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Calcium Phosphate Transfection of Eukaryotic Cells   

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Transfection of DNA into cells is an indispensible protocol in molecular biology. While plenty of lipid-based transfection reagents are commercially available nowadays, a quick, simple, efficient and inexpensive method is to transfect eukaryotic cells via calcium phosphate co-precipitation with DNA (Graham and van der Eb, 1973). The insoluble calcium phosphate precipitate with the attached DNA adheres to the cell surface and is brought into the cells by endocytosis. Calcium phosphate transfection has been optimized and widely used with many adherent and nonadherent cell lines (Jordan et al., 1996). Calcium phosphate transfection can result in transient expression of the delivered DNA in the target cell, or establishment of stable cell lines (the latter requires a drug selection process). This protocol is also widely used for co-expression of plasmids for packaging viruses. Efficiency of transfection can be close to 100% depending on the cell lines used. Here, a calcium phosphate transfection protocol is described using a GFP plasmid and the HEK293 cell line.

Materials and Reagents

  1. HEK-293 cells (ATCC, catalog number: CRL-1573 ™)
  2. Eagle's minimum essential medium (ATCC, catalog number: 30-2003 ™)
  3. Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
  4. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
  5. HEPES (Sigma-Aldrich, catalog number: H4034 )
  6. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
  7. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S5136 )
  8. pEGFP-Actin plasmid (this is an example plasmid; Clontech, catalog number: PT3265-5 )
    Note: The pEGFP-Actin Vector expresses the EGFP-Actin fusion protein in mammalian cells; this protein is incorporated into growing actin filaments and allows for visualization of actin-containing subcellular structures in living and fixed cells.
  9. 2x HBS buffer (see Recipes)
  10. Solution-A (see Recipes)
  11. Solution-B (see Recipes)


  1. Tissue culture plates (e.g., 35 mm polystyrene)
  2. Cell culture incubator: 37 ºC and 5% CO2


  1. Prepare 2 M CaCl2 solution in water, filter sterilize and keep at room temperature.
  2. Prepare the 2x HBS buffer (see Recipes).
  3. Carry HEK-293 cells in Eagle's Minimum Essential Medium with 10% FBS.
  4. 24 h before transfection, trypsinize and reseed log-phase cells into 35 mm tissue culture dishes. For seeding density, cells should reach 60-70% confluence for transfection.
    Note: Best confluence for different cell lines differ. For HEK-293 cells, 60-70% confluence is suggested.
  5. 3 h before transfection, replenish cells with fresh medium.
  6. For each DNA transfection, prepare mixtures in two separate tubes (Solution-A and Solution-B, see Recipes)
  7. Add Solution-B slowly (drop-wise) into Solution-A while mixing Solution-A.
    Note: This is the most important step for forming DNA/calcium phosphate co-precipitate. Mix gently but thoroughly to allow formation of precipitates evenly.
  8. After mixing the two solutions, incubate at room temperature for 20-30 min (a shorter incubation time may be used for different cell types; please determined empirically). The solution will become opaque while precipitates being formed.
  9. Gently tap the mixture. Add the mixture directly to cells by dripping slowly and evenly into medium (a good way is to let tip touch medium surface).
  10. Gently tilt the plate back-and-forth a couple of times to allow even distribution of added precipitate on the cell surface.
  11. Incubate the cells at 37 ºC with 5% CO2 for 24 h and then replenish medium.


  1. GFP expression, if used as your positive, can be detected usually after 24-48 h of cell growth.
  2. Transfection efficiency (when using HEK293 cells and the above mentioned sample plasmid) can reach up to 90-100%.


  1. 2x HBS buffer
    50 mM   

    280 mM 

    1.5 mM   

    Adjust pH to 7.0 using HCl. Filter sterilize. The solution can be freezed/thawed once for future use.
  2. Solution-A
    100 μl 2x HBS
  3. Solution-B
    1-5 μg DNA (e.g., pEGFP-Actin plasmid as suggested above)
    12.2 μl of 2 M CaCl2
    ddH2O to bring volume up to 100 μl, pipet gently to mix
    Note: Titration of DNA should be carried out to obtain the best efficiency of transfection.


This protocol was developed in the Department of Immunology, Scripps Research Institute, La Jolla, CA, USA and adapted from Graham and van der Eb (1973) and Jordan et al. (1996). The work was funded by NIH grants CA079871 and CA114059, and Tobacco-Related Disease, Research Program of the University of California, 15RT-0104 to Dr. Jiing-Dwan Lee  [see Chen et al. (2009)].


  1. Chen, Y., Lu, B., Yang, Q., Fearns, C., Yates, J. R., 3rd and Lee, J. D. (2009). Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration. Cancer Res 69(8): 3713-3720.
  2. 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(2): 456-67.
  3. Jordan, M., Schallhorn, A. and Wurm, F. M. (1996). Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 24(4): 596-601.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Chen, Y. (2012). Calcium Phosphate Transfection of Eukaryotic Cells. Bio-101: e86. DOI: 10.21769/BioProtoc.86.
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If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

Venkatesh Padmanabhan
Vellore Institute of Technology - Vellore
Thank you for the protocol :)
3/18/2021 12:55:41 AM Reply
Harry Danwonno
University of Ghana
Thank you for the protocol.
Any preferred storage temperature for 2xHBS?
-20 or -80
11/19/2020 2:41:30 AM Reply
Thanks for protocol, It is cost effective with high efficiency. However, I have one concern regarding the cellular toxicity of this method. Is it low toxic than lipid-mediated transfection or shows similar toxicity? Please let me know.
1/17/2019 3:33:51 AM Reply
Neoletix Lab, Inc
Hi, Thank you for your protocol, I have a question: in step "9" you mix the two solutions at room temperature for 20-30 minutes. but I see some other protocols including your references saying 1 minute is enough. prolonged incubation would decrease the DNA binding efficient.
What's your suggestion? please confirm.
Thanks a lot.
7/21/2014 11:14:29 AM Reply
Yanling Chen
The Scripps Research Institute

The incubation time needs to be determined empirically for different cell types. Yes, a shorter incubation time can be used. Thanks.

7/25/2014 8:43:11 AM Reply

Thanks a lot
10/17/2012 9:38:23 AM Reply
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