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| Product Name | Plus Multi-Fragment One-Step Cloning Kit |
| Catalog No. | RESK-0040 |
| Description | The Plus Multi-Fragment One-Step Cloning Kit is a simple, fast, and highly efficient solution for directional DNA cloning and multi-fragment assembly. PCR products from 50 bp to 10 kb can be cloned into any site of any vector, and up to five fragments can be joined in a defined order in a single reaction. The kit's 2× enzyme premix combines a recombinase, a ready-to-use reaction buffer, and a proprietary recombination enhancer for markedly improved cloning efficiency. Simply linearize the vector, add 15-25 bp homology arms matching the adjacent fragment or vector ends to the 5' ends of the PCR primers, and incubate at 50°C for 20 minutes before transformation. The positive clone rate exceeds 95%. |
| Intended Use | Directional cloning and ordered assembly of 2-5 PCR fragments (50 bp-10 kb) into any site of any vector; also suitable for single-fragment cloning and, in combination with a high-fidelity DNA polymerase, for site-directed mutagenesis. |
| Principle / Technology | Homologous recombination. The recombinase combines exonuclease, DNA polymerase, and ligase activities: it specifically recognizes 15-20 bp homologous sequences at the vector and insert ends and resects dsDNA in the 5'→3' direction to generate cohesive ends that anneal at 50°C; the polymerase activity fills remaining single-stranded gaps, and the ligase activity seals the nicks by forming phosphodiester bonds. |
| Sample Type | Purified PCR fragments and linearized cloning vectors |
| Performance Range / Specifications | Ordered assembly of up to 5 fragments (2-5 fragments) in one reaction Fragment length range: 50 bp-10 kb Positive clone rate: >95% Reaction time: 20-50 minutes (50°C for 20 minutes for standard reactions) |
| Specificity | Assembly order and insertion orientation are defined by 15-25 bp terminal homology arms shared between adjacent fragments and the vector ends; the recombinase specifically recognizes these homologous sequences. |
| Reaction Conditions / Protocol | 50°C for 20 minutes (up to 50 minutes for complex multi-fragment assemblies), then transform directly. Primer design: homology arm (15-25 bp, GC content 40-60%) + optional restriction site + gene-specific priming sequence; calculate the annealing temperature only from the gene-specific portion, excluding homology arms and added restriction sites. |
| Components / Formulation | 2× MultiS One-Step Enzyme Premix (recombinase, optimized reaction buffer, and recombination enhancer) |
| Storage Conditions | Store at -25 to -15°C. |
| Shelf Life | 1 year when stored at -25 to -15°C. |
| Package Specifications | 10 T |
| Shipping Conditions | Shipped with ice packs. |
| Expiration Date / Stability | Stable for 1 year when stored at -25 to -15°C. |
| Compatibility | Compatible with any vector and any cloning site; no restriction sites are required on the insert, and inserts with cohesive or blunt ends are accepted. Suitable for single-fragment and multi-fragment directional cloning; can be combined with a high-fidelity DNA polymerase for site-directed mutagenesis. |
| Recommended Buffer System | Optimized homologous recombination buffer, pre-mixed in the 2× enzyme premix. |
| Application Notes / Precautions | Primer design: use a 15-25 bp homology arm (GC content 40-60%) plus a gene-specific priming sequence; restriction sites are optional, and the annealing temperature should be calculated only from the gene-specific sequence. 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%. False-positive (empty-vector) colonies usually result from incomplete vector linearization or contamination with circular plasmid of the same resistance; verify by transforming the linearized vector alone. For incomplete linearization, increase restriction enzyme input, extend digestion time, and gel-purify the digested vector (double digestion is generally recommended). To remove residual circular template from PCR-linearized vectors or inserts, digest with DpnI and/or gel-purify before the recombination reaction. Compared with conventional cloning, no insert digestion, gel recovery of digested inserts, or overnight ligation is needed, saving approximately one day of work. |
For research use only, not for clinical use.
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