The Keys to Successful Transfection

Transfection, like many techniques in the lab, can be an art, especially for stubborn cell lines. To maximize transfection efficiency, you must have the right conditions and, for some cell lines it seems, the right touch. If you’re lucky, you are working with less finicky cells that transfect well regardless of conditions. If you’re not so lucky, you might want to spend a few minutes reading through this list of ways that you can improve your transfection results.

  1. Optimize the amount of DNA added to the cells.
    The amount of DNA per well of cells depends on the type of DNA and cell line and often requires optimization. For 60mm dishes, 1–10μg plasmid DNA is a good initial range. For different size plates, scale the amount of DNA in proportion to the relative growth area.
  2. Optimize the amount of transfection reagents (e.g., charge ratio).
    The ratio of transfection reagent:DNA often requires optimization and varies with the cell line transfected. For lipid-based reagents, the amount of positive charge contributed by the cationic lipid component should equal or exceed the negative charge contributed by the DNA. Ratios of 1.5:1 to 4:1 reagent:DNA work well with many cell lines, but ratios outside of this range (4.5:1 to 6:1) may be optimal for some cell types or applications.
  3. Be sure that the DNA is transfection-grade.
    Some cell lines are particularly sensitive to contaminants found in DNA preparations, such as endotoxins, salt and ethanol, leading to reduced transfection efficiency or cell viability. For best results, the purified DNA should be free of such contaminants.
  4. Optimize the transfection time.
    The transfection reagent:DNA complex is often left in contact with the cells for 30 minutes to 4 hours or even overnight before the medium is replaced. For some reagents, the medium does not need to be replaced after transfection. Be sure to monitor cell morphology during the transfection interval, particularly when cells are maintained in serum-free medium, as some cells will lose viability. Shorter transfection times may significantly reduce the risk of cell death.
  5. Determine the effect of serum in the transfection.
    Some reagents tolerate the presence of serum; some do not. For cells lines that require continuous exposure to serum for viability, use a reagent that tolerates serum to maintain cell health.
  6. Optimize the number of cells per well.
    The plating density for a specific cell line will depend on the growth rate. Adherent cells should be at 50–80% confluency the day of transfection. A general guideline is to plate about 5 × 105 adherent cells per 60mm culture dish or 106 suspension cells per assay. Scale the number of cells up or down proportionately if using different size plates.
  7. Calibrate the system using a test plasmid with reporter gene function. An ideal reporter gene product is not endogenous to the cell, can be expressed from a plasmid DNA and is usually an enzyme that can be assayed conveniently. Commonly used reporter genes are luciferase, chloramphenicol acetyltransferase and β-galactosidase.
  8. For calcium phosphate-mediated transfection, be sure the pH of the HEPES-buffered saline (HBS) solution is 7.05–7.1.
    If the pH is too high, the DNA:calcium phosphate precipitate will be too coarse, and if the pH is too low, the precipitate will not form; in both cases, the transfection efficiency will be reduced.
  9. Be sure that cells are healthy, actively growing and uncontaminated with Mycoplasma or bacteria prior to transfection. When possible, use cells that have a low passage number.

For more detailed information about transfection optimization, visit the Transfection chapter of the Protocols and Applications Guide.

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Terri Sundquist

Terri has worked as a Scientific Communications Specialist at Promega Corporation for more than 13 years, and prior to that, spent more than 5 years solving problems and answering questions as a Promega Technical Services Scientist. She graduated with B.S. degrees in Chemistry and Biology at the University of Wisconsin—River Falls, then earned her M.S. in Molecular Biology from the Mayo Graduate School in Rochester Minnesota.

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