Transfection is a powerful tool with a range of applications in molecular and cell biology, particularly in studying gene expression for drug discovery. Though there are many different transfection techniques, you can boost the reproducibility and reliability of your results by following these general practices:
Planning is paramount
Before you begin, familiarize yourself with the transfection protocol and make sure you have everything you need, including sufficient reagents, DNA and media.
Start with healthy cells
To give your transfection the highest chance of success, only use cells with over 90% viability. If your cells are frozen, make sure you passage them 3-4 times after thawing to give them time to recover and adjust to their normal rate of growth. If cells aren’t passaged regularly, they can overgrow, which can affect the outcome of transfection significantly.
Use high-quality DNA
In order to achieve reliable results, it’s crucial to use only the purest DNA. Purity can be measured by the OD 260/280 ratio, and should generally be between 1.7-1.8 – anything higher or lower could indicate contaminants and shouldn’t be used.
Plating your cells
Cell plating hugely impacts transfection efficiency, so it’s important to get this step right. Plate your cells no sooner than the day before you plan to transfect, aiming for 70-90% confluency at the time of transfection.
Prepare lipid DNA complexes
Lipid DNA complexes have a positively charged surface that aids the delivery of nucleic acids into cells. For optimal fusion, dilute both the lipid and DNA in medium separately, before adding them together to form your complex.
Use controls to assess efficiency
Following the procedure, you can measure the number of transfected cells against a positive control such as GFP or Lac7 reporter plasmid by microscopy or flow cytometry. Expression should be detectable approximately 24 to 48 hours following transfection.
The next time you use transfection in your research, we recommend incorporating the above practices into your workflow to make sure your transfected cells have optimal expression.
Looking for additional transfection tips? Find more valuable insights here.