The FuGENE HD volume-to-DNA mass ratio (µL/µg) determines the charge of the mix added to the cells (the negatively charged DNA must be balanced by the positively charged, cationic lipid of the reagent), and the volume of this mixture determines how much DNA is administered. More is not necessarily better and may lead to reduced protein expression and reduced cell health (Figure 3). Typical reagent:DNA ratios are between 1.5:1 and 4:1 with addition of 2–10 µL per well. In this experiment, optimal conditions for HEK293 transfection were 5 µL of a 2.5:1 mix.
Controls should be included in optimization experiments. Untransfected cells are used as an indication of maximum viability and no reporter expression; DNA- and reagent-only controls are included to monitor any unexpected effects of the transfection mix components on the cells.