General Transfection Procedure - Each Cell Line Must Be Optimized
- Recommended Lipofectamine (LF2K) to DNA ratio is 2μL : 1μg but can vary from 1:1 to 4:1.
- Make a solution of 2μL LF2K in 48μL Optimem (50μL total) for each dish transfected. Let sit for five minutes.
- 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.
- Combine DNA and LF2K solutions (now a total of 100μL) and let sit 25min at room temperature (can sit up to 6 hours).
- Meanwhile, split cells according to normal procedure. Make sure to resuspend the cells in Optimem following centrifugation!
- 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.
- Add the 100μL of the DNA/LF2K solution.
- Add excess normal Media (DMEM + 1% Pen/Strep + 10% FBS) 4-6 hours later.
- Change media the following day.
- 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
- 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.
- 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).
- Transfect a high confluency layer of cells and split in the next 24-48 hours into smaller dishes for microscopy.
- After transfecting leave in OptiMEM overnight.
- A list of alternative methodologies can be found here