Automated solid-phase subcloning based on beads brought into proximity by magnetic force.

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 g...

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Autores principales:	Elton P Hudson, Andrej Nikoshkov, Mathias Uhlen, Johan Rockberg
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/6b08f8dde85e454b8bdb2569e17ca94e
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id	oai:doaj.org-article:6b08f8dde85e454b8bdb2569e17ca94e
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spelling	oai:doaj.org-article:6b08f8dde85e454b8bdb2569e17ca94e2021-11-18T07:18:08ZAutomated solid-phase subcloning based on beads brought into proximity by magnetic force.1932-620310.1371/journal.pone.0037429https://doaj.org/article/6b08f8dde85e454b8bdb2569e17ca94e2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22624028/?tool=EBIhttps://doaj.org/toc/1932-6203In 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.Elton P HudsonAndrej NikoshkovMathias UhlenJohan RockbergPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 5, p e37429 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Elton P Hudson Andrej Nikoshkov Mathias Uhlen Johan Rockberg Automated solid-phase subcloning based on beads brought into proximity by magnetic force.
description	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.
format	article
author	Elton P Hudson Andrej Nikoshkov Mathias Uhlen Johan Rockberg
author_facet	Elton P Hudson Andrej Nikoshkov Mathias Uhlen Johan Rockberg
author_sort	Elton P Hudson
title	Automated solid-phase subcloning based on beads brought into proximity by magnetic force.
title_short	Automated solid-phase subcloning based on beads brought into proximity by magnetic force.
title_full	Automated solid-phase subcloning based on beads brought into proximity by magnetic force.
title_fullStr	Automated solid-phase subcloning based on beads brought into proximity by magnetic force.
title_full_unstemmed	Automated solid-phase subcloning based on beads brought into proximity by magnetic force.
title_sort	automated solid-phase subcloning based on beads brought into proximity by magnetic force.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/6b08f8dde85e454b8bdb2569e17ca94e
work_keys_str_mv	AT eltonphudson automatedsolidphasesubcloningbasedonbeadsbroughtintoproximitybymagneticforce AT andrejnikoshkov automatedsolidphasesubcloningbasedonbeadsbroughtintoproximitybymagneticforce AT mathiasuhlen automatedsolidphasesubcloningbasedonbeadsbroughtintoproximitybymagneticforce AT johanrockberg automatedsolidphasesubcloningbasedonbeadsbroughtintoproximitybymagneticforce
_version_	1718423669939634176

Automated solid-phase subcloning based on beads brought into proximity by magnetic force.

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