Calcium phosphate transfection is a commonly used method for the introduction of DNA into eukaryotic cells. This technique has been used to obtain both transient1 and stable2 transfections in a wide variety of cell types. The procedure is based on slow mixing of HEPES-buffered saline containing sodium phosphate with a CaCl2 solution containing the DNA. A DNA−calcium phosphate co-precipitate forms, which adheres to the cell surface and is taken up by the cell, presumably by endocytosis. Glycerol shock may increase the uptake of DNA in some cell types.
The reagents supplied in the Calcium Phosphate Transfection Kit (Product No. CAPHOS) are sterilized by 0.2 µM filter and aseptically filled. The kit allows for either:
The Calcium Phosphate Transfection Kit (Product No. CAPHOS) contains the following:
Store all components at -20 °C.
Thaw and equilibrate all kit components to room temperature before use.
The procedure stated below is designed for the transfection of CHO cells with 1 µg/µL pSV40-CAT plasmid (diluted in sterile molecular biology grade water). Culture cells in standard serum-containing or serum-free medium appropriate for the cell type. Antibiotics are not recommended. Use good aseptic technique and use only sterile materials.
DNA plasmids should be high-quality, ethanol-precipitated, resuspended in molecular biology grade water to a final concentration of 1 µg/µL.
This protocol can be optimized for use with a wide variety of cell types. Seeding density, amount of DNA used, incubation time and glycerol shock can easily be varied to achieve higher expression and lower toxicity when needed.
Day One: Plate Cells
Plate the cells according to the following chart:
Day Two: Transfection
2. Two hours later, prepare two tubes with transfection reagents as follows:
3. Bubble the HeBS (tube B) using an automatic pipette pump attached to a 1 mL serological pipette fitted with a 200 uL pipette tip.
4. While bubbling the HeBS (tube B), add contents of tube A (from step 2), dropwise.
5. Vortex for 2 – 4 seconds.
6. Allow the precipitate to sit undisturbed for 20 minutes.
7. Drop the solution evenly over the cell culture medium on the plate. Gently agitate the dish to distribute the precipitates evenly over the cells on the plate.
8. Incubate cells overnight (approximately 16 hours).
Day Three: Optional Glycerol Shock
2. Remove medium from the dish and replace with glycerol solution. Incubate 2 minutes.
3. Remove glycerol solution and wash twice with PBS:
Day Three: Change Medium