Streamlined high-throughput cloning protocol to generate arrayed mutant libraries

Kerry T Sun; Tark S Patel; Justin Kim; Helen S H Tang; Ghazaleh Eskandari-Sedighi; Haresh Sureshkumar; Dean Schieve; S A Mok

doi:10.1016/j.xpro.2022.101930

Streamlined high-throughput cloning protocol to generate arrayed mutant libraries

STAR Protoc. 2023 Mar 17;4(1):101930. doi: 10.1016/j.xpro.2022.101930. Epub 2022 Dec 13.

Authors

Kerry T Sun¹, Tark S Patel², Justin Kim², Helen S H Tang², Ghazaleh Eskandari-Sedighi², Haresh Sureshkumar², Dean Schieve², S A Mok³

Affiliations

¹ Department of Biochemistry, University of Alberta, Edmonton, AB, Canada. Electronic address: ksun4@ualberta.ca.
² Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.
³ Department of Biochemistry, University of Alberta, Edmonton, AB, Canada. Electronic address: sueann@ualberta.ca.

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.

Keywords: Genetics; High Throughput Screening; Molecular Biology; Sequencing.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cloning, Molecular
Gene Library
Mutagenesis
Mutagenesis, Site-Directed
Polymerase Chain Reaction* / methods