Eccles: DGG Protocols

siRNA Transfection

• Resuspending siRNA’s:

• • Add 100µL sterile MQ per 5nmol powdered siRNA.
  • Gives a final concentration of 50µM siRNA in the tube.
  • Vortex to ensure all the siRNA is completely dissolved.
  • Store at -20°C.

• siRNA Transfection:

• • For one 24-well.
  • 1µL Lipofectamine 2000 + 49µL serum free OptiMEM.
  • Incubate for 5 minutes.
  • 1µL 50µM siRNA + 49µL serum free OptiMEM.
  • Add diluted siRNA to the Lipofectamine.
  • Incubate for 25 minutes.
  • Add 400µL serum free OpitMEM.
  • 500µL per well.
  • Final concentration of siRNA per 24-well is 0.1µM.
  • After 4 hours add 50µL FCS to give a final concentration of 10% per well.
  • Culture for required length of time.