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| Product Name | Zero-Background TOPO Blunt Cloning Kit |
| Catalog No. | RESK-0044 |
| Description | The Zero-Background TOPO Blunt Cloning Kit from Alta DiagnoTech is developed on the principle of topoisomerase-mediated, high-efficiency DNA ligation and is designed for blunt-end PCR products, such as those generated by high-fidelity polymerases. Compared with conventional T4 ligase-based methods, the kit ligate inserts in only 1-5 minutes; exhibits no vector self-ligation, so the positive clone rate approaches 100% and blue-white screening is unnecessary; and streamlines the workflow to just 15-20 minutes from ligation to plating, eliminating ice incubation, heat shock, and the 1-hour recovery step. Target products up to 5 kb can be cloned. |
| Intended Use | Rapid cloning of blunt-end PCR products (e.g., fragments amplified with high-fidelity/proofreading DNA polymerases) for downstream molecular cloning applications. |
| Principle / Technology | The kit exploits topoisomerase-mediated ligation rather than T4 ligase. The linearized vector is pre-coupled with topoisomerase at the CCCTT sequence, enabling covalent joining of blunt-end inserts within minutes. A suicide gene (ccdB) in the vector expresses a protein that kills the host bacteria when no fragment is inserted, so only clones with an integrated insert survive; this zero-background design prevents self-ligation artifacts and removes the need for blue-white screening. |
| Sample Type | Blunt-end PCR products (amplified with high-fidelity/proofreading polymerases); inserts up to 5 kb. |
| Performance Range / Specifications | Ligation completed in 1-5 minutes No self-ligation; positive clone rate approaching 100%; no blue-white screening required Only 15-20 minutes from ligation to plating; no ice bath, heat shock, or 1-hour recovery needed Clones target products up to 5 kb |
| Reaction Conditions / Protocol | Mix the blunt-end PCR product with the topoisomerase-coupled vector as directed in the manual and incubate at room temperature for 1-5 minutes (do not exceed 5 minutes). Transform into competent cells and plate; the whole process from ligation to plating takes approximately 15-20 minutes. |
| Storage Conditions | Store at -25 to -15 C. |
| Shelf Life | 1 year when stored at -25 to -15 C. |
| Package Specifications | 20 T |
| Expiration Date / Stability | Valid for 1 year when stored at -25 to -15 C. |
| Application Notes / Precautions | Fragments larger than 5 kb have not been tested and are likely to be difficult to clone. Overnight reactions are not permitted: topoisomerase has restriction endonuclease-like activity and will re-cleave the recombinant plasmid over extended incubation. Keep the reaction within 5 minutes. Compared with T4 ligase-based T-vector kits, this topoisomerase-based kit is faster and more efficient and can serve as a replacement. The 10x Enhancer supplied in the TOPO-Blunt kit is identical to that in the Zero-Background TOPO-TA kit and the two are interchangeable. The vector's suicide gene (ccdB) expresses a protein that kills bacteria unless an insert disrupts the gene; the CCCTT sequence is the site to which the topoisomerase is coupled. |
For research use only, not for clinical use.
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