Streamlined high-throughput cloning protocol to generate arrayed mutant libraries
Summary: 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 acc...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-03-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166722008103 |
| _version_ | 1811295991362486272 |
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| author | Kerry T. Sun Tark S. Patel Justin Kim Helen S.H. Tang Ghazaleh Eskandari-Sedighi Haresh Sureshkumar Dean Schieve S.A. Mok |
| author_facet | Kerry T. Sun Tark S. Patel Justin Kim Helen S.H. Tang Ghazaleh Eskandari-Sedighi Haresh Sureshkumar Dean Schieve S.A. Mok |
| author_sort | Kerry T. Sun |
| collection | DOAJ |
| description | Summary: 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. |
| first_indexed | 2024-04-13T05:41:45Z |
| format | Article |
| id | doaj.art-eae1d0ee748540c2aceea13d55b5ec6c |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-04-13T05:41:45Z |
| publishDate | 2023-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-eae1d0ee748540c2aceea13d55b5ec6c2022-12-22T03:00:05ZengElsevierSTAR Protocols2666-16672023-03-0141101930Streamlined high-throughput cloning protocol to generate arrayed mutant librariesKerry T. Sun0Tark S. Patel1Justin Kim2Helen S.H. Tang3Ghazaleh Eskandari-Sedighi4Haresh Sureshkumar5Dean Schieve6S.A. Mok7Department of Biochemistry, University of Alberta, Edmonton, AB, Canada; Corresponding authorDepartment of Biochemistry, University of Alberta, Edmonton, AB, CanadaDepartment of Biochemistry, University of Alberta, Edmonton, AB, CanadaDepartment of Biochemistry, University of Alberta, Edmonton, AB, CanadaDepartment of Biochemistry, University of Alberta, Edmonton, AB, CanadaDepartment of Biochemistry, University of Alberta, Edmonton, AB, CanadaDepartment of Biochemistry, University of Alberta, Edmonton, AB, CanadaDepartment of Biochemistry, University of Alberta, Edmonton, AB, Canada; Corresponding authorSummary: 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.http://www.sciencedirect.com/science/article/pii/S2666166722008103GeneticsSequencingHigh Throughput ScreeningMolecular Biology |
| spellingShingle | Kerry T. Sun Tark S. Patel Justin Kim Helen S.H. Tang Ghazaleh Eskandari-Sedighi Haresh Sureshkumar Dean Schieve S.A. Mok Streamlined high-throughput cloning protocol to generate arrayed mutant libraries STAR Protocols Genetics Sequencing High Throughput Screening Molecular Biology |
| 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 | Genetics Sequencing High Throughput Screening Molecular Biology |
| url | http://www.sciencedirect.com/science/article/pii/S2666166722008103 |
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