High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.
Saccharomyces cerevisiae (budding yeast) is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc) transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids) or genome mutation (e.g., gene mutation, deletion, epitope tagging) is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.
Department of Molecular and Cellular Biology; University of Arizona, Tucson, Arizona, United States of America.
Guangbo
http://orcid.org/0000-0001-5623-8227
G
Liu
Department of Molecular and Cellular Biology; University of Arizona, Tucson, Arizona, United States of America.
Clayton
C
Lanham
Department of Molecular and Cellular Biology; University of Arizona, Tucson, Arizona, United States of America.
J Ross
JR
Buchan
Functional Genomics Core facility, University of Arizona, Tucson, Arizona, United States of America.
Matthew E
ME
Kaplan
D057205:Automation, Laboratory / Q000295:instrumentation* / Q000379:methods; D002172:Canavanine / Q000633:toxicity; D003470:Culture Media; D055786:Gene Knockout Techniques; D015698:Genomic Library; D006358:Hot Temperature; D012371:Robotics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D013997:Time Factors; D014170:Transformation, Genetic
D003470:Culture Media; D002172:Canavanine