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
Descripción
Sumario: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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