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siRNA Transfection

siRNA Transfection Protocol

 siRNA transfection protocol: Transfect 6 well plates

  1. apply first siRNA treatment when cells are 60% confluent (measure confluency with voltmeter); plate 1.5x10^6 cells on Friday, transfect on Wednesday. For each well:
  1. Dilute 2.5 ug siRNA (20uM is approximately 0.26ug/ul. 9.62 ul per well of a 6-well plate) with culture media to a final volume of 100 ul. Use barrier pipette tips. You can make a master mix if all the wells will be transfected with the same siRNA. Do this in a sterile autoclaved plastic tube.
  2. Add 12 ul RNAiFect (Qiagen - or any other RNA transfection reagent, see manufacturer's protocol) per siRNA mastermix tube and mix gently.
  3. Incubate for 15 minutes at room temperature to allow complex formation between siRNA and lipids.
  4. Aspirate media from cells and add 900ul fresh media to each well.
  5. Add siRNA mixture (112 ul per well) drop-wise while gently swirling plate.
  • Apply second siRNA treatment 2 days later, following the steps outlined above. The second transfection will add to decrease expression of the gene of interest.

Other siRNA protocols

Also see our siRNA and RNAi page


 

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