Pelling:Protocols/Transient Transfections

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General Transfection Procedure - Each Cell Line Must Be Optimized

  1. Recommended Lipofectamine (LF2K) to DNA ratio is 2μL : 1μg but can vary from 1:1 to 4:1.
  2. Make a solution of 2μL LF2K in 48μL Optimem (50μL total) for each dish transfected. Let sit for five minutes.
  3. Make solutions of 1μg DNA in Optimem to a final volume of 50μL. The volume of DNA added depends on the concentration measured with UV.
  4. Combine DNA and LF2K solutions (now a total of 100μL) and let sit 25min at room temperature (can sit up to 6 hours).
  5. Meanwhile, split cells according to normal procedure. Make sure to resuspend the cells in Optimem following centrifugation!
  6. In small dish (35mm or 6-well plate), place minimum amount of Optimem medium (probably about 1-1.5mL) and cells. Lets cells sit and adhere for about an hour in the incubator. Cell density does not generally effect results.
  7. Add the 100μL of the DNA/LF2K solution.
  8. Add excess normal Media (DMEM + 1% Pen/Strep + 10% FBS) 4-6 hours later.
  9. Change media the following day.
  10. Image cells 24-48 hours after transfection.

Optimem is not absolutely necessary, some cells are fine with your regular DMEM

Things That Can Be Optimized

  1. Vary the LF2K:DNA ratio. However 1μg DNA is usually best, adding more DNA usually results in more cell death rather than higher transfection efficiency.
  2. Plate cells the day before transfection or add LF2K:DNA solution directly to a fresh suspension of cells (ie. freshly split, before they adhere to the dish).
  3. Transfect a high confluency layer of cells and split in the next 24-48 hours into smaller dishes for microscopy.
  4. After transfecting leave in OptiMEM overnight.
  5. A list of alternative methodologies can be found here