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RNAi在细胞培养中的应用
点击次数:649 更新时间:2016-01-25

I.6-Well PlatesA. BathingII.384-Well PlatesA. BathingB. Transfection
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. 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.

 
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