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 |
6-Well Plates
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.
Bathing
384-Well Plates
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.
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.
Bathing
Transfection