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Bacterial DNA topoisomerase II (gyrase) converts relaxed circular DNA into supercoiled DNA (the DNA supercoiling reaction). The change in fluorescence is used to measure gyrase supercoiling and to screen gyrase inhibitors. Inhibition of E. coli gyrase by novobiocin, ciprofloxacin, enrofloxacin and levofloxacin is shown in the protocol.
Kit vs Kit Plus
DSA100K is the Kit without enzyme; DSA100KE (Kit Plus) adds 50 µl of 100 x E. coli gyrase. The H19 dye stock also differs: 8 µl of 4500 x dye in the Kit versus 20 µl of 1500 x dye in the Kit Plus; other components are the same, for 100 assays each.
| Kit (enzyme not included), 100 assays (DSA100K) | 100 assays |
|---|---|
| Kit Plus (enzyme included), 100 assays (DSA100KE) | 100 assays |
Kit Components
| Component | Kit (enzyme not included), 100 assays (DSA100K) | Kit Plus (enzyme included), 100 assays (DSA100KE) |
|---|---|---|
| 10 x Buffer T2 | 600 µl | 600 µl |
| 10 x Relaxed DNA | 405 µl | 405 µl |
| H19 dye | 8 µl (4500 x) | 20 µl (1500 x) |
| 10 x ATP | 450 µl | 450 µl |
| 10 x H19 dilution buffer | 3000 µl | 3000 µl |
| 100 x E. coli gyrase | Not included | 50 µl |
Final reaction conditions: 20 mM Tris-HCl pH 8, 35 mM NH4OAc, 4.6% glycerol, 1 mM DTT, 0.005% Brij35, 8 mM MgCl2, 25 µg/ml relaxed plasmid DNA, 1 mM ATP and 20 nM gyrase. Magnesium is essential; EDTA should be avoided. A reaction without ATP can serve as negative control.
Assay Principle
Relaxed and supercoiled DNA give different fluorescence with the H19 dye: in the presence of magnesium, relaxed DNA suppresses the dye signal much more than supercoiled DNA. As gyrase converts relaxed plasmid into its supercoiled form, fluorescence (Ex 485 nm / Em 535 nm) rises; inhibitors keep the signal low.
Specifications
| Assay Target | E. coli gyrase DNA supercoiling |
|---|---|
| Target Type | DNA Topoisomerase II (Gyrase) |
| Organism | E. coli |
| Assay Type | Enzyme Activity Assay; Inhibitor Screening 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) |
| Size | 100 assays |
| Storage | Please contact us to confirm storage conditions. |
| Shipping | Shipped frozen. |
Protocol
The manufacturer's protocol describes the procedure and reaction volumes.
- E. coli DNA Topoisomerase II (Gyrase) Assay (PDF, see Documents): Covers catalog # DSA100KE.
Safety & Handling
Can’t find the exact format you need? We can help you source or customize this E. coli gyrase DNA supercoiling 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.
