What factors affect transfection efficiency?

The four main factors that affect transfection efficiency include: 

1. Cell Health - To ensure cell viability, cells must be cultured in an appropriate medium supplemented with serum or growth factors. Using a medium lacking necessary factors can negatively impact cell growth. For efficient transfection, it’s just as important to avoid using contaminated cells or media, such as those contaminated with yeast or mycoplasma. If cells are compromised, it’s better to discard them and reseed from frozen, uncontaminated stock instead. 
2. DNA Quality and Quantity - Using plasmid DNA that is free from RNA, protein, and chemical and microbial contaminants is key to boosting transfection efficiency. The type of DNA, target cell line, transfection reagent, and number of cells will determine the optimal amount of DNA to use in the transfection.
3. Confluency - A general guideline for transfection is to use cells that are at 40-80% confluency. If too few cells are used, the limited contact between cells can prevent the culture from growing properly. If too many cells are used, the resultant contact inhibition causes the cells to become resistant to the uptake of foreign DNA.  
4. Number of Passages - For efficient transfection, it’s best to keep the number of passages low, ideally below 50. Maintaining consistency in the number of passages for cells used in various experiments is just as important. This is because cell characteristics can undergo changes over time and after repeated passages, cells may become unresponsive to the same transfection conditions. This will result in poor expression of the transfected gene.