Plasmid DNA Transfection Protocol

Clean the cell culture hood and work surface with 70% ethanol. Seed cells 24 hours prior so they reach 70-90% confluency at the time of transfection.

Assemble all required materials including lipofectamine LTX and plus reagent, Optimum reduced serum medium, plasmid DNA (1 microgram per microliter), microcentrifuge tubes, pipettes, marker, timer, and 24-well plate with confluent cells.

Prepare four tubes with 50 microliters of Optimum medium each. Add increasing volumes of lipofectamine LTX (2, 3, 4, and 5 microliters to tubes 1-4 respectively) and vortex each tube well.

Prepare a separate tube with 250 microliters of Optimum medium, add 5 microliters of plasmid DNA (5 micrograms), then add 5 microliters of plus reagent and mix well.

Add 50 microliters of the diluted DNA solution to each of the four lipofectamine LTX tubes (1-4). Incubate the complexes for 5 minutes at room temperature.

Remove the 24-well plate from the incubator and add 50 microliters of each DNA-reagent complex from tubes 1-4 to the corresponding wells 1-4. Return the plate to the incubator.

Grow cells for 1-3 days at 37°C. View GFP fluorescence using a fluorescent microscope or imaging station to assess transfection efficiency in each well and identify the optimal reagent concentration.