ArticlePloS one2015
Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies.
Article in PloS one, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 86 papers.
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Who cites it
86 citing papers in PubMed.
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- Simple and robust in vivo engineering of plasmid DNA at any copy number in Escherichia coli.Communications biology · 2025Article
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- Fatty acid synthesis promoted by PA1895-1897 operon delays quorum sensing activation in Pseudomonas aeruginosa.AMB Express · 2024Article
- In vivo cloning of PCR product via site-specific recombination in Escherichia coli.Applied microbiology and biotechnology · 2024Article
- Combined effect of SAR-endolysin LysKpV475 with polymyxin B andMicrobiology (Reading, England) · 2024Article
- Regulation of tricarboxylate transport and metabolism inApplied and environmental microbiology · 2024Article
26 more citing papers are in PubMed but not listed here.
Corrections and comments
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Authors and funding
4 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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 double-stranded 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. 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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