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

Authors
  • Chen, Fan1
  • Li, Yi-ya1
  • Yu, Yan-li1
  • Dai, Jie1
  • Huang, Jin-ling1
  • Lin, Jie1
  • 1 Minnan Normal University, Zhangzhou, 363000, P.R. China , Zhangzhou (China)
Type
Published Article
Journal
BMC Biotechnology
Publisher
Springer (Biomed Central Ltd.)
Publication Date
Mar 15, 2021
Volume
21
Issue
1
Identifiers
DOI: 10.1186/s12896-021-00679-6
Source
Springer Nature
Keywords
License
Green

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.

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