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DNA decatenation is an essential step of DNA replication in cells. In bacteria, topoisomerase IV (the parC-parE complex) carries out this reaction, converting concatenated DNA into decatenated DNA.
Kit Components
| Component | SDD96KE |
|---|---|
| 10 x Assay buffer | 600 µl |
| 10 x Concatenated DNA | 500 µl |
| 10 mM ATP | 110 µl |
| 1000 x S. aureus topo IV | 8 µl |
| Stop solution (0.4 M EDTA) | 600 µl |
| 20 x Fluorescence dye | 260 µl |
| 10 x Rinse buffer | 2 ml |
| V-bottom plate | 1 plate |
| TDD filter plate | 1 plate |
| Black 96-well plate | 1 plate |
Final reaction conditions: 20 mM Tris-HCl pH 8, 35 mM NH4OAc, 4.6% glycerol, 1 mM DTT, 0.005% Brij35, 8 mM MgCl2, 3 µg/ml concatenated DNA, 0.2 mM ATP and 5 nM S. aureus topoisomerase IV. A reaction without enzyme or ATP can serve as negative control.
Assay Principle
After the reaction, the sample is filtered through the TDD filter plate, which separates decatenated DNA from concatenated DNA. The decatenated DNA passes into a black 96-well plate, is stained with fluorescence dye and read at 535 nm (excitation 485 nm), so inhibitors lower the signal.
Specifications
| Assay Target | S. aureus Topo IV DNA decatenation |
|---|---|
| Target Type | DNA Topoisomerase IV |
| Organism | S. aureus |
| Assay Type | Enzyme Activity Assay; Inhibitor Screening Assay |
| Detection Method | Fluorescence |
| Detection Wavelength | Ex 485 nm / Em 535 nm |
| Plate Format | 96-well filter plate |
| Instrument Required | Fluorescence reader + vacuum device |
| Equipment Needed | Lab vacuum line or pump (up to 80 kPa or 600 mmHg), plate vacuum device, plate fluorescence reader (Ex 485 nm / Em 535 nm) |
| Size | 96 assays |
| Storage | Please contact us to confirm storage conditions. |
| Shipping | Shipped frozen. |
Protocol
The manufacturer's protocol describes the procedure and reaction volumes.
- 96-Well S. aureus Topo IV DNA Decatenation Assay (PDF, see Documents): Covers catalog # SDD96KE.
Safety & Handling
Can’t find the exact format you need? We can help you source or customize this S. aureus Topo IV DNA decatenation 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.