Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
Invitrogen™ Lipofectamine™ 2000 Transfection Reagent is a proprietary formulation for the transfection of nucleic acids (DNA and RNA) into eukaryotic cells providing the following advantages:
Guidelines for transfection
Use the procedure on below to transfect cells with short interfering RNA (siRNA) or Invitrogen™ Stealth RNAi™.
Note: For more tips for your RNAi experiment, refer to “Seven Steps to RNAi Success”. This manual is available from www.lifetechnologies.com/RNAi or Technical Service, as are cell-type specific RNAi transfection protocols (see “RNAi protocols”.)
Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, Sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of CHO-K1 cells with a reporter plasmid.
Use this brief procedure to transfect Stealth RNAi or siRNA into mammalian cells in a 24-well format. For other formats, see Scaling Up or Down Transfections. All amounts and volumes are given on a per well basis. Use this procedure as a starting point; optimize transfections as described in "Optimizing Stealth RNAi or siRNA transfection", especially if you are transfecting a mammalian cell line for the first time.
To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying RNA and Lipofectamine 2000 concentrations. Test 10-50 pmol RNA and 0.5-1.5 μl Lipofectamine 2000 for 24- well format. Depending on the nature of the target gene, transfecting cells at higher densities may also be considered when optimizing conditions.
Use the following procedure to transfect DNA into mammalian cells in a 24-well format. For other formats, see "Scaling up or down transfections". All amounts and volumes are given on a per well basis. Prepare complexes using a DNA (μg) to Lipofectamine 2000 (μl) ratio of 1:2 to 1:3 for most cell lines. Transfect cells at high cell density for high efficiency, high expression levels, and to minimize cytotoxicity. Optimization may be necessary (see "Optimizing plasmid DNA transfection").
To obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as DNA and Lipofectamine 2000 concentrations. Make sure that cells are greater than 90% confluent and vary DNA (μg): Lipofectamine 2000 (μl) ratios from 1:0.5 to 1:5.
To transfect cells in different tissue culture formats, vary the amounts of Lipofectamine 2000, nucleic acid, cells, and medium used in proportion to the relative surface area, as shown in the table. With automated, high-throughput systems, a complexing volume of 50 µl is recommended for transfections in 96-well plates.
Note: You may perform rapid 96-well plate transfections by plating cells directly into the transfection mix. Prepare complexes in the plate and directly 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 complexes.
Culture vessel | Surf. area per well1 | Shared reagents | DNA transfection | RNAi transfection | |||
Vol. of plating medium | Vol. of dilution medium2 | DNA | Lipofectamine 2000 | RNA | Lipofectamine 2000 | ||
96-well
|
0.3 cm
2 |
100 µl
|
2 x 25 µl
|
0.2 µg
|
0.5 µl
|
5 pmol
|
0.25 µl
|
24-well
|
2 cm
2 |
500 µl
|
2 x 50 µl
|
0.8 µg
|
2.0 µl
|
20 pmol
|
1.0 µl
|
12-well
|
4 cm
2 |
1 ml
|
2 x 100 µl
|
1.6 µg
|
4.0 µl
|
40 pmol
|
2.0 µl
|
6-well
|
10 cm
2 |
2 ml
|
2 x 250 µl
|
4.0 µg
|
10 µl
|
100 pmol
|
5 µl
|
60-mm
|
20 cm
2 |
5 ml
|
2 x 0.5 ml
|
8.0 µg
|
20 µl
|
200 pmol
|
10 µl
|
10-cm
|
60 cm
2 |
15 ml
|
2 x 1.5 ml
|
24 µg
|
60 µl
|
600 pmol
|
30 µl
|
¹ Surface areas may vary depending on the manufacturer.
² Volumes of dilution medium in Step 2a & 2b of DNA or RNAi transfection protocols.