When it comes to "vector construction," the first thing that might come to mind is the restriction enzyme method, which involves cutting with restriction endonucleases to produce sticky ends and then using T4 DNA ligase to join the fragments. This method requires a long ligation time, is cumbersome to operate, and has a low positive rate. Anyone who has done molecular experiments may have had this experience: after multiple rounds of PCR, restriction digestion, ligation, and transformation, the positive rate is still very low. What went wrong? After working for several days, there are still only a few positive clones, and one might have to start over accidentally.
Today, Hanbio will detail a method that can quickly construct homologous recombinant vectors in one step with a high positive rate: seamless cloning.
Seamless cloning technology is a new, fast, and straightforward cloning method that allows for the insertion of one or more target DNA fragments at any site within a plasmid without the need for any restriction endonucleases. This method breaks from the traditional double digestion and ligation process, requiring just one step of recombination to achieve high-efficiency recombinant vectors, thereby significantly improving work efficiency.
Restriction enzyme method: Select an appropriate restriction site on the target vector for single or double digestion, and purify the digested plasmid using gel recovery.
Inverse PCR method: Design a pair of primers oriented in opposite directions on either side of the insertion site to amplify the target vector and obtain the linearized plasmid fragment through gel recovery.
Introduce homologous sequences at the 5' ends of the primers to ensure that the amplification product has completely matching sequences (15-25 bp) with the linearized vector.
Mix the linearized vector, insertion fragment, and seamless cloning reaction mixture in proportion and incubate at a constant temperature for 5-60 minutes.
These three steps complete the plasmid recombination. Subsequent transformation, colony PCR, and other operations will yield positive clones.
The seamless cloning kit is a powerful experimental tool. First, it provides highly flexible site selection, allowing researchers to clone genes at any position on the vector, meeting diverse experimental needs. Second, the process of operating the seamless cloning kit is quick and simple, taking only about an hour from preparation to completion of vector construction, significantly saving experimental time. Furthermore, the cloning efficiency of the seamless cloning kit is very high, with the proportion of positive clones reaching over 90%, effectively increasing the success rate of the experiment. Finally, the seamless cloning kit supports the simultaneous assembly of multiple target gene fragments, making the construction of complex gene structures easier and more efficient. In summary, the seamless cloning kit, with its excellent performance, provides robust support for research in genetic engineering, molecular biology, and other fields.
