Eccles: DGG Protocols: Difference between revisions
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'''siRNA Transfection''' | [['''siRNA Transfection''']] | ||
'''Resuspending siRNA’s:''' | |||
*Add 100µL sterile MQ per 5nmol powdered siRNA. | *Add 100µL sterile MQ per 5nmol powdered siRNA. | ||
| Line 9: | Line 9: | ||
'''siRNA Transfection:''' | |||
*For one 24-well. | *For one 24-well. | ||
Revision as of 16:26, 19 July 2006
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.
