Calcium Phosphate Transfection
Calcium Phosphate Transfection
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DOI：
10.1002/0471142301.nsa01cs01
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发表时间：
1997-11
影响因子：
--
通讯作者：
R. Kingston;Claudia A. Chen;H. Okayama
中科院分区：
文献类型：
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作者：
R. Kingston;Claudia A. Chen;H. Okayama
本单元介绍了两种基于磷酸钙的真核细胞转染方法，可用于瞬时和稳定转染。在这些方案中，质粒DNA通过粘附于细胞表面的沉淀物引入单层细胞培养物。使用HEPES缓冲溶液形成磷酸钙沉淀，直接在细胞上分层。对于一些细胞，用甘油或DMSO冲击细胞提高转染效率。在另一种高效方法中，使用BES缓冲系统，使沉淀物在培养基中逐渐形成，然后滴到细胞上。虽然替代方法对于用环状质粒DNA稳定转化细胞特别有效，但是对于用线性质粒或基因组DNA转化或对于瞬时表达，两种方案都产生类似的结果。
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.