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During DNA replication, bacterial DNA topoisomerase IV (the ParC-ParE complex) converts concatenated, or interlinked, DNA into decatenated DNA. Some decatenation activity is also seen with other topoisomerases, such as topoisomerase I and bacterial gyrases.
Formats and Pack Sizes
DDC020K contains the buffer, concatenated DNA, ATP, EDTA, dye and 20 spin columns but no enzyme (bacterial topoisomerase IV is supplied separately). DDC020KE adds 12 µl of 100 x E. coli topoisomerase IV (500 nM) for 20 assays, and DDC100KE is the 100-assay size with E. coli topoisomerase IV included and volumes scaled up.
| Kit (enzyme not included), 20 assays (DDC020K) | 20 assays |
|---|---|
| Kit Plus (E. coli Topo IV included), 20 assays (DDC020KE) | 20 assays |
| Kit Plus (E. coli Topo IV included), 100 assays (DDC100KE) | 100 assays |
Kit Components
| Component | Kit (enzyme not included), 20 assays (DDC020K) | Kit Plus (E. coli Topo IV included), 20 assays (DDC020KE) | Kit Plus (E. coli Topo IV included), 100 assays (DDC100KE) |
|---|---|---|---|
| Assay buffer (Buffer T4) | 150 µl (10 x) | 150 µl (10 x) | 600 µl |
| 10 x Concatenated DNA | 105 µl | 105 µl | 520 µl |
| 50 x ATP (10 mM) | 30 µl | 30 µl | 120 µl |
| 0.4 M EDTA | 120 µl | 120 µl | 550 µl |
| 20 x Fluorescence dye | 160 µl | 160 µl | 780 µl |
| Spin columns | 20 columns | 20 columns | 100 columns |
| 100 x E. coli topoisomerase IV (500 nM) | Not included | 12 µl | 55 µl |
The assay buffer is optimized for bacterial topoisomerase IV. Final reaction conditions: 20 mM Tris-HCl pH 8, 35 mM NH4OAc, 4.6% glycerol, 1 mM DTT, 0.005% Brij35, 8 mM MgCl2, 2 µg/ml concatenated DNA, 0.2 mM ATP and 5 nM topoisomerase IV.
Assay Principle
After the decatenation reaction, a quick spin-column step separates the products: concatenated DNA stays on the column while decatenated DNA passes through. The eluted DNA is mixed with a fluorescence dye and read at Em 535 nm (Ex 485 nm), so the signal rises with decatenation activity.
Specifications
| Assay Target | Topo IV DNA decatenation activity |
|---|---|
| Target Type | DNA Topoisomerase IV |
| Organism | E. coli |
| Assay Type | Enzyme Activity Assay |
| Detection Method | Fluorescence |
| Detection Wavelength | Ex 485 nm / Em 535 nm |
| Instrument Required | Fluorescence reader + centrifuge |
| Assay Time | About 65 min (60 min reaction + 4.5 min spin-column steps) |
| Sizes | 20 assays, 100 assays |
| Storage | Please contact us to confirm storage conditions. |
| Shipping | Shipped frozen. |
Protocol
The manufacturer's protocol describes the procedure and reaction volumes.
- DNA Topoisomerase IV Assay (PDF, see Documents): Covers catalog # DDC020K, DDC020KE, DDC100KE.
Safety & Handling
Can’t find the exact format you need? We can help you source or customize this topo IV DNA decatenation activity 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.
