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Calcium phosphate–mediated transfection of eukaryotic cells

Nature Methods volume 2, pages 319320 (2005) | Download Citation

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The uptake of DNA is markedly enhanced when the nucleic acid is presented as a coprecipitate of calcium phosphate and DNA1. The insoluble precipitate attaches to the cell surface and is brought into the cells by endocytosis. Since the publication of the original method1, increases in efficiency have been achieved by including additional steps such as glycerol shock2 and/or chloroquine treatment3. This protocol is a modified version of a published method4, in which calcium phosphate–based transfection methods for Chinese hamster ovary cells and the 293 line of human embryonic kidney cells were rigorously optimized. The protocol is easily adapted for use with other types of cells, both adherent and nonadherent.

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References

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    & A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456–467 (1973).

  2. 2.

    & Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J. Virol. 31, 360–369 (1979).

  3. 3.

    & High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res. 11, 1295–1308 (1983).

  4. 4.

    , & Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 24, 596–601 (1996).

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https://doi.org/10.1038/nmeth0405-319

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