Transfection of mammalian cell lines with plasmids and siRNAs
Shawn M. Ferguson, Agnes Roczniak-Ferguson
Abstract
This protocol details the transfection of mammalian cell lines with plasmids and siRNAs.
Steps
Lipofectamine 2000 (Invitrogen) or Fugene HD (Promega) or Fugene 6 (Promega) transfection reagents
On the day before transfection, plate 100,000 HeLa cells per well in a 6 well dish. For other cell lines, the number of cells will need to be optimized to achieve 50-75% confluency on the day of transfection.
Warm Optimem (Gibco) and transfection reagent to Room temperature
Add 200µL Optimem + 6µL transfection reagent + 2µg plasmid DNA to a round bottom polystyrene tube and mix gently.
Incubate 0h 20m 0s at Room temperature.
Add dropwise to cells and mix gently. Return cells to incubator. Duration of transfection must be optimized for each plasmid and downstream application.
RNAiMax transfection reagent
Warm Optimem (Gibco) and RNAiMax (Invitrogen) transfection reagent to Room temperature.
Combine 200µL OPTiMEM, 5µL RNAiMAX transfection reagent and 5µL of 20 siRNA and mix gently.
Incubate 0h 20m 0s at Room temperature.
Add transfection mix to 1.8mL media containing 100,000 HeLa cells per well in 6-well dish and return to cell culture incubator for 24-72 hours (duration needs to be optimized for each target gene).
