Count cells prior to seeding to ensure that the same number of cells is seeded for each experiment. Keep incubation times between seeding and complex addition consistent between experiments.
Possible mycoplasma contamination
Mycoplasma contamination influences transfection efficiency. Variations in the growth behavior of mycoplasma-infected cells will lead to different transfection efficiencies between replicate experiments.
Cells have been passaged too many times
Cells that have been passaged for an extended number of times tend to change their growth behavior, morphology and transfectability. When cells with high passage numbers are used for replicate experiments, decreased transfection efficiencies may be observed in later experiments. We recommend using cells with low passage number (< 50 splitting cycles).
Variations in serum quality can lead to variation in transfection efficiency. In general, it is advisable to test a small lot of serum from a reputable supplier with a control cell line and assess it before performing transfection experiments. Once a given lot has yielded satisfactory and reproducible results, additional sera from the same lot should be purchased.