What is your Protocol for RNAi on Cell Cultures
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6-Well Plates
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Bathing
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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.
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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.
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Incubate 3 days and analyze.
Length of incubation may vary depending on assay.
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Prepare dsRNA suspended in water.
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Bathing
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384-Well Plates
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Bathing
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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.
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Incubate 3 days and analyze.
Length of incubation may vary depending on assay.
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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.
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Transfection
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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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Remove 384-well plates from freezer to thaw.
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Bathing
