Co-transfection of HEK293 with Lipofectamine 2000 (24 well plate)

From Invitrogen

Remember to include the proper positive and negative controls in your experiment.

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.
   1. Dilute 100-200 ng of DNA and 1-10 pmol of RNAi in 50uL of DMEM. Mix gently.
   2. Mix Lipofectamine 2000 gently before use, then dilute 0.5-1.5 uL in 50uL DMEM. Mix gently and incubate for 5 minutes at room temperature.
   3. 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. Removal of complexes or media change is not required; however, growth medium may be replaced after 4-6 hours without loss of transfection activity.