Can siRNA and plasmid be co-transfected into cells?
Yes, although you must use specific transfection methods and reagents to optimize the reaction. The procedure below is recommended to cotransfect your plasmid DNA and an RNAi molecule into mammalian cells using Lipofectamine 2000. A more detailed version can be found on our website by searching "RNAi Transfection Protocols".
1. One day before transfection, plate cells in the appropriate amount of growth medium without antibiotics such that they will be 80-90% confluent at the time of transfection.
2. For each transfection sample, prepare DNA-RNAi molecule-Lipofectamine 2000 complexes as follows:
a. Dilute the DNA and RNAi molecule in the appropriate amount of Opti-MEM I Medium without serum. Mix gently.
b. Mix Lipofectamine 2000 gently before use, then dilute the appropriate amount in Opti-MEM I Medium without serum. Mix gently and incubate for 5 minutes at room temperature.
c. After the 5 minute incubation, combine the diluted DNA and RNAi molecule with the diluted Lipofectamine 2000. Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur. The solution may appear cloudy, but this will not impede the transfection.
3. Add the DNA-RNAi molecule-Lipofectamine 2000 complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.
4. Incubate the cells at 37°C in a CO2 incubator until you are ready to harvest cells and assay for your target gene. Removal of complexes or media change is not required; however, growth medium may be replaced after 4-6 hours without loss of transfection activity.
1. One day before transfection, plate cells in the appropriate amount of growth medium without antibiotics such that they will be 80-90% confluent at the time of transfection.
2. For each transfection sample, prepare DNA-RNAi molecule-Lipofectamine 2000 complexes as follows:
a. Dilute the DNA and RNAi molecule in the appropriate amount of Opti-MEM I Medium without serum. Mix gently.
b. Mix Lipofectamine 2000 gently before use, then dilute the appropriate amount in Opti-MEM I Medium without serum. Mix gently and incubate for 5 minutes at room temperature.
c. After the 5 minute incubation, combine the diluted DNA and RNAi molecule with the diluted Lipofectamine 2000. Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur. The solution may appear cloudy, but this will not impede the transfection.
3. Add the DNA-RNAi molecule-Lipofectamine 2000 complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.
4. Incubate the cells at 37°C in a CO2 incubator until you are ready to harvest cells and assay for your target gene. Removal of complexes or media change is not required; however, growth medium may be replaced after 4-6 hours without loss of transfection activity.