Document Type
Journal ArticlePublication Date
2018-07-02Keywords
Biochemistry, Biophysics, and Structural BiologyGenetics and Genomics
Laboratory and Basic Science Research
Systems Biology
Metadata
Show full item recordAbstract
In yeast hybrid assays, the process of identifying preys that interact with the bait of interest involves several steps. First, in this protocol, the bait yeast strain is transformed with a library of activation domain (AD)-prey clones and plated on selective media containing 3-aminotriazole (3AT). This selects transformants containing an AD-prey clone that induces HIS3 reporter expression. Second, these "HIS-positive" colonies are analyzed for LacZ induction (and, optionally, URA3 induction in yeast two-hybrid (Y2H) assays). Third, yeast PCR is used on these "double-positive" colonies to amplify the insert from the AD-prey plasmid. Fourth, some of this PCR product is used to perform a gap-repair retest to confirm the interaction in fresh bait-strain yeast, and the remainder is used for DNA sequencing to determine prey identity for those that successfully retest. Finally, interactions are carefully examined to filter out likely false-positive interactions. This protocol takes 20-43 d plus sequence confirmation to complete.Source
Cold Spring Harb Protoc. 2018 Jul 2;2018(7):pdb.prot094987. doi: 10.1101/pdb.prot094987. Link to article on publisher's site
DOI
10.1101/pdb.prot094987Permanent Link to this Item
http://hdl.handle.net/20.500.14038/49870PubMed ID
29967273Related Resources
ae974a485f413a2113503eed53cd6c53
10.1101/pdb.prot094987