Rapid and Efficient Plasmid Construction by Homologous Recombination in Yeast

Cold Spring Harbor Protocols
Jolanda van LeeuwenGuihong Tan

Abstract

The cloning of DNA fragments is a fundamental aspect of molecular biology. Traditional DNA cloning techniques rely on the ligation of an insert and a linearized plasmid that have been digested with restriction enzymes and the subsequent introduction of the ligated DNA into Escherichia coli for propagation. However, this method is limited by the availability of restriction sites, which often becomes problematic when cloning multiple or large DNA fragments. Furthermore, using traditional methods to clone multiple DNA fragments requires experience and multiple laborious steps. In this protocol, we describe a simple and efficient cloning method that relies on homologous recombination in the yeast Saccharomyces cerevisiae to assemble multiple DNA fragments, with 30-bp homology regions between the fragments, into one sophisticated construct. This method can easily be extended to clone plasmids for other organisms, such as bacteria, plants, and mammalian cells.

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Sep 4, 2015·Cold Spring Harbor Protocols·Jolanda van LeeuwenGuihong Tan

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Citations

Aug 25, 2017·PloS One·Faqing HuangAllen Yang Huang
Jan 31, 2018·Scientific Reports·Fanli ZengYibin Lin
Mar 25, 2017·Microbial Cell Factories·Nora WeberHarald Heider
Feb 26, 2019·Proceedings of the National Academy of Sciences of the United States of America·Jing HouCharles Boone
Sep 16, 2019·The Journal of Biological Chemistry·Jake F Watson, Javier García-Nafría
Mar 12, 2020·Applied Microbiology and Biotechnology·Julia A BubisDmitry S Karpov
Nov 5, 2020·International Journal of Molecular Sciences·Daria S SpasskayaDmitry S Karpov
Aug 22, 2018·Fungal Genetics and Biology : FG & B·Brandon T PfannenstielNancy P Keller
Sep 4, 2015·Cold Spring Harbor Protocols·Jolanda van LeeuwenGuihong Tan

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