The optimal incubation time ranges from 2 to 6 days depending on various factors like the stability of the protein in the medium, potential toxicity of the protein, cell density and stability of the plasmid in the nucleus.

To check the timepoint for stopping the incubation, remove aliquots at 24, 48, 72, and 96 hours post-transfection to assay for recombinant transgene or protein expression.