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

PLoS One. 2012;7(5):e37429. doi: 10.1371/journal.pone.0037429. Epub 2012 May 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotechnology / methods*
  • Cloning, Molecular / methods*
  • Flow Cytometry
  • Genes / genetics*
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism*
  • Magnetics / methods*
  • Microscopy, Fluorescence
  • Microspheres*