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热门关键字：　细胞培养　 pcr 　琼脂糖凝胶电泳　质粒DNA的提取　凝胶加样缓冲液　 PCR inventor

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

6-Well Plates
1. 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.
384-Well Plates
1. 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 ul cells into wells of 384-well plate.
  6. Incubate dsRNA with cells at RT for 30'.
  7. Add 30 ul 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.
2. 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 ul cells into wells of 384-well plate.
  6. Incubate dsRNA with cells at RT for 30'.
  7. Add 30 ul 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.

上一篇：Rules of siRNA design for RNA&n 下一篇：RNAi实验中双链短RNA（dsRNA）制备过程

RNAi在细胞培养中的应用

点击： 作者： 来源： 时间： 2006-11-07 　本站论坛

The protocols listed here are for Drosophila cells in 6 well plates and our pre-aliquoted 384 well plates. 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

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