Yeast two-hybrid screen

The Y2H screen was performed by cotransformation of pGBKT7-Prdm9 construct and mouse testes cDNA library cloned in pGADT7 (3 × 10⁶ to 10⁷ clones) (638848; Clontech) in PJ69-4α strain. Positive interactions were selected by plating the transformants on three different selective media lacking combinations of –Trp, –Leu, –His, and –Ala and supplemented with 3 mM 3-amino-1,2,4-triazole for detection of more stringent interactions. Each positive clone was tested for autoactivation by crossing to a yeast strain containing empty pGBKT7 plasmid. Plasmid DNA was isolated and sequenced to identify the individual cDNA library clone.