The protocols listed here are for Drosophila cells in 6 well plates and our pre-aliquoted 384 well plates. RNAi experiments may be done in other size plates, just scale up or down. Adopted from Clemens et al., PNAS 2000 vol 97(12): 6499-6503 I. 6-Well Plates A. Bathing II. 384-Well Plates A. Bathing B. Transfection Ⅰ 6-Well Plates A. Bathing 1.Prepare dsRNA suspended in water. We use ~500 bp dsRNA. 2.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. 3.Count cells, then spin to pellet (~1200 rpm, 5'). 4.Resuspend cells at 1-5 x 106 cells/ml in serum free media. 5.Plate 1 ml cells into wells of 6-well plate. It doesn't seem to matter if dsRNA or cells are added first. 6.Incubate dsRNA with cells at RT for 30'. 7.Add 3 ml complete media with 10% FBS to each well. 8.Incubate 3 days and analyze. Length of incubation may vary depending on assay. Ⅱ. 384-Well Plates A. Bathing 1.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. 2.Spin plates at ~1200 rpm for 1'. before removing seals. 3.Count cells, then spin to pellet (~1200 rpm, 5'). 4.Resuspend cells at 1-5 x 106 cells/ml in serum free media. 5.Plate 10 μl cells into wells of 384-well plate. 6.Incubate dsRNA with cells at RT for 30'. 7.Add 30 μl of complete media to each well. 8.Seal the plates to prevent evaporation. 9.Incubate 3 days and analyze. Length of incubation may vary depending on assay. B. Transfection 1.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. 2.Spin plates at ~1200 rpm for 1'. before removing seals. 3.Count cells, then spin to pellet (~1200 rpm, 5'). 4.Resuspend cells at 1-5 x 106 cells/ml in serum free media. 5.Plate 10 μl cells into wells of 384-well plate. 6.Incubate dsRNA with cells at RT for 30'. 7.Add 30 μl of complete media to each well. 8.Seal the plates to prevent evaporation. 9.Incubate 3 days and analyze. Length of incubation may vary depending on assay.
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