Silver: miRNA screens
From OpenWetWare
Jump to navigationJump to search
- MiRNA are arrayed in 96-well plate and stored at –20C. Transfection is performed with siPORT-NeoFx reagent (Ambion #4510) in OPTI-MEM.
- OPTI-MEM and NeoFX should be at room temperature at time of transfection.
- For each transfection mix 0.5 microliter NeoFX with 29.5 µl OPTI-MEM. Incubate at room temperature for 10 minutes. Make master mix for large-scale transfection.
- Add 30 microliter of NeoFX/OPTI-MEM mix to each well containing lyophilized miRNA. Incubate at room temperature for 10 minutes.
- In the mean time, trypsinize cells and dilute them to ~ 85,000 cells/ml in DMEM/10% FBS/Pen-Strep (this will give you about 6000 cells per well at time of plating).
- Add 70 µl of the cell suspension (equivalent to 6000 cells) to miRNA/NeoFX/OPTI-MEM mixture.
- Mix and transfer to flat bottom 96-well Corning plate (#3614).
- Allow transfection to proceed for 48 hours.
- Screen cells using assay of choice.
- For further information on NeoFX consult http://www.ambion.com
