Plasmid DNA Transfection Protocol - 한국어

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

Prepare four tubes with 50 microliters of Optimem medium each, then add increasing volumes of Lipofectamine LTX reagent: 2 µL to tube 1, 3 µL to tube 2, 4 µL to tube 3, and 5 µL to tube 4. Mix each tube thoroughly by vortexing.

Combine 250 microliters of Optimem medium with 5 micrograms of plasmid DNA (5 microliters of 1 µg/µL stock), then add 5 microliters of Plus reagent and mix well.

Add 50 microliters of the diluted DNA solution to each of tubes 1, 2, 3, and 4 containing the Lipofectamine LTX dilutions. Incubate all complexes for 5 minutes at room temperature.

Remove the 24-well plate from the incubator and add 50 microliters of DNA-reagent complex from tubes 1, 2, 3, and 4 to wells 1, 2, 3, and 4 respectively. Return the plate to the 37°C incubator.

Grow cells for 1-3 days at 37°C to allow sufficient time for plasmid expression and protein production.

Examine each well using a fluorescence microscope or imaging station to visualize GFP expression and determine which reagent concentration achieved the highest transfection efficiency.