| Field | Specification |
|---|---|
| Organism | |
| Applications | |
| Assay type | |
| Assay target | |
| Assay area | |
| Target type | |
| Detection method | |
| Detection wavelength | |
| Instrument required | |
| Plate format | |
| Assay time | |
| Storage | |
| Shipping | |
| Catalog no. (Mfr.) | |
| Main SKU |
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 for high-throughput screening of gyrase inhibitors.
Kit Components
| Component | DSA100KP |
|---|---|
| 10 x Buffer T2 | 600 µl |
| 10 x Relaxed DNA | 405 µl |
| 1500 x H19 dye | 20 µl |
| 10 x ATP | 450 µl |
| 10 x H19 dilution buffer | 3000 µl |
| 100 x P. aeruginosa gyrase | 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 | P. aeruginosa gyrase supercoiling |
|---|---|
| Target Type | DNA Topoisomerase II (Gyrase) |
| Organism | P. aeruginosa |
| 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.
- P. aeruginosa Gyrase DNA Supercoiling Assay (PDF, see Documents): Covers catalog # DSA100KP.
Safety & Handling
Can’t find the exact format you need? We can help you source or customize this P. aeruginosa gyrase 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.