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| Product Name | Universal II One-Step Cloning Kit |
| Catalog No. | RESK-0037 |
| Description | The Universal II One-Step Cloning Kit is a next-generation homologous recombination cloning kit, now upgraded for greatly improved cloning positive rates. The carefully optimized second-generation 2x Universal II Enzyme Premix combines the recombinase and all buffers required for the recombination reaction, and is supplemented with a unique recombination enhancer that significantly improves recombination cloning efficiency. The kit directionally clones PCR products into any site of any vector, and is compatible with unpurified PCR products, directly recovered PCR products, and low-concentration gel-recovered products. It can recombine adapter fragments with homologous arms of 30-70% GC content. Simply linearize the vector completely and add 15-25 bp of sequence homologous to the linearized vector ends to the 5' ends of the forward and reverse PCR primers for the insert, so that the 5' and 3' termini of the insert PCR product exactly match the two ends of the linearized vector. Under the action of the recombinase, the recombination reaction between the PCR product and the linearized vector is completed in as little as 5 minutes at 50 degC, with cloning positive rates above 95%. |
| Intended Use | One-step, seamless, directional cloning of PCR products into any vector at any site, including single-fragment insertion, multi-fragment assembly, and point-mutation construction; for research use. |
| Principle / Technology | Homologous recombination cloning. The 2x enzyme premix contains a recombinase, reaction buffer, and a unique recombination enhancer. Homologous sequences of 15-25 bp matching the linearized vector ends are introduced at the 5' ends of the insert primers; the recombinase assembles insert and vector through these homologous arms at 50 degC, without restriction digestion or ligation. |
| Sample Type | PCR products (unpurified, directly recovered, or low-concentration gel-recovered) and linearized vectors; insert fragments of 50 bp-25 kb. |
| Performance Range / Specifications | Directional, seamless assembly of 1-7 DNA fragments in a single tube (validated for assemblies of up to 6 fragments / 7.6 kb total insert) Recombination completed in as little as 5 minutes at 50 degC (reaction time 5-50 min) Cloning positive rate above 95% (100% positive rate demonstrated in single-, two-, three-, and six-fragment assemblies) Vector-plus-insert ligation length over 25 kb (21 kb vector + 4.5 kb insert demonstrated) Insert fragment length range: 50 bp-25 kb Works in small reaction volumes as low as 6 uL and with low-input inserts (10 ng) Recombines fragments with homologous arms of 30-70% GC content Stable connection of low-concentration five- and six-fragment assemblies; remains stable after 7 days at 37 degC |
| Specificity | Directional cloning through 15-25 bp homologous arms ensures precise, sequence-defined insertion at any vector site; compatible with both sticky and blunt ends. |
| Reaction Conditions / Protocol | Completely linearize the vector (by restriction digestion or inverse PCR). Amplify the insert with primers carrying 15-25 bp 5' extensions homologous to the linearized vector ends. Mix the insert PCR product and linearized vector (recommended vector:insert molar ratio 1:2) with the 2x enzyme premix. Incubate at 50 degC; the recombination reaction completes in as little as 5 minutes (5-50 min depending on fragment number and length). Transform the reaction product into competent cells and plate on selective medium. Single-tube assembly of multiple fragments without prior digestion; reaction volumes as low as 6 uL are supported. |
| Components / Formulation | 2x Universal II Enzyme Premix (recombinase, recombination reaction buffer, and a unique recombination enhancer) Positive control (linearized vector with insert; positive colonies verified with universal M13 primers yielding a band of ~500 bp; ampicillin or carbenicillin resistance, carbenicillin recommended) |
| Storage Conditions | Store at -25 to -15 degC. |
| Shelf Life | 18 months when stored at -25 to -15 degC. |
| Package Specifications | 5 T 20 T 50 T 10x 50 T |
| Product Form | Kit (2x enzyme premix with positive control) |
| Quality Control | Performance validated by single-fragment (6 uL micro-volume, 10 ng input), two-, three-, five-, and six-fragment assemblies and >25 kb vector-fragment ligation, each achieving high colony counts and 100% positive rates; stability verified after 7 days at 37 degC and after freeze-thaw. |
| Source / Origin | Recombinant enzyme-based formulation. |
| Expiration Date / Stability | Stable for 7 days at 37 degC; freezing at -20 degC and repeated freeze-thaw do not affect product performance; shelf life 18 months at -25 to -15 degC. |
| Compatibility | Suitable for any vector and any insertion site; compatible with sticky and blunt ends; supports multi-fragment assembly and point-mutation cloning; accepts unpurified PCR products, directly recovered PCR products, and low-concentration gel-recovered products; recombines fragments with homologous arms of 30-70% GC content; positive control compatible with ampicillin or carbenicillin selection. |
| Recommended Buffer System | Recombination reaction buffer pre-mixed in the 2x enzyme premix (with recombination enhancer). |
| Application Notes / Precautions | Primer design: add 15-25 bp of sequence homologous to the linearized vector ends to the 5' ends of the insert forward and reverse primers; homologous arms of 30-70% GC content are supported. The vector must be completely linearized before the recombination reaction. Recommended vector:insert molar ratio is 1:2; maximum assembly size is 25 kb, and reaction volumes as low as 6 uL are supported. For colony PCR of the positive control, use universal M13 primers, which amplify a band of ~500 bp. The positive control carries ampicillin/carbenicillin resistance; carbenicillin is recommended because ampicillin loses activity quickly. If the kit freezes solid at -20 degC, performance is not affected; freeze-thaw has been verified not to impact cloning efficiency. |
| Batch-to-Batch Consistency | The upgraded formulation improves cloning positive rates and remains stable after freeze-thaw and 7 days at 37 degC, ensuring reproducible performance. |
For research use only, not for clinical use.
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