High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots

被引:8
|
作者
Liu, Guangbo [1 ]
Lanham, Clayton [1 ]
Buchan, J. Ross [1 ]
Kaplan, Matthew E. [2 ]
机构
[1] Univ Arizona, Dept Mol & Cellular Biol, Tucson, AZ 85721 USA
[2] Univ Arizona, Funct Genom Core Facil, Tucson, AZ 85721 USA
来源
PLOS ONE | 2017年 / 12卷 / 03期
关键词
P-BODIES; YEAST; TDP-43; EXPRESSION; EVENTS; SCREEN; GENOME; MODEL;
D O I
10.1371/journal.pone.0174128
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
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页数:15
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