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Home / Assay Kits

96-well Topoisomerase DNA Cleavage Assay Kits

SKU : BHE16300093

This product includes 300 ul of 10 x reaction buffer, 30 ul of 10 x DNA, 260 ul of SDS solution, 30 ul of 100 mM ATP, 260 ul of proteinase K solutions, 10 ml of TDC matrix, 6 ml of rinse buffer, 500 ul of 10 x dye, a black receiver plate, a V-bottom reaction plate, 26 ul of 100 x E. coli Gyrase and a TDC filter plate. A plate filtration device and a vacuum line are needed for the plate filtration process.

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Mfr.No.	T2C96KE, T4C96KE, T4C96KS, TDC96K
Description	Bacterial gyrase, topoisomerase IV (parC-parE) and mammalian topoisomerase II are type II topoisomerases. These topoisomerases change DNA topology by cleavage and religation of both DNA strands. When an inhibitor such as a fluoroquinolone drug blocks the religation step of the topoisomerase reaction, the covalent complex between the cleaved or linearized DNA and the topoisomerase (cleavage complex) is accumulated. Formation of the DNA cleavage complex causes DNA damage in the cells (topoisomerase poison). Testing formation of DNA cleavage complexes is useful for understanding the topoisomerase inhibition mechanism of novel topoisomerase inhibitors. The 96-well topoisomerase DNA cleavage assay kits are based on the principle that the linearized DNA has much greater permeability through a solution matrix and a filter than the closed circular DNA. For example, both novobiocin and ciprofloxacin inhibit DNA gyrase. However, novobiocin does not cause formation of cleavage complex. Ciprofloxacin does. The assay kit is in 96-well plate format and can be used for testing topoisomerase poison compounds in a high throughput setting. The 96-well E. coli Gyrase DNA Cleavage Assay Kit Plus (Catalog No. T2C96KE) includes all the components in TDC96K plus 26 µl of 100 x E. coli Gyrase.
Drug target assays	Antibacterial drug targets
Materials Required But Not Provided	A lab vacuum system: A lab vacuum line or pump (vacuum up to 80 kpa or 600 mmHg). A vacuum device: A plate vacuum device: Pall Corporation, Catalog No. 5017. A fluorescence reader: A plate fluorescence reader with excitation at 485 nm and emission at 535 nm.

Storage Temperature	Store the TDC filter plate and the TDC matrix at 4ºC and the other regents at -20 ºC

Product Manual	Datasheet
Protocols Description	Assay Protocol 1.Reaction and sample preparation: The total volume of each reaction mixture is 25µl including: 0.5µl of inhibitor, 19µl of H2O, 2.5µl of 10 x TDC buffer , 0.25µl of 100 x DNA, 0.25µl of 0.1 M ATP, 2.5µl of 1µM topoisomerase. Incubate the reaction mixture at 37°C for 60 min. Add 2.5µl of SDS and mix the solution. Then add 2.5µl of proteinase K and mix the solution and incubate the solution at 37°C for 60 min. Add 100µl of the TDC matrix and mix for 5 min. 2. Assay Place a 96-well black plate in the filtration device as a receiver of the filtration. Place the TDC filter plate on the top of the device. Load 110µl of the sample onto the filter. Apply the vacuum (80 kpa or 600 mmHg) until the solution goes through the filter (about 5 min). Add 50µl 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 (diluted from the 10 x dye with water) into each well. Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.

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Combination SKU	Supplier No.	Specifications	Your price	Quantity
BHE16300093-1	T2C96KE	E. coli Gyrase	$747.13		Add to Cart
BHE16300093-2	T4C96KE	E. coli Topo IV	$747.13		Add to Cart
BHE16300093-3	T4C96KS	S. aureus Topo IV	$971.83		Add to Cart
BHE16300093-4	TDC96K	Topoisomerase	$638.15		Add to Cart

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