Bacterial Transformation and Plasmid Isolation for FLAG-KRAS Exogenous Expression

The bacterial transformation of the plasmids for exogenous expression of FLAG-KRAS (pMDS-TetOn3G-kozak-FLAG-KRAS WT/mutants) was performed using the One-Shot Stbl3 (C737303; Invitrogen) chemically competent bacterial cells to replicate each plasmid following the manufacturer’s instructions. Subsequently, 100 μl of the bacteria–plasmid solutions was plated into LB selective agar plates containing the antibiotic spectinomycin (50 μg/ml). Plates were incubated at 37°C, overnight. The next day, individual bacterial colonies were selected from a LB agar plate and grown in 4 ml LB broth with the corresponding antibiotics for 6–12 h at 37°C in a shaker-incubator at 250 rpm. After incubation, several aliquots of this original starter culture were used to generate a bacterial glycerol stock for long-term storage at −80°C (1 ml transformed bacteria in 1 ml 50% glycerol). The remainder of the original starter culture was then used to grow at a large scale the transformed bacteria under selective antibiotics overnight in 500 ml of LB medium at 37°C in a shaker-incubator at 250 rpm. The HiSpeed Plasmid Maxi Kit (QIAGEN) was used to generate a larger amount of the FLAG-KRAS plasmids. The kit was used following the manufacturer’s instructions. The final DNA was eluted in 400 μl of TE buffer and allowed to resuspend overnight to ensure homogeneity. The next day, concentrations and purities were measured on the Implen NanoPhotometer NP80, and plasmid DNA was stored at −20°C.