Why is calcium phosphate used in transfection?

Calcium phosphate facilitates the binding of the DNA to the cell surface. DNA then enters the cell by endocytosis. The procedure is routinely used to transfect a wide variety of cell types for transient expression or for producing stable transformants.

What is calcium phosphate transfection?

Calcium phosphate transfection is a commonly used method for the introduction of DNA into eukaryotic cells. A DNA−calcium phosphate co-precipitate forms, which adheres to the cell surface and is taken up by the cell, presumably by endocytosis. Glycerol shock may increase the uptake of DNA in some cell types.

What is the DNA in calcium phosphate precipitation method?

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.

What is calcium phosphate method?

Abstract. The calcium phosphate transfection is a widely used method for introducing foreign DNA plasmids into cells. Mechanisms underlying this transfection method are not yet defined; however, DNA-calcium phosphate precipitates are internalized by the cells and DNA is efficiently expressed in almost all cell types.

How do you do calcium phosphate transfection?

The steps for transfection with Calcium Phophate are very straight forward:

  1. Generate DNA strand (circular DNA is much easier to introduce)
  2. Mix calcium phosphate with DNA and generate nanoparticulate precipitates.
  3. Incubate with cells.
  4. Select cells expressing the DNA of interest.

How does transfection reagent work?

Upon cellular uptake, transfection complexes are sequestrated into intracellular vesicles. Our transfection reagents are able to induce the release of the nucleic acids into the cytoplasm through vesicle membrane rupture or fusion.

What type of compound is calcium phosphate?

Calcium phosphate is a calcium salt of phosphoric acid with a chemical formula Ca3(PO4)2. It is also known as Calcium phosphate tribasic or Tricalcium Phosphate. Calcium phosphate appears as a white amorphous or crystalline powder that is odourless and tasteless.

How calcium phosphate is formed?

10.4. Calcium phosphates include various salts of tribasic phosphoric acid (H3PO4). H2PO4−, HPO42− or PO43− ions can all be formed through progressive removal of H+ ions from this acid. Their natural occurrence in skeletal tissues and teeth makes them of particular interest to both clinicians and biomedical scientists.

Does calcium chloride precipitate DNA?

Calcium chloride (CaCl2) is an inexpensive reagent. It is also known to precipitate RNA at the concentration of around 1 M, but DNA is not precipitated in this condition [8]. Thus, this reagent also works in the plasmid purification process like LiCl.

Is calcium phosphate basic?

Calcium phosphate is basic salt since it is a source of weak phosphoric acid and a slightly stronger base of calcium hydroxide.

How is calcium phosphate precipitate used in cell transfection?

A HEPES-buffered solution is used to form a calcium phosphate precipitate that is directly layered onto the cells. For some cells, shocking the cells with glycerol or DMSO improves transfection efficiency.

When was calcium phosphate co-precipitation first used?

Calcium phosphate co-precipitation has been a popular transfection method since its introduction in the early 1970s (Graham and van der Eb, 1973) because the components it requires are easily available and inexpensive.

Where does the precipitate go after the transfection?

This step generates a precipitate that is dispersed onto the cultured cells. The precipitate is taken up by the cells via endocytosis or phagocytosis.

How are transfection methods used in Bio-Rad?

Transfection Methods www.bio-rad.com/transfection Calcium Phosphate The protocol involves mixing DNA with calcium chloride, adding this in a controlled manner to a buffered saline/ phosphate solution, and allowing the mixture to incubate at room temperature. This step generates a precipitate that is dispersed onto the cultured cells.