What is your Protocol for RNAi on Cell Cultures?
2004-8-3 8:48:00 信息来源：本站原创
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What is your Protocol for RNAi on Cell Cultures?
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- 6-Well Plates
- Bathing
- Prepare dsRNA suspended in water.
We use ~500 bp dsRNA.
- Add ~10-30 µg dsRNA to wells of 6-well tissue culture plate.
We use 0.1-0.3 µg in 384-well plates for 25-50 nM final concentration.
- Count cells, then spin to pellet (~1200 rpm, 5').
- Resuspend cells at 1-5 x 106 cells/ml in serum free media.
- Plate 1 ml cells into wells of 6-well plate.
It doesn't seem to matter if dsRNA or cells are added first.
- Incubate dsRNA with cells at RT for 30'.
- Add 3 ml complete media with 10% FBS to each well.
- Incubate 3 days and analyze.
Length of incubation may vary depending on assay.
- 384-Well Plates
- Bathing
- Remove 384-well plates pre-aliquoted with dsRNA from freezer to thaw. The 384-well plates contain 5ul of ~0.05ug/ul dsRNA in water for ~0.25ug dsRNA/well.
The dsRNAs are ~500 bp.
- Spin plates at ~1200 rpm for 1'. before removing seals.
- Count cells, then spin to pellet (~1200 rpm, 5').
- Resuspend cells at 1-5 x 106 cells/ml in serum free media.
- Plate 10 ul cells into wells of 384-well plate.
- Incubate dsRNA with cells at RT for 30'.
- Add 30 ul of complete media to each well.
- Seal the plates to prevent evaporation.
- Incubate 3 days and analyze.
Length of incubation may vary depending on assay.
- Transfection
- Remove 384-well plates from freezer to thaw.
The 384-well plates contain 5ul of ~0.016ug/ul dsRNA in water for ~0.08ug dsRNA/well.
The dsRNAs are ~500 bp.
- Spin plates at ~1200 rpm for 1'. before removing seals.
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