Haynes:TransfectionPlasmid Lipo

<- Back to Protocols

Mammalian Cell Transfection - Lipofectamine LTX Reagent

Karmella Haynes, 2012

Materials

• 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.

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. Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells.
6. Incubate cells at 37°C in a CO₂ incubator
7. (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.