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

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.
 [1] ;  [2] ;  [2] ;  [1] ;  [3]
  1. J. Craig Venter Institute, La Jolla, CA (United States)
  2. Cornell Univ., Ithaca, NY (United States)
  3. Imperial College, London (United Kingdom)
Publication Date:
OSTI Identifier:
Grant/Contract Number:
EE0006109; SC0006644
Accepted Manuscript
Journal Name:
Additional Journal Information:
Journal Volume: 10; Journal Issue: 9; Journal ID: ISSN 1932-6203
Public Library of Science
Research Org:
J. Craig Venter Institute Inc., La Jolla, CA (United States); Cornell Univ., Ithaca, NY (United States)
Sponsoring Org:
USDOE Office of Energy Efficiency and Renewable Energy (EERE); USDOE Office of Science (SC)
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES polymerase chain reaction; DNA cloning; plasmid construction; cloning; DNA recombination; DNA; homologous recombination; sequence assembly tools