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Transfection of DNA into cell is an indispensible protocol in molecular biology. While plenty of lipid-based transfection reagents are commercially available, a quick, simple, efficient and inexpensive method is to transfect eukaryotic cells via Calcium Phosphate co-precipitation with DNA (1). The insoluble Calcium phosphate precipitate, together with attached DNA, adheres to the cell surface and both are brought into the cells by endocytosis. Calcium phosphate transfection has been optimized and widely used in many adherent and nonadherent cell lines (2). Delivering of DNA into the target cells can be transient or for establishing stable cell lines; besides, this protocol is widely used for packaging virus. Efficiency of transfection can be close to 100% depending on different cell lines. Here an example protocol of CaPO4 transfection is described using a GFP plasmid and the HEK293 cell.
Materials, reagents and equipments
1. HEK-293 cells (ATCC # CRL-1573)
2. Eagle's Minimum Essential Medium (ATCC #30-2003)
3. Fetal Bovine Serum (ATCC #30-2020)
4. Calcium chloride, CaCl2 (Sigma-Aldrich #C5670)
5. HEPES (Sigma-Aldrich #H4034)
6. Sodium chloride, NaCl (Sigma-Aldrich #S5886)
7. Sodium phosphate dibasic, Na2HPO4 (Sigma-Aldrich #S5136)
8. Tissue culture plates (e.g., 35mm polystyrene)
9. Cell culture incubator: 37 oC and 5% CO2
10. pEGFP-Actin plasmid (This is an example plasmid; Clontech #PT3265-5)
Company 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. http://www.clontech.com/images/pt/dis_vectors/PT3265-5.pdf
1. Prepare 2 M CaCl2 solution in water, filter sterilize and keep at room temperature.
2. Prepare the 2x HBS buffer consists of:
50 mM HEPES
280 mM NaCl
1.5 mM Na2HPO4
Adjust pH to 7.0 using HCl. Filter sterilize. The solution can be freeze/thaw once for future use.
3. Carry HEK-293 cells in Eagle's Minimum Essential Medium with 10% FBS.
4. 24 hours before transfection, trypsinize and reseed log-phase cells into 35 mm tissue culture dishes. Seeding density should be that the cells 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 hours before transfection, replenish cells with fresh medium.
6. For each DNA transfection, prepare following mixtures in two separate tubes:
Solution-A: 100 μl 2x HBS
Solution-B: 1-5 ug 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.
7. Add Solution-B slowly (drop-wise) into Solution-A while mixing Solution-A.
8. Note: This is the most important step for forming DNA/Calcium phosphate co-precipitate. Mix gently but thoroughly to allow formation of precipitates evenly.
9. After mixing the two solutions, incubate at room temperature for 20-30 minutes. The solution will become opaque while precipitates form.
10. 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).
11. Gently tilt the plate back and forth a couple of times to allow evenly distribution of added precipitate on cell surface.
12. Incubate the cells at 37 oC with 5% CO2 for 24 hours, replenish medium.
Note: a. GFP expression will be very obvious after 24-48 hours of cell growth.
b. Efficiency can reach up to 90~100%.
1. Graham F.L., van der Eb A.J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52(2): 456-67.
2. Jordan M., Schallhorn A., 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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