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

Elton P. Hudson, Andrej Nikoshkov, Mathias Uhlén, Johan Rockberg

Research output: Contribution to journalJournal articleResearchpeer-review

308 Downloads (Pure)

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.
Original languageEnglish
JournalP L o S One
Volume7
Issue number5
Pages (from-to)-
ISSN1932-6203
DOIs
Publication statusPublished - 2012

Keywords

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

Cite this