4.7. Positive Clone Rotation Verification

The screened prey plasmids, the empty pGADT7 vector (negative control), and the empty p53-pGADT7 vector (positive control) were transferred into competent yeast cells containing the bait vector. The transformed yeast strains were plated onto SD/-Leu and SD/-Leu/50 ng/mL AbA media and incubated for 3–5 days at 30 °C. The positive clones, which exhibited sizes ranging between 2 and 3 mm, were subsequently suspended in 20 μL of ddH₂O and diluted 1, 10, 100, and 1000 times to enable further investigation.

Next, 4 μL of the diluted yeast strains was streaked onto both SD/-Leu and SD/-Leu/50 ng/mL AbA media, followed by incubation for 3–5 days at 30 °C. The colony’s growth was observed and recorded for further analysis.