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| Product Name | Zero-Background TOPO Blunt Simple Cloning Kit (MCS-Depleted) |
| Catalog No. | RESK-0045 |
| Description | The Zero-Background TOPO Blunt Simple Cloning Kit (MCS-Depleted) from Alta DiagnoTech applies the rapid, high-efficiency DNA-joining activity of topoisomerase to the cloning of blunt-end PCR products, such as those produced by high-fidelity polymerases. Versus traditional T4 ligase-based cloning, ligation is completed in only 1-5 minutes; there is no vector self-ligation, yielding a positive clone rate of nearly 100% without blue-white screening; and the workflow from ligation to plating takes just 15-20 minutes, with no ice bath, heat shock, or 1-hour recovery. Target products up to 5 kb can be cloned. The TOPO-Blunt simple vector contains no multiple cloning site (MCS), so downstream restriction digests at user-introduced sites are not affected by MCS sequences, increasing both digestion efficiency and cloning success. |
| Intended Use | Rapid cloning of blunt-end PCR products when downstream restriction digestion at user-defined sites is planned without interference from an MCS region. The simple vector is convenient for subsequent subcloning manipulations. |
| Principle / Technology | The kit is based on topoisomerase-mediated ligation: the approximately 1.8 kb linearized simple vector is pre-coupled with topoisomerase, which covalently joins blunt-end inserts to the vector within minutes with no ligase and no restriction digestion of the vector required. A suicide gene (ccdB) in the vector backbone eliminates non-recombinant background by killing host bacteria that take up vector without an insert. This simple vector lacks the MCS region and carries ampicillin resistance. |
| 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 MCS-depleted simple vector (~1.8 kb, modified; migrates above 2 kb on gel electrophoresis): downstream digestion is unaffected by MCS sites, increasing digestion efficiency and cloning success |
| Reaction Conditions / Protocol | No restriction digestion of the vector is needed; combine the blunt-end PCR product directly with the topoisomerase-coupled simple vector and incubate at room temperature for 1-5 minutes. Transform into competent cells and plate on LB or SOC agar containing ampicillin (generally 100 ug/mL). The entire process from ligation to plating takes approximately 15-20 minutes. |
| 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 | User-supplied vectors cannot be used with this kit; the supplied vector is pre-coupled with topoisomerase. Choose the MCS-depleted simple vector when subsequent subcloning at custom restriction sites is planned; without an MCS, downstream ligation and digestion are more convenient. No restriction digestion of the vector is required; the PCR product is ligated directly. The TOPO vector is approximately 1.8 kb but is modified, so it migrates as a band larger than 2 kb on gel electrophoresis. Use LB or SOC solid medium containing ampicillin at a general working concentration of 100 ug/mL for selection. |
For research use only, not for clinical use.
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