Yeast Two-Hybrid Protein Interaction

The fusion protein pBD-MCM7 contained 719 amino acids from MCM7 and 219 amino acids from bait domain [58 (link)]. The construct was transformed into One ShotTM competent cells (Invitrogen, Carlsbad, CA). The pAD-LMNB2 vector was generated from pACT2 in 0.5 ml of polyethylene glycol/LiAc at 30°C for 30minutes. After this initial incubation with plasmid DNA, the cell solution was combined with 20 mL of DMSO and incubated for 15 minutes at 42°C. The cells were pelleted, resuspended in 1 mL YPD medium, and shaken at 30°C for 40 minutes. The transformed cells were then pelleted, resuspended in 0.5 mL 0.9% NaCl, and plated onto the appropriate SD agar plate. The transformants were first plated on low stringency SD-Leu/−Trp and medium stringency SD-Leu/−Trp/− His plates. The colonies that grew on those plates were then subjected to the β-galactosidase assay as previously described for 24 and then allowed to grow further on the high stringency SD-Ade/−His/−Leu/−Trp plate.