ArticleJournal of structural and functional genomics2006
Enzyme free cloning for high throughput gene cloning and expression.
Article in Journal of structural and functional genomics, 2006. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 21 papers.
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Who cites it
21 citing papers in PubMed.
- A small novel A-kinase anchoring protein (AKAP) that localizes specifically protein kinase A-regulatory subunit I (PKA-RI) to the plasma membrane.The Journal of biological chemistry · 2012Trial
- Host defence peptide plectasin targets bacterial cell wall precursor lipid II by a calcium-sensitive supramolecular mechanism.Nature microbiology · 2024Article
- DNA origami signposts for identifying proteins on cell membranes by electron cryotomography.Cell · 2021Article
- Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy.Nature communications · 2020Article
- Article
- DNA repair factor APLF acts as a H2A-H2B histone chaperone through binding its DNA interaction surface.Nucleic acids research · 2018Article
- Successful completion of a semi-automated enzyme-free cloning method.Journal of structural and functional genomics · 2016Article
- Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris.Microbial cell factories · 2015Article
- Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling.Journal of biomolecular NMR · 2015Article
- Insight into the conformational stability of membrane-embedded BamA using a combined solution and solid-state NMR approach.Journal of biomolecular NMR · 2015Article
- Homologous recombinatorial cloning without the creation of single-stranded ends: exonuclease and ligation-independent cloning (ELIC).Molecular biotechnology · 2015Article
- Functional analysis of the anti-adalimumab response using patient-derived monoclonal antibodies.The Journal of biological chemistry · 2014Review
- Structural basis of nucleic acid binding by Nicotiana tabacum glycine-rich RNA-binding protein: implications for its RNA chaperone function.Nucleic acids research · 2014Article
- The Fanconi anemia associated protein FAAP24 uses two substrate specific binding surfaces for DNA recognition.Nucleic acids research · 2013Article
- Review
- One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies.Applied and environmental microbiology · 2012Article
- Immunity to intracellular Salmonella depends on surface-associated antigens.PLoS pathogens · 2012Article
- Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR.Journal of biomolecular NMR · 2011Article
- Article
- Flexi vector cloning.Methods in molecular biology (Clifton, N.J.) · 2009Article
Corrections and comments
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Authors and funding
4 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EFC) procedure, a PCR-only method that eliminates all variables other than PCR efficiency by circumventing enzymatic treatments. We compared the cloning efficiency of EFC with that of Ligation Independent Cloning (LIC). Both methods are well suited for HTP cloning, but EFC yields three times more transformants and a cloning efficiency of 91%, comparable with recombinational cloning methods and significantly better than LIC (79%). EFC requires only nanogram amounts of both vector and insert, does not require highly competent cells and is, in contrast to LIC, largely insensitive to variations in PCR product concentration. Automated protein expression screening of expression strains directly transformed with EFC reactions showed, that the traditional preceding step via a cloning strain can be circumvented. EFC proves an efficient and robust HTP cloning method, that is compatible with existing Ligation Independent Cloning vectors, and highly suitable for automation.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.