title: Automated solid-phase subcloning based on beads brought into proximity by magnetic force.
abstract: 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.
authors.affiliation: School of Biotechnology, KTH-Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Elton P
authors.initials: EP
authors.lastname: Hudson
authors.forename: Andrej
authors.initials: A
authors.lastname: Nikoshkov
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlen
authors.forename: Johan
authors.initials: J
authors.lastname: Rockberg
mesh_terms: D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D005434:Flow Cytometry; D005796:Genes / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D008280:Magnetics / Q000379:methods*; D008856:Microscopy, Fluorescence; D008863:Microspheres