Yeast Two-Hybrid Screening for MCM7 Interactors

The fusion protein pGBKT7-MCM7 contained 719 amino acids from MCM7 and 219 amino acids from bait domain [37 (link)]. The construct was transformed into One ShotTM competent cells (Invitrogen, Carlsbad, CA). pAD-RACK1 was constructed in pACT2 in 0.5 mL of polyethylene glycol/LiAc and incubated at 30°C for 30 minutes. 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.