Eccles: DGG Protocols: Difference between revisions
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siRNA Transfection | siRNA Transfection | ||
Resuspending siRNA’s: | *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: | *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 16:17, 19 July 2006
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.
