting the filtration device to a vacuum line, placing the black 96-well plate in the chamber of the filtration device as a receiver of the filtration and the TDD filter plate on the top of the device.
 
2. Reaction and sample preparation:
The reactions are carried out in a V-bottom plate. The total volume of each reaction mixture is 50µl including: 1µl of inhibitor, 34.5µl of H2O, 5µl of 10 x assay buffer, 5µl of 10 x concatenated DNA, 5µl of 2 mM ATP, 0.5µl of 100 x topoisomerase. Incubate the reaction mixture at 37°C for 60 min. Add 5µl of Stop Solution to stop the reaction.
 
Note: The final concentrations for bacterial topoisomerase DNA decatenation assays are 20 mM Tris-HCl, pH 8, 35 mM NH4OAc, 4.6 % glycerol, 1 mM DTT, 0.005% Brij35, 8 mM MgCl2, 3µg/ml concatenated DNA, 0.2 mM ATP and 5 nM E. coli topoisomerase IV. A negative control reaction can be the reaction mixture without enzyme or ATP.
 
3. Assay
Load 50µl of the sample onto the filter plate. Apply the vacuum (80 kpa or 600 mmHg) until the solution goes though the filter. Add 150µl of the Rinse Buffer and let the buffer completely go through the filter. Stop the vacuum and take out the receiver plate. Add 50µl of the 1 x dye into each well. Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.
SKU Supplier No. Size Your price Quantity
BHE16300109 EDD96KE 96 assays $776.23
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