The Best Practices of Transfection


Click here to return to the Molecular Biology Guide


The work of scientific research is an art which requires good attention to detail.  The quality of transfection is highest when these commonly used best practices and notes on optimization are employed. 

The Cells

  • Cells should be healthy, actively dividing, of relatively low passage number, and morphologically normal.
  • Cultures ready for transfection should be >70% confluent (if adherent), or about 5x105 – 2x106 cells/ml (if in suspension). 
  • Optimize the seeding density and count carefully.

The Medium

  • Transfect cells in their usual growth medium, whether it is serum-free or serum-supplemented. 
  • Avoid the use of antibiotics during plating and transfection. Enhanced permeability cause by some reagents can allow low levels of antibiotics to cause high cell death.
  • Don’t change the medium before adding transfection complexes. Many cells benefit from the growth factors in conditioned medium.

The DNA

  • High quality plasmid DNA should be purified using standard protocol or popular kits like Sigma’s GenElute Endotoxin-free Plasmid Purification Kits.
  • Ensure your DNA is pure by measuring the OD 260/280 ratio.  Good DNA scores 1.7 – 1.9.
  • Prepare the plasmid DNA at 1 µg/µL in DNase/RNase-free water or TE buffer and store frozen.

 

The Procedure

  • Do not form complexes in the presence of serum. Use serum-free medium or un-supplemented basal medium (DMEM or F-12 is recommended).
  • Do not complex the lipids for longer than the recommended time. Complexes form quickly!
  • Add complexes dropwise to the culture to ensure even distribution.
  • Use a GFP or LacZ reporter plasmid as a positive control to assess transfection efficiency.
  • For best transfection efficiency, run the optimization experiment.