CALCIUM PHOSPATE TRANSFECTION OF HUMAN CELLS

DNA Preparati on
Use between 10-20 µg of DNA to transfect 3x106 cells. Q.S the desired amount of DNA in 220 µl of 0.1x TE . Add 250 µl 2x HEPES-buffered saline and vortex briefly to mix. Add 31 µl 2 M CaCl2 slowly over a 30 second period to the DNA solution. Incubate at room temperature for 30 minutes to form a precipitate.

Cell Preparation
Wash 3x106 cells once in PBS. Resuspend the cells in 0.5 ml PBS.

Transfection
Pipet the DNA solution once to reduce the size of any large clumps before transfering the DNA to the cells. Incubate the cells and DNA for 20 minutes at room temperature. Add 5.5 ml of complete medium, bringing the cells to a 6 ml volume. Dispense 2 ml of cells per well (1x106 ce lls/well; 0.5x106 cells/ml) in a Costar 24 well plate. Incubate at 37°C in a 5% CO2 incubator overnight.

The next day, collect the cells and wash once in PBS. Resuspend the cells in 6 ml medium and return to 3 wells of a new 24 well plate. There will still be a moderate amount of the precipitate on the surface of the cells. Selective reagents(G418, etc) may be added at this time. Return cells to the incubator. Feed and split the cells as needed.

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RECIPES


0.1x TE (1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA)
For 100 ml:
Transfer 0.1 ml of 1 M Tris-HCl, pH 8.0 to 9.9 ml of sterile water to make a 10 mM solution.
Transfer 0.1 ml of 0.5M EDTA to 9.9 ml of sterile water to make a 5 mM EDTA solution. Transfer 2 ml of this 5 mM EDTA solution to the 10 mM Tris-HCl solution.
Add 88 ml sterile water to bring the solution to 100 ml.
Filter sterilize and autoclave.


2x HEPES Buffered Saline (280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4, 12 mM dextrose, 50 mM HEPES, pH 7.5)


2 M Calcium Chloride
Dissolve 14.7 g CaCl2 in 100 ml H2O. Filter sterilize and autoclave.

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Proper citation for data acquired from this document is: "Frank, M. B. Calcium Phosphate Transfection of Human Cell Lines. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/transfrm.html). 1997. Oklahoma City. Revision Date: May 5, 2000."