Yeast minimal media synthetic defined media

Yeast two-hybridization assays were performed using the Matchmaker^TM GAL4 Two-Hybrid System 3 (Clontech, USA), according to the manufacturer’s manual. The lithium acetate method was used to introduce pGADT7 (encoding the activation domain) and pGBKT7 (encoding the binding domain) plasmids into yeast (Saccharomyces cerevisiae strain AH109). The yeast were grown on the Yeast Minimal Media/Synthetic Defined (SD) media (Clontech, USA) without leucine and tryptophan, then transferred to selection media lacking leucine, tryptophan, and histidine but supplemented with 2 mM 3-amino-1,2,4-triazole (3-AT) (Sigma-Aldrich, USA). For the yeast spot assays, exponentially grown yeast cells were harvested and adjusted to OD₆₀₀ = 0.5 using sterilized water, and then further diluted (1:10 and 1:100). Yeast cells were spotted onto the SD medium without leucine and tryptophan, and the SD medium without leucine, tryptophan, or histidine. Their growth was observed after three days.

Yeast two-hybridization assays were performed using the Matchmaker™ GAL4 Two-Hybrid System 3 (Clontech, USA) according to the manufacturer’s manual. The lithium acetate method was used to introduce pGADT7 (as the yeast activation domain) and pGBKT7 (as the yeast binding domain) plasmids into yeast strain Saccharomyces cerevisiae strain AH109. Yeast were plated on Yeast Minimal Media/Synthetic Defined (SD) media (Clontech, USA) without leucine and tryptophan, then transferred to selection media without leucine, tryptophan and histidine supplemented with 15 mM 3-amino-1, 2, 4-triazole (3-AT) (Sigma, USA). For yeast spotting assays, exponentially grown yeast cells were harvested and adjusted to OD₆₀₀ 0.5 with sterilized water and diluted 1/10 and 1/100. Yeast cells were spotted onto SD medium without leucine and tryptophan and SD medium without leucine, tryptophan and histidine. Growth was assessed 3 days after spotting.