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.

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.