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

Publication: Research - peer-reviewJournal article – Annual report year: 2012

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Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force. / Hudson, Elton P.; Nikoshkov, Andrej; Uhlén, Mathias; Rockberg, Johan.

In: P L o S One, Vol. 7, No. 5, 2012, p. -.

Publication: Research - peer-reviewJournal article – Annual report year: 2012

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Author

Hudson, Elton P.; Nikoshkov, Andrej; Uhlén, Mathias; Rockberg, Johan / Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force.

In: P L o S One, Vol. 7, No. 5, 2012, p. -.

Publication: Research - peer-reviewJournal article – Annual report year: 2012

Bibtex

@article{19ce9860ad274aa28fa539d5c8d68ac1,
title = "Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force",
keywords = "BIOLOGY, DNA-MOLECULES, PCR PRODUCTS, PROTEIN EXPRESSION, PICHIA-PASTORIS, IN-VITRO, CLONING, RECOMBINATION, HYBRIDIZATION, EMULSION, TARGETS",
publisher = "Public Library of Science",
author = "Hudson, {Elton P.} and Andrej Nikoshkov and Mathias Uhlén and Johan Rockberg",
year = "2012",
doi = "10.1371/journal.pone.0037429",
volume = "7",
number = "5",
pages = "--",
journal = "P L o S One",
issn = "1932-6203",

}

RIS

TY - JOUR

T1 - Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force

A1 - Hudson,Elton P.

A1 - Nikoshkov,Andrej

A1 - Uhlén,Mathias

A1 - Rockberg,Johan

AU - Hudson,Elton P.

AU - Nikoshkov,Andrej

AU - Uhlén,Mathias

AU - Rockberg,Johan

PB - Public Library of Science

PY - 2012

Y1 - 2012

N2 - 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.

AB - 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.

KW - BIOLOGY

KW - DNA-MOLECULES

KW - PCR PRODUCTS

KW - PROTEIN EXPRESSION

KW - PICHIA-PASTORIS

KW - IN-VITRO

KW - CLONING

KW - RECOMBINATION

KW - HYBRIDIZATION

KW - EMULSION

KW - TARGETS

U2 - 10.1371/journal.pone.0037429

DO - 10.1371/journal.pone.0037429

JO - P L o S One

JF - P L o S One

SN - 1932-6203

IS - 5

VL - 7

SP - -

ER -