Transformation and transfection are core techniques for introducing foreign DNA into cells. They are the foundation of recombinant protein production, gene therapy, functional genomics, and synthetic biology.

Bacterial Transformation

Transformation is the uptake of exogenous DNA by bacterial cells. Most laboratory strains of Escherichia coli are not naturally competent and must be made artificially competent.

Chemical transformation uses calcium chloride (CaCl₂) treatment to make the bacterial cell membrane permeable to DNA. The cells are incubated on ice with the DNA, heat-shocked at 42 °C for 45–90 seconds, then returned to ice. A brief outgrowth in non-selective medium allows expression of the antibiotic resistance marker before plating on selective agar. Chemical transformation typically yields 10⁶–10⁸ colony-forming units per microgram of plasmid DNA.

Electrocompetent cells are prepared by washing log-phase bacteria in ice-cold 10% glycerol to remove ions. A brief high-voltage pulse (1.5–2.5 kV) transiently porates the cell membrane, allowing DNA entry. Electroporation yields 10⁹–10¹⁰ CFU/µg for small plasmids and works with ligation products and larger constructs.

Transfection of Eukaryotic Cells

Transfection introduces DNA into mammalian or insect cells. The term distinguishes it from transformation, which refers to bacterial uptake.

• Lipid-mediated transfection: cationic lipids form liposomes that encapsulate the DNA and fuse with the cell membrane. This method is efficient for most adherent cell lines (HeLa, HEK 293) but is toxic to some primary cells.
• Calcium phosphate co-precipitation: DNA forms a precipitate with calcium phosphate that settles onto cells and is taken up by endocytosis. It is inexpensive but less efficient and requires careful pH control.
• Electroporation: as in bacteria, a brief electric pulse porates the membrane. It works for hard-to-transfect cells (primary cells, suspension cells) but causes significant cell death.
• Viral transduction: using modified lentivirus, retrovirus, or adeno-associated virus (AAV) to deliver genetic material. This is the most efficient method for primary cells and for stable integration into the genome.

Stable vs. Transient Transfection

Transient transfection produces expression for 2–7 days as the plasmid remains episomal. Stable transfection uses a selectable marker (e.g., puromycin, G418, hygromycin) to select cells that have integrated the DNA into their genome, producing permanent cell lines.