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| Product Name | Universal Zero-Background TOPO TA/Blunt Cloning Kit |
| Catalog No. | RESK-0041 |
| Description | The Universal Zero-Background TOPO TA/Blunt Cloning Kit is built on next-generation topoisomerase technology for fast, high-efficiency DNA fragment ligation. A highly efficient blunt-ending factor makes the kit compatible with both blunt-end and A-tailed (sticky-end) PCR products. Compared with conventional ligase-based cloning, it offers clear advantages: ligation is complete in just 5 minutes; there is no vector self-ligation, so the positive clone rate approaches 100% and blue-white screening is unnecessary; and target products up to 5 kb can be ligated. Simply add the purified target fragment to start the reaction. |
| Intended Use | Rapid TOPO-based cloning of A-tailed or blunt-end PCR products (150 bp-5 kb) without blue-white screening. |
| Principle / Technology | Topoisomerase-mediated ligation. The vector is pre-activated with topoisomerase, which joins DNA fragments carrying 5'-hydroxyl ends to the vector in minutes. A blunt-ending factor enables both TA cloning and blunt-end cloning. Vector self-ligation is eliminated, giving near-100% positive clones without blue-white screening. |
| Sample Type | A-tailed PCR products and blunt-end PCR products |
| Performance Range / Specifications | Compatible fragment length: 150 bp-5 kb (up to 5 kb) Positive clone rate: approaching 100% (100% positive rate validated with 200 bp, 1 kb, 2 kb, and 4 kb fragments at 10-200 ng input) Ligation time: 5 minutes No vector self-ligation; no blue-white screening required Stable for 7 days at 37°C and after 50 freeze-thaw cycles |
| Specificity | No vector self-ligation, yielding near-zero background. The TOPO reaction requires 5'-hydroxyl ends, so non-phosphorylated PCR products ligate directly; restriction-digested products with 5' phosphates must be dephosphorylated (e.g., with calf intestinal alkaline phosphatase) before TOPO cloning. |
| Reaction Conditions / Protocol | Incubate at room temperature (20-37°C; 25°C in a thermocycler recommended) for no more than 5 minutes; for low-concentration PCR products or longer fragments, extend incubation but do not exceed 15 minutes. Do not perform the reaction on ice. Keep the reaction volume at or below 10 μL; concentrate dilute target DNA before ligation. Use fresh PCR products whenever possible; products stored at -20°C for up to one week remain usable, but avoid repeated freeze-thaw cycles and degradation. Run a gel to check PCR products first: a single, strong band without primer dimers can be ligated directly; purify products with nonspecific bands or primer dimers before ligation. Select on plates containing 100 μg/mL ampicillin. |
| Components / Formulation | TOPO TA/Blunt cloning vector (ampicillin resistance) Blunt-ending factor |
| Storage Conditions | Store at -25 to -15°C. |
| Shelf Life | 1 year when stored at -25 to -15°C. |
| Package Specifications | 5 T, 20 T, 50 T |
| Quality Control | Stability-tested: the kit remains fully functional after 7 days at 37°C and after 50 freeze-thaw cycles. Ligation performance validated with blunt-end and A-tailed fragments from 200 bp to 4 kb, achieving 100% positive clones. |
| Expiration Date / Stability | Stable for 1 year when stored at -25 to -15°C; remains stable after 7 days at 37°C or 50 freeze-thaw cycles. |
| Compatibility | Compatible with both A-tailed and blunt-end PCR products (TA cloning and blunt-end cloning). Restriction-digested products carry 5' phosphates and are suitable for TA cloning but not TOPO cloning unless dephosphorylated first. The vector carries ampicillin resistance; use 100 μg/mL ampicillin for selection. Blue-white screening is not applicable because the kit contains no β-galactosidase system. |
| Recommended Buffer System | Optimized topoisomerase reaction buffer (included in the kit). |
| Application Notes / Precautions | Blue-white screening is not possible: the kit contains no β-galactosidase system, and the near-100% positive rate makes it unnecessary. Restriction-digested fragments cannot be ligated directly into the TOPO vector because their 5' ends are phosphorylated; dephosphorylate with calf intestinal alkaline phosphatase if TOPO cloning is required. Keep the reaction volume at or below 10 μL; overly small volumes impair ligation, and dilute target DNA should be concentrated first. Do not incubate on ice; incubate at room temperature (20-37°C) for no more than 5 minutes, extendable to 15 minutes for low-concentration or long fragments (25°C recommended). Use fresh PCR products for the highest ligation efficiency; products stored at -20°C for up to one week are acceptable, but avoid repeated freeze-thaw cycles and degradation. Check PCR products on a gel before ligation: a single strong band without primer dimers can be used directly; otherwise purify the product first. The vector is ampicillin-resistant; use 100 μg/mL ampicillin on selection plates. |
| Batch-to-Batch Consistency | The kit retains full performance after 7 days at 37°C and after 50 freeze-thaw cycles, demonstrating robust lot stability. |
For research use only, not for clinical use.
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