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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.
- High WeiPoot Red top Heels Women's Round Solid Material Toe Soft Boots Closed Low 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.
- 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.
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.