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Calcium Phosphate Transfection

2003; Wiley; Volume: 63; Issue: 1 Linguagem: Inglês

10.1002/0471142727.mb0901s63

1934-3639

Robert E. Kingston, Claudia A. Chen, John K. Rose,

RNA Interference and Gene Delivery

Abstract This unit presents two methods of calcium phosphate‐based eukaryotic cell transfection that can be used for both transient and stable transfections. In these protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. 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. In the alternate high‐efficiency method, a BES‐buffered system is used that allows the precipitate to form gradually in the medium and then drop onto the cells. While the alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, both protocols yield similar results for transformation with linear plasmid or genomic DNA, or for transient expression.