Calcium Phosphate Transfection

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

1 Massachusetts General Hospital  and Harvard Medical School, Boston, Massachusetts, 2 National Institute of Mental Health, Bethesda, Maryland, 3 Osaka University, Osaka, Japan
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.13
DOI:  10.1002/0471142735.im1013s31
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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 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 . Transfection efficiency in some cell lines can be increased by shocking the cells with glycerol or dimethyl sulfoxide (DMSO) as described in the first .

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Transfection and Expression of cloned DNA
  • Basic Protocol 1: Transfection Using Calcium Phosphate–DNA Precipitate Formed in HEPES
  • Support Protocol 1: Glycerol/DMSO Shock of Mammalian Cells
  • Alternate Protocol 1: High‐Efficiency Transfection Using Calcium Phosphate–DNA Precipitate Formed in BES
  • Support Protocol 2: CsCl Plasmid DNA Purification
  • Reagents and Solutions
  • Commentary
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: 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)
  • protocol 4CsCl‐purified plasmid DNA (10 to 50 µg per transfection, see protocol 4support protocol)
  • recipe2.5 M CaCl 2
  • recipe2× 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 1: 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 1: High‐Efficiency Transfection Using Calcium Phosphate–DNA Precipitate Formed in BES

  Additional Materials
  • TE buffer ( appendix 2A)
  • recipe2× BES‐buffered solution (BBS)
  • Humidified 35°C, 3% CO 2 incubator with Fyrite gas analyzer (Fisher or Curtin Matheson)
  • Humidified 35° to 37°C, 5% CO 2 incubator

Support Protocol 2: CsCl Plasmid DNA Purification

  Additional Materials
  • Plasmid‐bearing E. coli strain
  • 10 mg/ml chloramphenicol
  • recipeSucrose/Tris/EDTA solution
  • 10 mg/ml chicken egg white lysozyme, prepared fresh in sucrose/Tris/EDTA solution (Sigma)
  • recipeTriton 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
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library