Eccles:siRNA Transfection

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Resuspending siRNA’s

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

siRNA Transfection

Per well of a 24-well plate

  1. 1µL Lipofectamine 2000 + 49µL serum free OptiMEM.
  2. Incubate for 5 minutes.
  3. 1µL 50µM siRNA + 49µL serum free OptiMEM.
  4. Add diluted siRNA to the Lipofectamine.
  5. Incubate for 25 minutes.
  6. Add 400µL serum free OpitMEM.
  7. 500µL per well.
  8. Final concentration of siRNA per 24-well is 0.1µM.
  9. After 4 hours add 50µL FCS to give a final concentration of 10% per well.
  10. Culture for required length of time.