Haynes:TransfectionPlasmid Lipo

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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

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. Use 1.0 mL antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep) per well.

Transfection:

1. Warm plasmid DNA, Lipofectamine LTX, PLUS reagent, and Opti-MEM I Reduced Serum Medium to room temperature in the biosafety cabinet.
2. Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
   1. Label sterile microfuge (1.5 ml) tubes.
   2. For each transfection, bring 2.0 μg plasmid DNA up to 20 μL total volume with sterile DNase-free water. Take the DNA samples to the biosafety cabinet.
   3. Add 570 μL Opti-MEM to each 20 μL DNA sample.
   4. Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
   5. Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
3. Add DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each well of cells.
4. Incubate cells at 37°C in a CO₂ incubator
5. (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 24 hours.

For stable cells lines: Passage cells at a 1:10 (or higher) dilution into fresh growth medium 24 hours after transfection. Add selective medium the following day. Grow until isolated colonies appear. Transfer single colonies into 24-well plates. Expand the cells for further testing.