Plasmid DNA Transfection Protocols

Use the following procedure to transfect DNA into eukaryotic cells in a 35 mm dish or a 6-well plate. For other size of cell culture dish, refer to Table 1 for scaling up and down. All amounts and volumes are given on a per well basis. Prepare complexes using a DNA (ug) to Transfection Reagent (uL) ratio of 1:3 for most cell lines or primary cells. Use a DNA (ug) to Transfection Reagent (uL) ratio of 1:2 for HEK293. Optimization may be necessary (see Optimizing Plasmid DNA Transfection).

• Transfection of adherent cells (35 mm dishes or 6-well plate)

Split the cells one day before before transfection to reach 80-95% confluence in the day of transfection.

• 1. For each transfection sample, prepare the transfection complexes as follows:



In a sterile 1.5 ml microtube, mix the following reagents:

2.5 ug Plasmid DNA

7.5 uL Transfection Reagent

X uL Serum-free* DMEM (or any other serum free-medium, PBS, DPBS etc.)

The total reaction is 100 uL.


Absolutely no Opti-MEM and serum in the transfection mixture



• 2. Pipetting up and down the mixture a few times. Spin briefly in a centrifuge. Leave the mixture at room temperature for 20-40 mins.
• 3. Add the entire mixture evenly to cells in the 35 mm culture dish or 6-well plate. Mix the reagent with the cells by tilting the dish or plate several times. Return the dish to a CO2 incubator.



Note: Serum concentration in the growth medium has no effect on the transfection efficiency. Up to 20% of fetal bovine serum has been tested without significantly changing the transfection efficiency.



• 4. Incubate cells for 18-48 hours prior to testing for transgene expression. At any time between 18-48 hours after the transfection, replace the medium in the dish with 2 mL (or any volume you