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Calcium Phosphate Transfection

Robert E. Kingston¹, Claudia A. Chen², Hiroto Okayama³

¹Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
²National Institute of Mental Health, Bethesda, Maryland
³Osaka University, Osaka, Japan, Japan

Publication Name:

Current Protocols in Immunology

Unit Number:

Unit 10.13

DOI:

10.1002/0471142735.im1013s31

Online Posting Date:

May, 2001

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Abstract

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.

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Transfection and Expression of cloned DNA
Unit Introduction
Basic Protocol: Transfection Using Calcium Phosphate–DNA Precipitate Formed in HEPES
Support Protocol: Glycerol/DMSO Shock of Mammalian Cells
Alternate Protocol: High-Efficiency Transfection Using Calcium Phosphate–DNA Precipitate Formed in BES
Support Protocol: CsCl Plasmid DNA Purification
Reagents and Solutions
Commentary
Bibliography

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Materials

Basic Protocol: Transfection Using Calcium Phosphate–DNA Precipitate Formed in HEPES

Materials

Exponentially growing eukaryotic cells (e.g., HeLa, BALB/c 3T3, NIH 3T3, CHO, or rat embryo fibroblasts)
Complete medium (appropriate for the cell line used)
CsCl-purified plasmid DNA (10 to 50 µg per transfection, see support protocol)
2.5 M CaCl₂
2× HEPES-buffered saline (HeBS)
Phosphate-buffered saline (PBS; appendix 2A)

10-cm tissue culture dishes
15-ml conical tube, sterile

Additional reagents and equipment for ethanol precipitation (unit 10.1)

Support Protocol: Glycerol/DMSO Shock of Mammalian Cells

Additional Materials

Sterile 10% (v/v) glycerol or 10% or 20% DMSO (v/v) in complete medium

Alternate Protocol: High-Efficiency Transfection Using Calcium Phosphate–DNA Precipitate Formed in BES

Additional Materials

TE buffer (appendix 2A)
2× BES-buffered solution (BBS)

Humidified 35°C, 3% CO₂ incubator with Fyrite gas analyzer (Fisher or Curtin Matheson)
Humidified 35° to 37°C, 5% CO₂ incubator

Support Protocol: CsCl Plasmid DNA Purification

Additional Materials

Plasmid-bearing E. coli strain
10 mg/ml chloramphenicol
Sucrose/Tris/EDTA solution
10 mg/ml chicken egg white lysozyme, prepared fresh in sucrose/Tris/EDTA solution (Sigma)
Triton lytic mix
Cesium chloride
10 mg/ml ethidium bromide
CsCl/TE solution (with and without 0.2 mg/ml ethidium bromide; unit 10.3)
1:1 (v/v) phenol/chloroform
100% ethanol 0.5% (v/v) SDS in TE buffer

37°C water bath
Beckman JS-4.2, type 35, VTI65, and Sorvall SS-34 rotors (or equivalents)
Beckman 1-liter centrifuge bottles
5-ml quick-seal ultracentrifuge tubes

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Literature Cited

Literature Cited
	Graham, F.L. and van der Eb, A.J. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456.
	Ishiura, M., Hirose, S., Uchida, T., Hamada, Y., Suzuki, Y., and Okada, Y. 1982. Phage particle–mediated gene transfer to cultured mammalian cells. Mol. Cell. Biol. 2:607-616.
	Wigler, M., Pellicer, A., Silverstein, S., and Axel, R. 1978. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell 14:725.
Key References
	Chen, C. and Okayama, H. 1987. High efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:2745-2752.
	Chen, C. and Okayama, H. 1988. Calcium phosphate–mediated gene transfer: A highly efficient system for stably transforming cells with plasmid DNA. BioTechniques 6:632-638.
	Ishiura et al., 1982. See above.