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| Product Name | Zero-Background TOPO TA Simple Cloning Kit (MCS-Depleted) |
| Catalog No. | RESK-0043 |
| Description | The Zero-Background TOPO TA Simple Cloning Kit (MCS-Depleted) from Alta DiagnoTech is built on the rapid, high-efficiency DNA-joining activity of topoisomerase and offers distinct advantages over traditional T4 ligase-based cloning: ligation finishes in just 1-5 minutes; there is no vector self-ligation, giving a positive clone rate of nearly 100% with no need for blue-white screening; and the entire workflow from ligation to plating takes only 15-20 minutes, with no ice bath, heat shock, or 1-hour recovery required. Inserts up to 5 kb can be cloned. Unlike standard TOPO vectors, the simple vector in this kit contains no multiple cloning site (MCS), so downstream restriction digests performed at user-introduced sites are not affected by MCS sequences, improving both digestion efficiency and cloning success. |
| Intended Use | Construction of cloning vectors for replication, storage, or sequencing of target genes. Ideal for A-tailed PCR products when downstream restriction analysis is planned at user-defined sites without interference from an MCS region. |
| Principle / Technology | The kit uses topoisomerase I-mediated ligation: the linearized vector is pre-charged with topoisomerase, which covalently joins the 5' hydroxyl ends of the insert to the vector within minutes without T4 ligase. A suicide gene (ccdB) in the vector backbone kills host bacteria unless disrupted by an inserted fragment, yielding a zero-background system with almost exclusively recombinant colonies. The TOPO vector is an ampicillin-resistant cloning vector without a promoter, and this simple version lacks the multiple cloning site (MCS) region. |
| Sample Type | A-tailed PCR products; inserts up to 5 kb (single fragment only; multi-fragment insertion is not supported). |
| 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 MCS-depleted simple vector: downstream restriction digestion is unaffected by MCS sites, increasing digestion efficiency and cloning success |
| Reaction Conditions / Protocol | Combine the PCR product with the topoisomerase-coupled vector according to the manual and incubate at room temperature for 1-5 minutes (do not exceed 5 minutes; overnight reactions are not permitted because the topoisomerase has restriction endonuclease-like activity that can re-cleave the recombinant plasmid over extended incubation). Transform into competent cells and plate on ampicillin-containing medium. The vector carries ampicillin resistance and contains no promoter. |
| Storage Conditions | Store at -20 C. Avoid repeated freeze-thaw cycles. |
| Shelf Life | 1 year. |
| Package Specifications | 20 T |
| Shipping Conditions | Shipped with ice packs. |
| Expiration Date / Stability | Valid for 1 year when stored at -20 C; avoid repeated freeze-thaw cycles. |
| Application Notes / Precautions | TOPO cloning is intended mainly for building cloning vectors for replication, storage, or sequencing of target genes; for expression vector construction, site-directed mutagenesis, or multi-fragment assembly, one-step cloning methods are recommended. The TOPO vector contains no promoter and carries ampicillin resistance; the full vector sequence is available on request. User-supplied vectors cannot be used with this kit, because the kit vector is pre-coupled with topoisomerase. PCR products require no special treatment, but gel purification is recommended to ensure a high positive rate. Do not mix the Enhancer of the Blunt and TA cloning kits; they are not recommended for interchangeable use. Overnight reactions are not allowed: topoisomerase has restriction endonuclease-like activity and will re-cleave the recombinant plasmid over time. Keep the reaction within 5 minutes. Inserts up to 5 kb have been validated; larger fragments are progressively more difficult. Only single-fragment insertion is supported. |
For research use only, not for clinical use.
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