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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).ORCID iD: 0000-0003-1899-7649
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).ORCID iD: 0000-0002-9977-5724
2012 (English)In: PLoS ONE, ISSN 1932-6203, E-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
Identifiers
URN: urn:nbn:se:kth:diva-99093DOI: 10.1371/journal.pone.0037429ISI: 000305343500113Scopus ID: 2-s2.0-84862064886OAI: oai:DiVA.org:kth-99093DiVA: diva2:541466
Funder
Knut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20120718

Available from: 2012-07-18 Created: 2012-07-13 Last updated: 2017-12-07Bibliographically approved

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Uhlén, Mathias

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