Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force

Publication: Research - peer-reviewJournal article – Annual report year: 2012

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In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.
Original languageEnglish
JournalP L o S One
Publication date2012
Volume7
Issue5
Pages-
ISSN1932-6203
DOIs
StatePublished
CitationsWeb of Science® Times Cited: 1

Keywords

  • BIOLOGY, DNA-MOLECULES, PCR PRODUCTS, PROTEIN EXPRESSION, PICHIA-PASTORIS, IN-VITRO, CLONING, RECOMBINATION, HYBRIDIZATION, EMULSION, TARGETS
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