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Transfection Procedure

Use the following procedure to transfect mammalian cells in a 24-well format. To transfect cells in other formats, see Scaling

Up or Down Transfections.

Adherent cells: One day before transfection, plate 0.5-2 x 105 cells in 500

µl 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 4-8 x 105 cells in 500

µl of growth

medium without antibiotics per well.

For each transfection sample, prepare DNA-Lipofectamine™ 2000 complexes as follows:

Dilute DNA in 50

µl 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 50

µl of Opti-MEM® I Medium (or

other medium without serum). Mix gently and incubate for 5 minutes at room temperature. Note: Combine the

diluted Lipofectamine™ 2000 with the diluted DNA within 30 minutes. Longer incubation times may decrease

activity. If D-MEM is used as a diluent for the Lipofectamine™ 2000, mix with the diluted DNA within 5 minutes.

After the 5 minute incubation, combine the diluted DNA with the diluted Lipofectamine™ 2000 (total volume is

100

µl). Mix gently and incubate for 20 minutes at room temperature to allow the DNA-Lipofectamine™ 2000

complexes to form. The solution may appear cloudy, but this will not inhibit the transfection. Note: DNA-

Lipofectamine™ 2000 complexes are stable for 6 hours at room temperature.

Add the 100

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 24-48 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.

For suspension cells: Add PMA and/or PHA (if desired) 4 hours after adding the DNA-Lipofectamine™ 2000 complexes

to the cells. Tip: For Jurkat cells, adding PHA-L and PMA at final concentrations of 1

µg/ml and 50 ng/ml, respectively,

enhances CMV promoter activity and gene expression. For K562 cells, adding PMA alone is sufficient to enhance

promoter activity.

Scaling Up or Down Transfections

To transfect cells in different tissue culture formats, vary the amounts of Lipofectamine™ 2000, DNA, cells, and medium used

in proportion to the difference in surface area (see table). With automated, high-throughput systems, larger complexing

volumes are recommended for transfections in 96-well plates. Note: You may perform rapid 96-well plate transfections (plate

cells and transfect simultaneously) by adding a suspension of cells directly to complexes prepared in the plate. Prepare

complexes and add cells at twice the cell density as in the basic protocol in a 100

µl volume. Cells will adhere as usual in the

presence of DNA-Lipofectamine™ 2000 complexes.

Culture

Vessel

Surface Area

per Well (cm2)

Relative Surface

Area (vs. 24-well)

Volume of Plating

Medium

DNA (

µg) and

Dilution Volume (

µl)

Lipofectamine™ 2000 (

µl)

and Dilution Volume (

µl)

96-well

0.3

0.2

100

µl

0.2

µg in 25 µl

0.5

µl in 25 µl

24-well

500

µl

0.8

µg in 50 µl

2.0

µl in 50 µl

12-well

1 ml

1.6

µg in 100 µl

4.0

µl in 100 µl

35-mm

2 ml

4.0

µg in 250 µl

µl in 250 µl

6-well

2 ml

4.0

µg in 250 µl

µl in 250 µl

60-mm

5 ml

8.0

µg in 0.5 ml

µl in 0.5 ml

10-cm

15 ml

µg in 1.5 ml

µl in 1.5 ml

Note: Surface areas are determined from actual measurements of tissue culture vessels.

Optimizing Transfection

To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying DNA

and Lipofectamine™ 2000 concentrations, and cell density. Make sure that cells are greater than 90% confluent and vary

DNA (

µg):Lipofectamine™ 2000 (µl) ratios from 1:0.5 to 1:5.

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