What is calcium phosphate precipitation?

Calcium phosphate co-precipitation has been a popular transfection method since its introduction in the early 1970s [1] because it requires components that are easily available and inexpensive. The principle of calcium phosphate co-precipitation involves mixing DNA with calcium chloride in a buffered saline/phosphate solution to generate a calcium-phosphate-DNA co-precipitate, which is then dispersed onto cultured cells.

In the calcium phosphate precipitation method, the calcium phosphate facilitates the binding of the condensed DNA in the co-precipitate to the cell surface, and the DNA enters the cell by endocytosis. Aeration of the phosphate buffer while adding the DNA-calcium chloride solution helps ensure the precipitate that forms is as fine as possible, which is important because clumped DNA will not adhere to or enter the cell as efficiently.

Figure 1. Mechanisms of chemical transfection, including calcium phosphate precipitation. In calcium phosphate precipitation specifically, a calcium-phosphate-DNA co-precipitate is formed which facilitates binding to the cell surface and entry of the nucleic acid into the cell via endocytosis.

Basic steps for performing calcium phosphate co-precipitation

Mix DNA with calcium chloride and add in a controlled manner to a buffered saline/phosphate solution.

Incubate at room temperature to generate a precipitate of extremely small, insoluble particles containing condensed DNA.

Add the DNA-calcium phosphate co-precipitate to cells, which adhere to the cell membrane. The co-precipitate enters into the cytoplasm via endocytosis.

Assay cells for transient gene expression or select for stable transfection.

Step 1 : Mix DNA

Mix DNA with calcium chloride and add in a controlled manner to a buffered saline/phosphate solution.

Step 2 : Incubate

Incubate at room temperature to generate a precipitate of extremely small, insoluble particles containing condensed DNA.

Step 3 : Add the DNA-calcium phosphate

Add the DNA-calcium phosphate co-precipitate to cells, which adhere to the cell membrane. The co-precipitate enters into the cytoplasm via endocytosis.

Step 4 : Assay cells

Assay cells for transient gene expression or select for stable transfection.


Advantages and disadvantages of the calcium phosphate gene transfer method

The calcium phosphate precipitation method offers many advantages, including its simplicity and economical materials; however, there are several drawbacks to using this method as well, including its cytotoxicity and sensitivity to environmental factors (e.g., pH, temperature).

Advantages

  • Easily available and economical components
  • Simplistic technique
  • Effective in many cell types
  • Can be used for both transient and stable transfection

Disadvantages

  • Prone to variability due to sensitivity to slight changes in pH, temperature, and buffer salt concentrations
  • Potential issues with reproducibility
  • Cytotoxic to certain cells, including primary cells
  • Unsuitable for in vivo transfer of nucleic acids to whole animals
  • Cannot be used with RPMI media
  • Relatively poor transfection efficiency compared to other chemical transfection methods

Explore the Invitrogen Calcium Phosphate Transfection Kit

Visit Transfection Basics to learn more about performing transfection in your lab.

References

  1. Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. PMID: 4705382.

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