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E. coli DNA Topoisomerase IV Assay Kit Plus (E. coli Topo IV included)

BHE16300115

This product includes all the components in DNA Topoisomerase IV Assay Kit (Catalog No. DDC020K)plus 12ul of 100 x E.coli topoisomerase IV (500 nM) for 20 E. coli topoisomerase IV DNA decatenation assays.

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Reference Narayanan S. et al. A cell cycle-controlled redox switch regulates the topoisomerase IV activity. Genes Dev. 29(11):1175-87 (2015).  
Product Manual Datasheet
Protocols Description Protocol for Bacterial Topoisomerase IV DNA Decatenation Assays
1. Reaction and sample preparation:
(1) The total volume of each reaction mixture is 50 µl including: 38.5 µl of H2O, 5 µl of 10 x buffer, 5 µl of 10 x concatenated DNA, 0.5 µl of 10 x enzyme, 1 µl of 10 mM ATP. Incubate the reaction mixture at 37°C for 60 min. Add 5 µl 0.4 M EDTA to stop the reaction.
 
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, 2 µg/ml concatenated DNA, 0.2 mM ATP and 5 nM topoisomerase IV. A negative control reaction can be the reaction mixture without addition of ATP.
 
2. Column preparation:
(1) Spin the column at 13000 rpm using a bench top Eppendorf centrifuge for 30 seconds to set down the resin.
(2) Remove the column cap and bottom tip. Cut off the cap of a 1.5-Eppendorf tube. Place the column into the tube. Spin the column at 13000 rpm for 2 min. Transfer the column into a fresh Eppendorf tube.
 
3. Assay
(1) Load the 50 µl of the reaction mixture onto the column. Spin the column at 13000 rpm for 2 min. Collect the solution eluted from the column.
(2) Dilute the 20 x fluorescence dye with water to make the 1 x fluorescence dye. Mix 150 µl of the 1x fluorescence dye with the solution eluted from the column.
(3) Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.
Combination SKU Supplier No. Specifications Size Your price Quantity
BHE16300115-1 DDC020KE enzyme included 20 assays $302.17
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BHE16300115-2 DDC100KE enzyme included 100 assays $894.19
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BHE16300115-3 DDC100 enzyme not included 100 assays $529.67
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BHE16300115-4 DDC020K enzyme not included 20 assays $250.74
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