Transfection Best Practices

Successful transfection of nucleic acids and proteins depends on numerous factors that range from reagent and cell type selection to protocol optimization. Ensure optimal quality in your experiment by checking the following list of commonly used best practices for performing transfection. Getting back to basics? View our Introduction to Cell Transfection to learn the fundamentals of this method and explore additional resources.

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, such as our 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.

Need help getting started? View our Transfection Reagent Selection Guide to find the right product for you.

Click here to return to the Molecular Biology Guide

Materials