Eccles:siRNA Transfection: Difference between revisions
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Back to [[Eccles Lab]] > [[Eccles:_DGG_Protocols | DGG Protocols]] | Back to [[Eccles Lab]] > [[Eccles:_DGG_Protocols | DGG Protocols]] | ||
==Resuspending siRNA’s== | |||
#Add 100µL sterile MQ per 5nmol powdered siRNA. | #Add 100µL sterile MQ per 5nmol powdered siRNA. | ||
#Gives a final concentration of 50µM siRNA in the tube. | #Gives a final concentration of 50µM siRNA in the tube. | ||
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== | ==siRNA Transfection== | ||
===For one 24-well=== | |||
#1µL Lipofectamine 2000 + 49µL serum free OptiMEM. | #1µL Lipofectamine 2000 + 49µL serum free OptiMEM. | ||
#ncubate for 5 minutes. | #ncubate for 5 minutes. | ||
Revision as of 21:03, 19 July 2006
Back to Eccles Lab > DGG Protocols
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.
- ncubate 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.
