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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| Published in: | STAR protocols Vol. 4; no. 1; p. 101930 |
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| Main Authors: | , , , , , , , |
| Format: | Journal Article |
| Language: | English |
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Elsevier Inc
17-03-2023
Elsevier |
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| Abstract | 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.
[Display omitted]
•Web app for automated generation of arrayed mutagenesis primer libraries•Optimized high-throughput site-directed mutagenesis PCR protocol•Streamlined bacterial transformation in a 96-well plate format•Automated magnetic-bead-based plasmid purification from transformed E. coli colonies
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
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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| AbstractList | 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. 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. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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. [Display omitted] •Web app for automated generation of arrayed mutagenesis primer libraries•Optimized high-throughput site-directed mutagenesis PCR protocol•Streamlined bacterial transformation in a 96-well plate format•Automated magnetic-bead-based plasmid purification from transformed E. coli colonies Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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. 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. • Web app for automated generation of arrayed mutagenesis primer libraries • Optimized high-throughput site-directed mutagenesis PCR protocol • Streamlined bacterial transformation in a 96-well plate format • Automated magnetic-bead-based plasmid purification from transformed E. coli colonies Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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. |
| ArticleNumber | 101930 |
| Author | Sureshkumar, Haresh Mok, S.A. Eskandari-Sedighi, Ghazaleh Sun, Kerry T. Schieve, Dean Patel, Tark S. Tang, Helen S.H. Kim, Justin |
| Author_xml | – sequence: 1 givenname: Kerry T. surname: Sun fullname: Sun, Kerry T. email: ksun4@ualberta.ca organization: Department of Biochemistry, University of Alberta, Edmonton, AB, Canada – sequence: 2 givenname: Tark S. surname: Patel fullname: Patel, Tark S. organization: Department of Biochemistry, University of Alberta, Edmonton, AB, Canada – sequence: 3 givenname: Justin surname: Kim fullname: Kim, Justin organization: Department of Biochemistry, University of Alberta, Edmonton, AB, Canada – sequence: 4 givenname: Helen S.H. surname: Tang fullname: Tang, Helen S.H. organization: Department of Biochemistry, University of Alberta, Edmonton, AB, Canada – sequence: 5 givenname: Ghazaleh surname: Eskandari-Sedighi fullname: Eskandari-Sedighi, Ghazaleh organization: Department of Biochemistry, University of Alberta, Edmonton, AB, Canada – sequence: 6 givenname: Haresh surname: Sureshkumar fullname: Sureshkumar, Haresh organization: Department of Biochemistry, University of Alberta, Edmonton, AB, Canada – sequence: 7 givenname: Dean surname: Schieve fullname: Schieve, Dean organization: Department of Biochemistry, University of Alberta, Edmonton, AB, Canada – sequence: 8 givenname: S.A. orcidid: 0000-0003-4593-3368 surname: Mok fullname: Mok, S.A. email: sueann@ualberta.ca organization: Department of Biochemistry, University of Alberta, Edmonton, AB, Canada |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/36520626$$D View this record in MEDLINE/PubMed |
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| Cites_doi | 10.1016/0378-1119(90)90336-P 10.1038/s41594-018-0057-1 10.1021/cb3002599 10.1074/jbc.M113.521997 |
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| Copyright | 2022 The Author(s) Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved. 2022 The Author(s) 2022 |
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| Keywords | High Throughput Screening Genetics Molecular Biology Sequencing |
| Language | English |
| License | This is an open access article under the CC BY-NC-ND license. Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
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| References | Inoue, Nojima, Okayama (bib1) 1990; 96 Thompson, Scaglione, Prensner, Gillies, Chinnaiyan, Paulson, Jinwal, Dickey, Gestwicki (bib4) 2012; 7 Sambrook, Russell (bib2) 2001 Mok, Condello, Freilich, Gillies, Arhar, Oroz, Kadavath, Julien, Assimon, Rauch (bib3) 2018; 25 Rauch, Gestwicki (bib5) 2014; 289 Inoue (10.1016/j.xpro.2022.101930_bib1) 1990; 96 Rauch (10.1016/j.xpro.2022.101930_bib5) 2014; 289 Mok (10.1016/j.xpro.2022.101930_bib3) 2018; 25 Thompson (10.1016/j.xpro.2022.101930_bib4) 2012; 7 Sambrook (10.1016/j.xpro.2022.101930_bib2) 2001 |
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| Title | Streamlined high-throughput cloning protocol to generate arrayed mutant libraries |
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