BioTechniques · 2010 · 641 citations · 13 references
Life SciencesBiotechnologyMolecular BiologyGenetic EngineeringMolecular BiotechnologyRecombinant Dna TechnologyPlasmid BiologyPlasmid EngineeringPlasmid GeneticsPolymerase Chain ReactionRecombinant Plasmid ConstructionRestriction Enzyme TechnologyEscherichia ColiInsert SequencesOverlap Extension CloningOverlap Extension PcrOverlap-extension Polymerase Chain ReactionPlasmid Insertion SequencesRestriction EndonucleasesT4 Dna Ligase
The study presents a simple, efficient, and reliable method for cloning any insert into any plasmid without restriction enzymes or ligase. The technique uses chimeric primers to PCR‑amplify the insert and plasmid fragments, performs a second overlap‑extension PCR with a circular plasmid template, removes the template with DpnI, and transforms E.
Here we describe a straightforward, efficient, and reliable way to clone an insert of choice into a plasmid of choice without restriction endonucleases or T4 DNA ligase. Chimeric primers containing plasmid sequence at the 5′ ends and insert sequence at the 3′ ends were used to PCR-amplify insertion sequences of various sizes, namely the genes for GFP (gfp), β-d-glucuronidase (gusA), and β-galactosidase (lacZ), as well as the entire luxABCDE operon. These inserts were employed as mega-primers in a second PCR with a circular plasmid template. The original plasmid templates were then destroyed in restriction digests with DpnI, and the overlap extension PCR products were used to transform competent Escherichia coli cells. Phusion DNA polymerase was used for the amplification and fusion reactions, so both reactions were easy to monitor and optimize.
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High efficiency transformation of Escherichia coli with plasmids
Hiroaki Inoue, Hiroshi Nojima, Hiroto Okayama · Gene · 1990 · 2.3K citations
Albertha J.M. Walhout, Gary F. Temple, Michael A. Brasch et al. · Methods in enzymology on CD-ROM/Methods in enzymology · 2000 · 677 citations