Transfecting 293T cells.

Dissolve PEI powder in water and adjust pH to 7 with hydrochloric acid until the cloudy solution becomes clear. Filter through a 0.22 micron filter for sterility, aliquot into 1 mL volumes, and store at -80°C. Test each batch's transfection efficiency using different DNA-PEI ratios with a GFP-expressing plasmid.

Plate 293T cells in 14 cm plates using RPMI 1640 with 5% bovine serum, penicillin, streptomycin, and glutamine. Allow cells to grow to approximately 80% confluence, which indicates readiness for transfection.

Remove the growth medium from confluent cells and replace with 12 mL of Optimem medium per plate. Incubate for 2-4 hours to deprive cells of serum and increase their pinocytic uptake capacity for the DNA-PEI complex.

In an Eppendorf tube, combine 16 micrograms of plasmid DNA with 0.75 mL of Optimem medium. Add 1.25 mL of 1 mg/mL PEI and incubate the mixture for 15 minutes to allow complex formation, which should produce a cloudy suspension as the cationic polymer encapsulates the DNA.

Add the prepared DNA-PEI complex to each plate drop-wise in a sterile manner, distributing evenly across the starved cell culture. Incubate overnight to allow cellular uptake and nuclear delivery of the plasmid.

After overnight incubation, add 12 mL of complete RPMI 1640 medium containing 5% bovine serum, penicillin, streptomycin, and glutamax to each plate to restore nutrients and suppress bacterial contamination.

Continue incubating the transfected cells for 2-7 days at 37°C with 5% CO₂ to allow maximum protein expression from the transfected plasmid. Harvest supernatant for secreted proteins or prepare cellular lysates and membrane preparations for intracellular and membrane-bound proteins.