DNA Transfection Protocol

A sample protocol is listed here for transfection experiments performed in 6-well plates. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. GenScript recommends using Lipofectamine 2000 for all transfections. It consistently produces high transfection efficiency and high protein overexpression.

• Adherent cells: One day before transfection, plate 0.25-1 x 10⁶ cells in 2 ml of growth medium without antibiotics per well so that they will be 90-95% confluent at the time of transfection.
• Suspension cells: On the day of transfection just prior to preparing complexes, plate 1.0–3.5 x10⁶ cells in 2 ml of growth medium without antibiotics per well.
• For each transfection sample, prepare DNA-Lipofectamine 2000 complexes as follows:
• Dilute DNA in 250 l of Opti-MEM I Reduced Serum Medium without serum (or other medium without serum). Mix gently.
• Mix Lipofectamine 2000 gently before use, then dilute the appropriate amount in 250 μl of Opti-MEM I Medium (or other medium without serum). Mix gently and incubate for 5 minutes at room temperature.
• After 5 minutes incubation, combine the diluted DNA with the diluted Lipofectamine 2000 (total volume is 500 μl). Mix gently and incubate for 20 minutes at room temperature to allow the DNA-Lipofectamine 2000 complexes to form.

Add the 500 μl of DNA-Lipofectamine 2000 complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.
Incubate the cells at 37°C in a CO₂ incubator for 24-72 hours until they are ready to assay for transgene expression. It is not necessary to remove the complexes or change the medium; however, growth medium may be replaced after 4-6 hours without loss of transfection activity.

• For stable cell lines: Passage the cells at a 1:10 or higher dilution into fresh growth medium 24 hours after transfection. Add selective medium the following day.