Protocol I (Transient transfection) (E3314)


Use good quality plasmid DNA, i.e., CsCl or standard maxiprep. Do not use mini-prep DNA. 

Use proliferating mammalian cultures, i.e., regularly passaged cells. 

Use complete growth medium without antibiotics and antimycotics to plate cells for transfection. Use pSV40-CLuc to establish the transfection efficiency in a particular cell line. 

Titrate the amount of plasmid and transfection reagent to achieve optimal transfection efficiency.


  1. Plate cells to obtain 70–80% cell density on the day of transfection.
  2. Prepare transfection complex mixtures (e.g. 1 μg plasmid and 1–3 μl TransPass D2 (NEB #M2554) in 100 μl serum-free medium per transfection of a 12-well format) (Table 1), and incubate at room temperature for 20–30 minutes before adding to the cells. 

    Table 1: Plasmid DNA transfection in the presence of serum
    Culture Vessel Surface
    Volume of
    Plating Medium
    (per well)
    DNA in Serum-free Mixture TransPass D2
    in Transfection
    96 well 0.32 75 µl 0.1 μg in 10 μl 0.1–0.3 μl
    48 well 0.95 125 µl 0.3 μg in 25 μl 0.3–0.9 μl
    24 well 1.9 250 µl 0.7 μg in 50 μl 0.7–2.0 μl
    12 well 3.8 500 µl 1.5 μg in 100 μl 1.5–4.0 μl
    6 well 9.5 1 ml 3 μg in 250 μl 6–12 μl
    60 mm dish 21 2 ml 6 μg in 500 μl 12–20 μl
    100 mm dish 55 7 ml 15–20 μg in 1 ml 34–50 μl
  3. Return the cells to the incubator and incubate for 24 hours.
  4. Harvest some or all of the supernatant (to get rid of floating cells in the supernatant, centrifuge at 900–1500 rpm for 30 seconds) and store at -20˚C until ready to assay the CLuc activity.
  5. (Optional) Replace the medium, return the cells to the incubator and continue with the incubation. 

    Note: If the experiment requires incubating cells for several days, replacing the media daily is highly recommended. However, some experiments may require allowing luciferase to accumulate over several days. In that case, replacing the medium is not necessary.