VellixClonTMAmbient Seamless Cloning Mix is a new generation of seamless cloning reagent specially designed for single-fragment directed cloning. Based on the independently developed room-temperature homologous recombinase system, the reaction can be carried out at room temperature (23-30℃), reducing the reliance of traditional seamless cloning on temperature control equipment such as PCR instruments and water baths, and allowing the cloning reaction to be directly completed on the laboratory bench.
This product is in the form of a premixed solution. Compared with the traditional two-component system of buffer solution and enzyme, it simplifies the sample addition steps and reduces the sample addition error simultaneously, so as to obtain stable and repeatable experimental results. The 10 μL reaction system design reduces reagent consumption by half compared to the traditional 20 μL reaction system, making it particularly suitable for small-batch pre-experiments and high-throughput screening scenarios.
This product is suitable for directed cloning of 0.1-10 kb fragments. Homologous recombination can be completed within 5 minutes at room temperature (23-30℃), which is significantly faster than the traditional reaction time of 30 minutes. It can complete the recombination reaction of conventional fragments in a relatively short time, greatly simplifying the cloning process. In addition, this product has a wide range of applicable DNA input amounts. When the amplification product is single and has no obvious non-specific bands, it can be compatible with unpurified PCR products, thereby simplifying the experimental steps. Precise cloning can be achieved on any carrier and at any site. VellixClonTM Ambient Seamless Cloning Mix can be applied in fields such as rapid cloning, high-throughput cloning, seamless cloning and site-directed DNA mutagenesis.
1. Room temperature quick
This product is suitable for directed cloning of 0.1-10 kb fragments. Homologous recombination can be completed within 5 minutes at room temperature (23-30℃), which is significantly faster than the traditional reaction time of 30 minutes. It can complete the recombination reaction of conventional fragments in a relatively short time, greatly simplifying the cloning process.
2. Compatible with low investment and unpurified:
It has a wide range of applicable DNA input amounts. When the amplification product is single and there is no obvious non-specific band, it can be compatible with the unpurified PCR product, thereby simplifying the experimental steps.
3. Applicable to single-segment scenarios:
A new generation of seamless cloning reagent specially designed for single-fragment targeted cloning, optimized for single fragments, especially suitable for short fragments, with clear application boundaries.
Fields such as rapid cloning, high-throughput cloning, seamless cloning and DNA site-directed mutagenesis.
1. Reaction temperature test
Conclusion: The single-fragment seamless cloning reagent BR1C103 has a wide reaction temperature range and can react within the room temperature range of 23 ° C to 30 ° C. Considering the number of colonies and positive rate of different fragment lengths, 25 ° C is taken as the more universal reaction temperature for this reagent.
2. Reaction time test
Conclusion: The single-fragment seamless cloning reagent BR1C103 can complete the single-fragment cloning of inserted fragments of different lengths under rapid reaction conditions at room temperature. The 1 kb fragment maintained efficient cloning within 5 to 30 minutes. Effective cloning results can also be obtained for the 4 kb and 10 kb fragments in a short response time. Data shows that BR1C103 is suitable for rapid and seamless cloning of fragments of different lengths at room temperature.
3. Low-input test
Conclusion: The single-fragment seamless cloning reagent BR1C103 has good compatibility with low input. When the vector or inserted fragment is reduced alone to 1/10 of the conventional input amount, sufficient colonies can still be obtained and a relatively high positive rate can be maintained. Under normal and synchronous low-investment conditions, its overall performance is comparable to that of the comparison products. For extremely low input amounts, especially when the input of longer inserted fragments is insufficient, it is recommended to appropriately increase the amount of DNA used to ensure stable cloning efficiency.
4. Testing of unpurified PCR products
Conclusion: The single-fragment seamless cloning reagent BR1C103 has good compatibility with PCR products that have not been purified and recovered. The 1 kb and 2 kb PCR products can be directly used for seamless cloning. Sufficient colonies were obtained under the input conditions of 1 μL and 0.25 μL respectively, and the positive rate reached 100%. The overall performance was comparable to that of similar products. This product supports direct assembly of PCR products without recycling. While ensuring the accuracy of cloning, it simplifies the operation process, shortens the experimental time, and reduces sample loss caused by the purification process. It is particularly suitable for application scenarios such as limited sample volume and rapid cloning.
