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Home / Assay Kits

Human Topoisomerase I DNA Relaxation Assay kit

SKU : BHA16300009

This product includes 160 ul of 10 x Buffer HT1, 84 ul of 10 x supercoiled plasmid DNA, 8 ul of 1500 x Dye H19 and 500 ul 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. Human topo I enzyme in not included and can be purchased separately.

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Mfr.No.	HRA020K, HRA100K, HRA100KE, HRA1000KE
Assay Specifications	(1) For each 10 assays, freshly dilute 2 µl of 1500 x H19 dye into 3 ml of 1x H19 dilution buffer. The 1x H19 dilution buffer is prepared by 10 fold dilution of the 10x H19 dilution buffer with water. Mix the solution evenly by inverting the tube a few times. (2) Mix 250 µl of the freshly prepared 1 x 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.
Description	Human DNA topoisomerase I converts supercoiled circular DNA into relaxed DNA (DNA relaxation reaction). The Human 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. Human Topoisomerase I DNA Relaxation Assay Kit (Catalog No. HRA020K) includes 160 µl of 10 x Buffer HT1, 84 µl of 10 x supercoiled plasmid DNA, 8 µl of 1500 x Dye H19 and 550 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. Human topo I enzyme in not included and can be purchased separately.
Drug target assays	Anticancer drug targets
Notes	/

Reference	Abdellatif, Khaled R.A. et al, Design, Synthesis and Cytotoxicity Evaluation of New 3, 5-Disubstituted-2- Thioxoimidazolidinones. Anti-Cancer Agents in Medicinal Chemistry, Volume 18, 573 (2018).

Product Manual	Datasheet
Protocols Description	DNA Relaxation Assay Protocol 1. Reaction and sample preparation: The total volume of each reaction mixture is 40 µl including: 28 µl of H2O, 4 µl of 10 x Buffer HT1, 4 µl of 10 x supercoiled DNA (250 µg/ml), 4 µl of 10 x enzyme (1 U/µl). Incubate the reaction mixture at 37°C for 60 min. Note: The final concentrations are 25 mM Tris-HCl, pH 7.4, 58 mM KCl, 0.25 mM DTT, 5 mM MgCl2, 0.25 mM EDTA, 15 µg/ml BSA, 25 µg/ml supercoiled plasmid DNA and 0.1 U/µl topoisomerase I. A negative control reaction can be the reaction mixture without the enzyme. 2. Assay (1) For each 10 assays, freshly dilute 2 µl of 1500 x H19 dye into 3 ml of 1x H19 dilution buffer. The 1x H19 dilution buffer is prepared by 10 fold dilution of the 10x H19 dilution buffer with water. Mix the solution evenly by inverting the tube a few times. (2) Mix 250 µl of the freshly prepared 1 x 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.

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Combination SKU	Supplier No.	Specifications	Size	Your price	Quantity
BHA16300009-1	HRA020K	enzyme not included	20 assays	$250.74		Add to Cart
BHA16300009-2	HRA100K	enzyme not included	100 assays	$604.47		Add to Cart
BHA16300009-3	HRA100KE	enzyme included	100 assays	$775.05		Add to Cart
BHA16300009-4	HRA1000KE	enzyme included	1000 assays	$4068.99		Add to Cart

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