Haynes:TransfectionPlasmid Lipo

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Mammalian Cell Transfection - Lipofectamine LTX Reagent

Karmella Haynes, 2012
Updated by Ben Nyer, 2014


  • plasmid DNA
  • Lipofectamine LTX
  • Opti-MEM I Reduced serum medium
  • Antibiotic-free growth medium (no pen/strep or other antibiotics)
  • Cells

Procedure - 6-well Format

1 day before transfection:

  1. Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection.
  • NOTE: if you are using a cell line that does not adhere strongly to the plate (eg, HEK cells), seed the cultures using antibiotic-free medium the day before to avoid having to replace medium the day of transfection.


  1. At your bench, bring 2.0 μg total plasmid DNA up to 20 μL total volume with fresh, sterile DNase-free water in fresh, sterile 1.5 mL tubes. Take the DNA samples to the tissue culture room.
  2. In the tissue culture room, warm antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep) at 37°C.
  3. Warm the Lipofectamine LTX vial, PLUS reagent vial, and and a 6 mL aliquot of Opti-MEM I Reduced Serum Medium to room temperature in the biosafety cabinet.
  4. Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
    1. Label sterile microfuge (1.5 ml) tubes.
    2. Add 570 μL Opti-MEM to each 20 μL DNA sample.
    3. Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
    4. Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
  5. Remove the 6-well plate containing your cultures from the incubator and place it in the biological safety cabinet.
  6. Aspirate off the old antibiotic-containing medium in the wells. Do NOT scrape the bottom of the wells, as the cells need to remain adherent to the plate.
  7. Add 4mL of warm antibiotic-free growth medium to each well.
  8. Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells.
  9. Incubate cells at 37°C in a CO2 incubator
  10. (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.

Transgene expression should be detectable after 18 hours.

For stable cells lines: Passage cells at a 1:10 (or higher, e.g. 1:20, 1:40, etc.) dilution into fresh antibiotic-free growth medium 48 hours after transfection. The next day (after 18 -24 hours later), add selective medium (based on the resistance marker for your plasmid). Grow the cells for 1 - 2 weeks until isolated colonies (about 100 cells per colony) appear. Transfer single colonies into 24-well plates. Grow the cells until they reach 100% confluency. Expand the cells in larger growth vessels (e.g., 6-well plates or 10 cm plates) for further testing.
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