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Human topoisomerase I relaxes supercoiled circular DNA. In this kit, relaxed DNA gives much weaker fluorescence with dye H19 (with magnesium present) than supercoiled DNA does, so the signal drops as the enzyme works.
Formats and Pack Sizes
HRA100K has no enzyme (human topo I is bought separately). HRA100KE adds 10 µl of 500 U/µl human topoisomerase I for 100 assays, and HRA1000KE is the larger enzyme-included kit for 1000 assays in 96-well format (2000 in 384-well).
| Kit (enzyme not included), 100 assays (HRA100K) | 100 assays (96-well) or 200 assays (384-well) |
|---|---|
| Kit Plus (enzyme included), 100 assays (HRA100KE) | 100 assays (96-well) or 200 assays (384-well) |
| Kit Plus (enzyme included), 1000 assays (HRA1000KE) | 1000 assays (96-well) or 2000 assays (384-well) |
Kit Components
| Component | Kit (enzyme not included), 100 assays (HRA100K) | Kit Plus (enzyme included), 100 assays (HRA100KE) | Kit Plus (enzyme included), 1000 assays (HRA1000KE) |
|---|---|---|---|
| 10 x Buffer HT1 | 480 µl | 480 µl | 4800 µl |
| 10 x Supercoiled plasmid DNA | 405 µl | 405 µl | 4050 µl |
| 1500 x Dye H19 | 20 µl | 20 µl | 170 µl |
| 10 x H19 dilution buffer | 3000 µl | 3000 µl | 30 ml |
| 1000 x human topoisomerase I (500 U/µl) | Not included | 10 µl | 40 µl |
Final reaction concentrations: 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.
Assay Principle
With magnesium present, relaxed DNA suppresses the fluorescence of dye H19 much more than supercoiled DNA does. As topo I converts supercoiled DNA into its relaxed form, the fluorescence signal at 535 nm (excitation 485 nm) goes down.
Specifications
| Assay Target | Human topo I DNA relaxation |
|---|---|
| Target Type | Topoisomerase |
| Organism | Human |
| Assay Type | Enzyme Activity Assay |
| Detection Method | Fluorescence |
| Detection Wavelength | Ex 485 nm / Em 535 nm |
| Plate Format | 96-well or 384-well plate |
| Instrument Required | Fluorescence microplate reader |
| Assay Time | About 65 min (60 min reaction + 5 min dye incubation) |
| Sizes | 100 assays (96-well) or 200 assays (384-well), 1000 assays (96-well) or 2000 assays (384-well) |
| Storage | Please contact us to confirm storage conditions. |
| Shipping | Shipped frozen. |
Protocol
The manufacturer's protocol describes the procedure and reaction volumes.
- Human Topo I DNA Relaxation Assay (PDF, see Documents): Covers catalog # HRA100K, HRA100KE, HRA1000KE.
Safety & Handling
Can’t find the exact format you need? We can help you source or customize this human topo I DNA relaxation assay — including larger pack sizes, bulk reagents, bulk enzyme, or a protocol adapted to your samples. Click Talk to a Scientist, email support@biohippo.com, or explore our Research Services. Our team will be in touch shortly.
