Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies

Kostylev, Maxim; Otwell, Anne E.; Richardson, Ruth E.; Suzuki, Yo; Isalan, Mark

doi:10.1371/journal.pone.0137466

Title: Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies

Journal Article · Tue Sep 08 00:00:00 EDT 2015 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0137466· OSTI ID:1326190

Kostylev, Maxim ^[1]; Otwell, Anne E. ^[2]; Richardson, Ruth E. ^[2]; Suzuki, Yo ^[1]; Isalan, Mark ^[3]

J. Craig Venter Institute, La Jolla, CA (United States)
Cornell Univ., Ithaca, NY (United States)
Imperial College, London (United Kingdom)

Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six doublestranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. In addition, we demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.

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Research Organization:: J. Craig Venter Institute Inc., La Jolla, CA (United States); Cornell Univ., Ithaca, NY (United States)

Sponsoring Organization:: USDOE Office of Energy Efficiency and Renewable Energy (EERE); USDOE Office of Science (SC)

Grant/Contract Number:: EE0006109; SC0006644

OSTI ID:: 1326190

Journal Information:: PLoS ONE, Vol. 10, Issue 9; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 75 works

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59 BASIC BIOLOGICAL SCIENCES
polymerase chain reaction
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plasmid construction
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DNA recombination
DNA
homologous recombination
sequence assembly tools

Title: Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies

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