Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force
2012 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 7, no 5, e37429- p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2012. Vol. 7, no 5, e37429- p.
IdentifiersURN: urn:nbn:se:kth:diva-99093DOI: 10.1371/journal.pone.0037429ISI: 000305343500113ScopusID: 2-s2.0-84862064886OAI: oai:DiVA.org:kth-99093DiVA: diva2:541466
FunderKnut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
QC 201207182012-07-182012-07-132013-04-15Bibliographically approved