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Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-8993-048X
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
2012 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 7, no 5, e37429- p.Article in journal (Refereed) Published
Abstract [en]

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.
National Category
Medical Biotechnology
URN: urn:nbn:se:kth:diva-99093DOI: 10.1371/journal.pone.0037429ISI: 000305343500113ScopusID: 2-s2.0-84862064886OAI: diva2:541466
Knut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience

QC 20120718

Available from: 2012-07-18 Created: 2012-07-13 Last updated: 2013-04-15Bibliographically approved

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Uhlén, MathiasRockberg, Johan
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Proteomics (closed 20130101)Science for Life Laboratory, SciLifeLab
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