Simplified plasmid cloning with a universal MCS design and bacterial in vivo assembly

Background: The ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning technologies has made significant improvements in plasmid construction, but simple and reliable tools are always desirable for time- and labor-saving purposes. Results: We developed and standardized a plasmid cloning protocol based on a universal MCS (Multiple Cloning Site) design and bacterial in vivo assembly. With this method, the vector is linearized first by PCR (Polymerase Chain Reaction) or restriction digestion. Then a small amount (10 similar to 20 ng) of this linear vector can be mixed with a PCR-amplified insert (5x molar ratio against vector) and transformed directly into competent E. coli cells to obtain the desired clones through in vivo assembly. Since we used a 36-bp universal MCS as the homologous linker, any PCR-amplified insert with similar to 15 bp compatible termini can be cloned into the vector with high fidelity and efficiency. Thus, the need for redesigning insert-amplifying primers according to various vector sequences and the following PCR procedures was eliminated. Conclusions: Our protocol significantly reduced hands-on time for preparing transformation reactions, had excellent reliability, and was confirmed to be a rapid and versatile plasmid cloning technique. The protocol contains mostly mixing steps, making it an extremely automation-friendly and promising tool in modern biology studies.

Automated summary

This plasmid cloning protocol based on universal MCS design and bacterial in vivo assembly eliminates the need for redesigning insert-amplifying primers and PCR procedures, significantly reducing hands-on time, with excellent reliability, making it a rapid and versatile tool for modern biology studies.