There are many steps of the lipid transfection protocol that can be optimized if the initial transfection attempt is not successful. You may boost transfection efficiency and/or lower toxicity by optimizing the health of the cell culture, seeding density, plasmid quality and quantity, lipid reagent concentration, complexing time, and transfection time. The best increase in efficiency comes from appropriate cell confluency (seeding density), plasmid DNA quantity, and transfection time.
Plate the cells one day prior to transfection. Plate each well of a 6 well plate with 1 x 106 cells. Plate each well of another 6 well plate with 2 x 106 cells. You will run the same protocol on each plate to test which seeding density is better for your cell line.
1. Dilute plasmid DNA (with reporter gene) and lipid reagent for complexing. Use only unsupplemented basal medium for dilution. You will test 0, 1, and 2 µL DNA at each cell density. Prepare the tubes as indicated in the chart below.
2. Add tube A to tube B and mix gently. Add tube C to tube D and mix gently. Add tube E to tube F and mix gently. Allow complexes to form 15-30 minutes at room temperature.
3. Remove half the medium from each cell culture dish and discard.
4. Add complexes drop-wise to the cell cultures on each dish, according to the diagram below. Swirl the plates gently to distribute the complexes evenly over the cells.
5. Incubate the cells 8 hours at 37 °C, and then change the medium (use regular growth medium). llow the cells in continue incubating 48 more hours.
Assess each well for transfection efficiency and use the best one as your standard set of conditions going forward. If you wish to also optimize incubation time before and after medium change in step five, just double the number of plates you seed and reagents you prepare.