The calcium phosphate method is a widely used technique for introducing foreign DNA into eukaryotic cells. This method, developed by Frank Graham and Peter van der Eb in the early 1970s, allows for the stable or transient expression of a desired gene within the target cells, making it an essential tool in molecular biology and genetic engineering.
The method works by forming a calcium phosphate-DNA precipitate, which is then taken up by the cells. Here are the basic steps involved in the calcium phosphate method:
- Preparation of DNA: The DNA of interest (plasmid, linear, or genomic) is isolated and purified. It is important to use high-quality DNA for efficient transfection.
- Formation of calcium phosphate-DNA complex: The DNA is mixed with a solution containing calcium chloride. A separate solution containing phosphate ions is then added dropwise to the DNA-calcium mixture. The calcium and phosphate ions react to form a fine precipitate of calcium phosphate, which binds to the DNA molecules.
- Incubation: The mixture is incubated at room temperature for 20-30 minutes to allow the calcium phosphate-DNA complexes to form completely.
- Cell treatment: The calcium phosphate-DNA precipitate is then added to the target cells, usually cultured in a monolayer in a tissue culture plate or flask. The cells take up the precipitate by endocytosis, a process in which the cell membrane engulfs and internalizes the particles.
- Incubation: The cells are incubated with the calcium phosphate-DNA precipitate for several hours to allow the DNA to be internalized and processed by the cells.
- Recovery and analysis: After a suitable incubation period, the cells are washed to remove any remaining calcium phosphate-DNA precipitate. Depending on the experiment, the transfected cells can be analyzed for gene expression, protein production, or other cellular responses.
The calcium phosphate method is a relatively simple and inexpensive technique for introducing DNA into cells. However, it has some limitations, such as a lower transfection efficiency compared to other methods (e.g., lipofection or electroporation) and sensitivity to the specific conditions of the cell culture. Despite these limitations, the calcium phosphate method remains an important tool in molecular biology research.