To begin, obtain HEK 293T cells and plate 800, 000 cells per well in a 6-well tissue culture plate in supplemented DMEM media. Incubate the plate overnight at 37 degrees Celsius with 5%carbon dioxide to achieve 60 to 80%confluency. Next, mix one microgram of each plasmid DNA with 200 microliters of serum-free DMEM and six microliters of transfection reagent.
After 15 minutes of incubation, dispense 100 microliters of transfection mixture per well of cells. Incubate overnight in a 37 degrees Celsius and 5%carbon dioxide incubator. On day three, add an appropriate amount of either rapamycin or ethanol separately to the cells and incubate them for one hour in a 37 degrees Celsius and 5%carbon dioxide incubator.
Then place the plate on ice and gently wash the cells with three milliliters of cold PBS. After removing the PBS add 300 microliters of lysis buffer containing either rapamycin or ethanol, and scrape the cells with a cell scraper. Transfer the sample to a 1.5 milliliter tube and microcentrifuge it for 10 minutes at 1, 000 g at four degrees Celsius.
To check for expression levels, aliquot 20 microliters of the supernatant into a new 1.5 milliliter tube. Incubate the sample with 20 microliters of 2x Laemmli buffer containing 5%beta mercaptoethanol at 95 to 100 degrees Celsius for five minutes.
