SiRNA Transfection
Reagents:
Lipofectamine 2000 (Invitrogen, Cat# 11668-027) is for plamid DNA transfection.
Lipofectamine RNAiMAX (Invitrogene, Cat# 13778-075 or 13778-150) is proprietary formulation specifically developed for the transfection of siRNA and Stealth RNAi duplexes into eukaryotic cells. High transfection efficiencies, minimal cytotoxicity, requiring low concentration of RNAi duplexes, broad peak of optimal transfection activity.
Oligofectamine (Invitrogen Cat# 12252-011) is proprietary formulation for the transfection of oligonucleotides and short interfering RNA (siRNA) into eukaryotic cells.
Transfect cells at 30-50% confluence,
Procedures:
1. Plate cells in a 6-well plate at a density of 3x10^5 cells/well.
2. Prepare siRNA： Stock siRNA concentration： 20 uM (20 nmol siRNA in 1 ml Rnase-free water).
3. Before cells attach, transfect cells with siRNA using Lipofectamine 2000 or Oligofectamine:
10 ul c-myc siRNA (synthesis, 20 pmol/ul)/250 ul OptiMen
10 ul Oligofectamine or Lipofectamine 2000/250 ul OptiMen
(siRNA final concentration: 100nM in 2 ml media)
15 ul c-myc siRNA (synthesis, 20 pmol/ul)/250 ul Opti-Men
10 ul Oligofectamine or Lipofectamine 2000/250 ul OptiMen
(siRNA final concentration: 150nM in 2 ml media)
4. Change media after overnight incubation. Incubate for another 12 hours.
5. Transfect cells again. Incubate for 24 hours.
6. Harvest protein or RNA.
