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.