Streamlined high-throughput cloning protocol to generate arrayed mutant libraries

Large-scale, site-directed mutagenesis enables rapid characterization of the biochemical and biological properties of proteins. Here, we present a cost-effective and adaptable cloning pipeline to generate arrayed gene libraries for a construct of interest. We detail steps to use an open access web a...

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Detalles Bibliográficos
Autores principales: Sun, Kerry T., Patel, Tark S., Kim, Justin, Tang, Helen S.H., Eskandari-Sedighi, Ghazaleh, Sureshkumar, Haresh, Schieve, Dean, Mok, S.A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9758493/
https://www.ncbi.nlm.nih.gov/pubmed/36520626
http://dx.doi.org/10.1016/j.xpro.2022.101930
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author Sun, Kerry T.
Patel, Tark S.
Kim, Justin
Tang, Helen S.H.
Eskandari-Sedighi, Ghazaleh
Sureshkumar, Haresh
Schieve, Dean
Mok, S.A.
author_facet Sun, Kerry T.
Patel, Tark S.
Kim, Justin
Tang, Helen S.H.
Eskandari-Sedighi, Ghazaleh
Sureshkumar, Haresh
Schieve, Dean
Mok, S.A.
author_sort Sun, Kerry T.
collection PubMed
description Large-scale, site-directed mutagenesis enables rapid characterization of the biochemical and biological properties of proteins. Here, we present a cost-effective and adaptable cloning pipeline to generate arrayed gene libraries for a construct of interest. We detail steps to use an open access web app to automate the design of mutagenesis primers optimized for our cloning protocols in a 96-well plate format. The protocol allows most molecular biology labs to clone 96 mutants (from PCR to sequence ready plasmid) in 3 days.
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spelling pubmed-97584932022-12-18 Streamlined high-throughput cloning protocol to generate arrayed mutant libraries Sun, Kerry T. Patel, Tark S. Kim, Justin Tang, Helen S.H. Eskandari-Sedighi, Ghazaleh Sureshkumar, Haresh Schieve, Dean Mok, S.A. STAR Protoc Protocol Large-scale, site-directed mutagenesis enables rapid characterization of the biochemical and biological properties of proteins. Here, we present a cost-effective and adaptable cloning pipeline to generate arrayed gene libraries for a construct of interest. We detail steps to use an open access web app to automate the design of mutagenesis primers optimized for our cloning protocols in a 96-well plate format. The protocol allows most molecular biology labs to clone 96 mutants (from PCR to sequence ready plasmid) in 3 days. Elsevier 2022-12-13 /pmc/articles/PMC9758493/ /pubmed/36520626 http://dx.doi.org/10.1016/j.xpro.2022.101930 Text en © 2022 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Sun, Kerry T.
Patel, Tark S.
Kim, Justin
Tang, Helen S.H.
Eskandari-Sedighi, Ghazaleh
Sureshkumar, Haresh
Schieve, Dean
Mok, S.A.
Streamlined high-throughput cloning protocol to generate arrayed mutant libraries
title Streamlined high-throughput cloning protocol to generate arrayed mutant libraries
title_full Streamlined high-throughput cloning protocol to generate arrayed mutant libraries
title_fullStr Streamlined high-throughput cloning protocol to generate arrayed mutant libraries
title_full_unstemmed Streamlined high-throughput cloning protocol to generate arrayed mutant libraries
title_short Streamlined high-throughput cloning protocol to generate arrayed mutant libraries
title_sort streamlined high-throughput cloning protocol to generate arrayed mutant libraries
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9758493/
https://www.ncbi.nlm.nih.gov/pubmed/36520626
http://dx.doi.org/10.1016/j.xpro.2022.101930
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