Unit

UNIT 9.1 Calcium Phosphate Transfection

  1. Robert E. Kingston (HEPES method)1,
  2. Claudia A. Chen (BES method)2,3,
  3. John K. Rose (optimization)4

Published Online: 1 AUG 2003

DOI: 10.1002/0471142727.mb0901s63

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Kingston, R. E., Chen, C. A. and Rose, J. K. 2003. Calcium Phosphate Transfection. Current Protocols in Molecular Biology. 63:I:9.1:9.1.1–9.1.11.

Author Information

  1. 1

    Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts

  2. 2

    National Institute of Mental Health, Bethesda, Maryland

  3. 3

    (BES method) Osaka University, Osaka, Japan

  4. 4

    Yale University School of Medicine, New Haven, Connecticut

Publication History

  1. Published Online: 1 AUG 2003
  2. Published Print: JUL 2003

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.