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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Bibliographic Details
Published in:	STAR protocols Vol. 4; no. 1; p. 101930
Main Authors:	Sun, Kerry T., Patel, Tark S., Kim, Justin, Tang, Helen S.H., Eskandari-Sedighi, Ghazaleh, Sureshkumar, Haresh, Schieve, Dean, Mok, S.A.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 17-03-2023 Elsevier
Subjects:	Genetics High Throughput Screening Molecular Biology Protocol Sequencing High Throughput Screening Genetics Molecular Biology Sequencing
Online Access:	Get full text
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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. [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.
Bibliography:	Technical contact These authors contributed equally Lead contact
ISSN:	2666-1667 2666-1667
DOI:	10.1016/j.xpro.2022.101930