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| Product Name | Zero-Background TOPO TA Cloning Kit |
| Catalog No. | RESK-0042 |
| Description | The Zero-Background TOPO TA Cloning Kit from Alta DiagnoTech harnesses the highly efficient DNA-joining activity of topoisomerase to deliver rapid, reliable cloning of A-tailed PCR products, such as those generated by Taq DNA polymerase. Compared with conventional T4 ligase-based methods, the kit offers clear advantages: ligation is completed in as little as 1-5 minutes; there is virtually no vector self-ligation, so the positive clone rate approaches 100% and blue-white screening is unnecessary; and the workflow is remarkably simple, taking only 15-20 minutes from ligation to plating while eliminating ice incubation, heat shock, and the 1-hour recovery step. Inserts up to 5 kb in length can be cloned efficiently. |
| Intended Use | Rapid cloning of A-tailed PCR products (e.g., fragments amplified with Taq DNA polymerase) for molecular cloning applications such as propagation, storage, and sequencing of target genes. |
| Principle / Technology | The kit is based on topoisomerase-mediated DNA ligation. The vector is pre-coupled with topoisomerase at the CCCTT recognition sequence, which covalently joins the insert to the vector within minutes without any ligase. A suicide gene (ccdB) in the vector is disrupted upon insert integration; when no fragment is inserted, ccdB is expressed and kills the host bacteria, so only recombinant clones can grow. This zero-background design eliminates vector self-ligation and removes the need for blue-white screening. |
| Sample Type | A-tailed PCR products (amplified with Taq DNA polymerase); 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 | Set up the ligation reaction in a total volume of no more than 10 uL, following the insert amount recommended in the manual. Incubate at room temperature for 1-5 minutes (typically 1-2 minutes; extend up to 5 minutes, but never longer, to obtain more colonies). Transform the reaction into competent cells and plate directly; ice incubation, heat shock, and extended recovery steps are not required. The entire 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 50 T |
| Expiration Date / Stability | Valid for 1 year when stored at -25 to -15 C. |
| Application Notes / Precautions | Restriction-digested plasmid products cannot be cloned directly into the TOPO vector because their 5' ends are phosphorylated; TOPO ligation requires a 5' hydroxyl end. If needed, dephosphorylate digested products with calf intestinal alkaline phosphatase (CIP) before ligation. Unphosphorylated PCR products can be used directly. Keep the reaction volume within 10 uL; volumes that are too small compromise ligation. If the target DNA concentration is low, concentrate it before ligation to improve the success rate. Ligation is generally complete within 1-2 minutes; it may be extended to 5 minutes for more colonies but must not exceed 5 minutes. Perform the reaction at room temperature. Choose the vector according to the polymerase used: TOPO TA vectors for Taq-amplified products, Blunt vectors for high-fidelity polymerase products. Simple vectors lack an MCS region, allowing custom restriction sites to be added during PCR amplification. Fresh PCR products give the highest ligation efficiency; products stored at -20 C remain usable within one week. Avoid repeated freeze-thaw cycles and degradation of PCR products. PCR products with a single clean band, high concentration, and no primer dimers can be ligated directly; purify products with nonspecific bands or primer dimers before ligation. |
For research use only, not for clinical use.
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