Calcium phosphate transfection.
This unit contains two methods of calcium phosphate-based eukaryotic cell transfection, protocols that can be used for both transient and stable transfections. In the protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. The Basic Protocol uses a HEPES-buffered solution to form a calcium phosphate precipitate that is directly layered onto the cells. In the alternate high-efficiency method, a BES-buffered system is used that allows the precipitate to form gradually in the medium and is then dropped onto the cells. The alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, and may be helpful with linear or genomic DNA. Both methods of transfection require very high-quality plasmid DNA, which can be prepared as described in the second Support Protocol. Transfection efficiency in some cell lines can be increased by shocking the cells with glycerol or dimethyl sulfoxide (DMSO) as described in the first Support Protocol.
Kingston
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Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Chen
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Okayama
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H
D000818:Animals; D002130:Calcium Phosphates / Q000737:chemistry*; D002478:Cells, Cultured; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D009997:Osmotic Pressure; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics; D014162:Transfection / Q000379:methods*
D002130:Calcium Phosphates; C020243:calcium phosphate; D005990:Glycerol; D004121:Dimethyl Sulfoxide