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

Hudson EP, Nikoshkov A, Uhlen M, Rockberg J

PLoS ONE 7 (5) e37429 [2012-05-18; online 2012-05-18]

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.

Affiliated researcher

Paul Hudson

SciLifeLab Fellow

PubMed 22624028

DOI 10.1371/journal.pone.0037429

Crossref 10.1371/journal.pone.0037429

Publications 9.5.0