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Home / Assay Kits

DNA Topoisomerase I Assay Kit (enzyme not included)

SKU : BHE16300088

This product includes the reaction buffer (T1 Buffer), supercoiled plasmid DNA and fluorescence dye H19 for assaying 20 DNA relaxation reactions in a 96-well plate assay format or 40 assays in a 384-well assay format. The reaction buffer is optimized for bacterial topoisomerase I.

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Mfr.No.	DRA020K
Description	DNA topoisomerases such as bacterial topoisomerase I convert supercoiled circular DNA into relaxed DNA (DNA relaxation reaction). The DNA Topoisomerase I Assay Kit is based on the principle that the relaxed DNA suppresses the fluorescent intensity much more than the supercoiled DNA when the DNA interacts with fluorescence dye H19 (a fluorescence dye for DNA relaxation / supercoiling assay) in the presence of magnesium. When the supercoiled DNA is converted into its relaxed form, the fluorescent signal decreases. The DNA Topoisomerase I Assay Kit (Catalog number DRA020K) includes 160 µl of 10 x Buffer T1, 84 µl of 10 x supercoiled plasmid DNA, 8 µl of 1500 x Dye H19 and 500 µl of 10 x H19 dilution buffer. It is for 20 DNA relaxation assays in a 96-well plate format or 40 assays in a 384-well assay format.
Drug target assays	Antibacterial drug targets

Product Manual	Datasheet
Protocols Description	Assay Protocol 1.Reaction and sample preparation: The total volume of each reaction mixture is 40µl including: 28µl of H2O, 4µl of 10 x Buffer, 4µl of 10 x supercoiled DNA, 4µl of 10 x Topoisomerase I. The 10 x enzyme solution is freshly prepared by dilution of the enzyme stock solution with the 1 x Buffer. Incubate the reaction mixture at 37°C for 60 min. Note: The final concentrations for the bacterial topoisomerase I assay are 16 mM Tris-HCl, pH 8, 6 mM MgCl2, 25µg/ml supercoiled plasmid DNA and 40 nM topoisomerase I. Free magnesium is required for the H19 detection of the DNA topology. High concentrations of EDTA should be avoided in the reaction and detection solution. 2. Assay (1) Make 1x 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 1500x H19 dye stock solution with 1.5 ml of 1x H19 Dilution Buffer (1500 x dilution). Mix the solution evenly by inverting the tube a few times. (2) Mix 250µl of the diluted H19 dye with each reaction solution. Incubate the mixture for 5 min. (3) Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm.

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Combination SKU	Supplier No.	Specifications	Size	Your price	Quantity
BHE16300088-1	DRA020K	enzyme not included	20 assays	$250.74		Add to Cart
BHE16300088-2	DRA020KE	enzyme included	20 assays	$302.17		Add to Cart
BHE16300088-3	DRA100KE	enzyme included	100 assays	$775.05		Add to Cart

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