NOTICE: You are viewing a page of the openwetware wiki. Our "dewikify" feature makes a wiki page appear as a normal web page. On September 1st 2017, this feature will GO AWAY and this URL will redirect to the source URL on our wiki. We're sorry for the inconvenience.

siRNA transfection

  1. ANKH siRNA transfection optimization ppt: Media:ANKH siRNA transfection optimize.pptx‎

Part 1: Ratio Optimization (variable based on cell type)

Materials:

  1. Lipfectamine 2000
  2. Opti-MEM I Reduced Serum Medium
  3. Stealth RNA/siRNA oligomers
  4. Stealth scramble siRNA
  5. BLOCK-it Fluorescent Oligo (ex=494nm, em=519nm) (positive control for transfection reagent)
  6. Fibronectin
  7. PBS (4C)
  8. Glass/Plastic Bottom Dishes
  1. Coat glass with 1mL 1:100 Fibronectin:PBS before seeding

*let sit for 1 hour before aspirating off extra

  1. Plate cells 24 hours prior to transfection to reach ~50-60% confluency

*need to balance time in culture following transfection with confluency at transfection (don’t want cells to grow to overconfluency but cells will die if transfection reagents are strong) *transfection may slow cell division

      For MC3T3s on glass slides in culture for multiple days past transfection:

Start with: *1 day: 325,000 cells/slide *2 days: 150,000 cells/slide *3 days: 80,000 cells/slide *4 days: 50,000 cells/slide

       For MC3T3s on tissue culture dishes in culture for multiple days past transfection:

Start with: *1 day: 400,000 cells/dish *2 days: 200,000 cells/dish *3 days: 100,000 cells/dish *4 days: 50,000 cells/dish

Follow: http://iccb.med.harvard.edu/screening/RNAi%20Libraries/Lipid%20Panel/16%20-%20Invitrogen%20Lipofectamine2000.pdf

*use the best ratio

Part 2: Verify Knock-down

*lyse the cells using RIPA buffer and run a BCA then Western blot to probe for the knockdown of your target protein. *make sure to use perform transfection in duplicate with negative (scramble siRNA)

       *Western blot should include both negative and positive controls (untransfected cell protein lysate)