Eccles: DGG Protocols: Difference between revisions

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[['''siRNA Transfection''']]
*[[Eccles:siRNA_Transfection | 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.

Revision as of 16:35, 19 July 2006

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