Abstract
BackgroundThe 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.ResultsWe 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 ~ 20 ng) of this linear vector can be mixed with a PCR-amplified insert (5× 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 ~ 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.ConclusionsOur 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.
Highlights
The ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies
Plasmid cloning is one of the most commonly used techniques in molecular biology research. It plays a crucial role in studying the structure, function, and evolution of genes [1, 2] while serving as an essential tool in genetic, protein, and metabolic engineering [3, 4]
In this study, inspired by previous reports [6, 8, 20, 21], we developed a novel cloning strategy based on a UMCS design and bacterial in vivo assembly
Summary
Bacterial strains and reagents Chemically competent cell strains of E. coli DH5α, Top, BL21(DE3), XL10-Gold, and JM109(DE3) were obtained from Weidi Biotechnology (Shanghai, China) with transformation efficiency of ~ 5 × 108, ~ 5 × 108, ~ 107, ~ 2 × 109, and ~ 108 CFU/μg pUC19, respectively. The SanPrep Column Plasmid Mini-Preps Kit (Sangon Biotech, Shanghai, China) was used for plasmid extraction from bacteria, the SanPrep Column PCR Product Purification Kit (Sangon Biotech) was used for PCR product purification, and the SanPrep Column DNA Gel Extraction Kit (Sangon Biotech) was used for PCR product or enzyme-digested DNA purification after agarose gel electrophoresis, all following the manufacturer’s instructions. Both the 4S Red Plus (Sangon Biotech) stained 1% agarose gel and DNA Marker (250 ~ 10,000 bp, Sangon Biotech) were used for all gel electrophoresis experiments. Q5® High-Fidelity DNA Polymerase from NEB (Ipswich, MA, USA) was used to amplify the vector and insert, while Taq PCR Master Mix (Sangon Biotech) was used for colony PCR
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