Mirus TransIT-LT1 Transfection Reagent Protocol

Divide cultured COS-7 cells 18-24 hours before transfection to reach optimal density of 50-70% confluence. Bring TransIT-LT1 reagent, DNA, and serum-free medium (OptiMEM) to room temperature, ensuring the reagent is clear and precipitate-free.

In a sterile tube, pipette 500 µL of serum-free OptiMEM medium, then add 5 µg of purified DNA and mix. Add 15 µL of TransIT-LT1 reagent, mix thoroughly, and incubate at room temperature for 20 minutes to allow complex formation.

Maintain the recommended 3:1 reagent-to-DNA ratio and ensure incubation time is between 15-30 minutes for consistent, reproducible results. Consistency in these parameters is critical for achieving optimal transfection efficiency.

Add the prepared transfection complex dropwise directly to cells in complete growth medium and gently mix by rocking the plate back and forth. Transfer the culture plate to a 37°C, 5% CO₂ incubator and incubate overnight until ready for harvest.