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| Product Name | Plus One-Step Cloning Kit |
| Catalog No. | RESK-0039 |
| Description | The Plus One-Step Cloning Kit is a simple, fast, and highly efficient solution for directional DNA cloning. It allows PCR products from 50 bp to 10 kb to be cloned into any site of any vector without restriction digestion of the insert or overnight ligation. To clone, linearize the vector and add 15-25 bp homology arms matching the linearized vector ends to the 5' ends of the insert PCR primers, so the PCR product carries terminal sequences identical to the vector ends. The insert and linearized vector are then recombined at 50°C for just 20 minutes and are ready for transformation, with a positive clone rate of over 95%. |
| Intended Use | Directional cloning of a single PCR fragment (50 bp-10 kb) into any site of any vector by homologous recombination. |
| Principle / Technology | Homologous recombination. PCR primers with 15-25 bp 5' homology arms generate inserts whose ends match the linearized vector ends; a recombinase then joins the insert and vector in a single 50°C incubation, eliminating the need for restriction sites on the insert. |
| Sample Type | Purified PCR fragments and linearized cloning vectors |
| Performance Range / Specifications | Efficient cloning of 50 bp-10 kb fragments (inserts of 100 bp-10 kb recommended) Positive clone rate: >95% Fragment number: 1 Reaction time: 20 minutes Validated with 1.2 kb, 3 kb, and 5 kb inserts in a 4.7 kb vector (vector:insert molar ratio 1:3) |
| Specificity | Terminal 15-25 bp homology arms identical to the linearized vector ends direct site- and orientation-specific insertion; avoid cloning sites with repetitive sequences within 50 bp up- or downstream, and keep GC content within 20 bp of the cloning site at 40-60%. |
| Reaction Conditions / Protocol | 50°C for 20 minutes, then transform directly. Vector input should exceed 0.01 pmol (0.03 pmol recommended); vector:insert molar ratio 2:1-1:5, optimal 1:2-1:3. Linearize the vector by restriction digestion (double digestion recommended) or inverse PCR. Gel purification of the vector and insert is recommended for best results. |
| Components / Formulation | Linearized control vector (ampicillin resistance, prepared by inverse PCR) |
| Storage Conditions | Store at -20°C. |
| Shelf Life | 1 year when stored at -20°C. |
| Package Specifications | 20 T, 50 T |
| Shipping Conditions | Shipped with ice packs. |
| Expiration Date / Stability | Stable for 1 year when stored at -20°C. |
| Compatibility | Compatible with any vector and any cloning site. Competent cells with a transformation efficiency of at least 10^7 cfu/μg are recommended; electroporation is possible using electrocompetent cells. Suitable for multi-gene expression constructs using polycistronic, IRES, or 2A peptide elements. |
| Recommended Buffer System | Optimized recombination buffer (included in the kit). |
| Application Notes / Precautions | Avoid cloning sites with repetitive sequences within 50 bp up- or downstream; keep the GC content within 20 bp of the cloning site at 40-60%. Linearize the vector by restriction digestion (single or double; double digestion recommended) or inverse PCR. Use more than 0.01 pmol of vector (0.03 pmol recommended) and a vector:insert molar ratio of 2:1-1:5, optimally 1:2-1:3. Poor-quality linearized vector or insert causes false positives or inhibits the reaction; gel-purify the products when necessary. Recommended insert size is 100 bp-10 kb; fragments shorter than 100 bp may be degraded by exonuclease activity. Use competent cells with a transformation efficiency of at least 10^7 cfu/μg; electrocompetent cells are required for electroporation. The included linearized vector carries ampicillin resistance and is ready to use; the final expression vector is not provided. |
For research use only, not for clinical use.
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