Calcium Phosphate Transfection of HeLa cells
(in Promega protocols, pp241)
1. Plate the cells the day before the transfection experiment such that they are approximately 80% confluent by the time they are transfected (one 6 well plate (total) corresponds to one 10 cm dish in terms of surface area. Split HeLa 1:8 to 1:12, or 2 x 105 cells per 6 well). Cells should not be clumpy. To avoid this the trypsinised cells may be pelleted for 5 min at 1500 rpm and resuspended before plating (although MC is sceptical of the efficacy of this - its best avoided by following good cell culture practice beforehand).
2. 1-3 hours before transfection, replace the cell medium with fresh medium. Thaw one aliquot 2xHBS (-20°C freezer in TC) and place together with sterile water and sterile 1MCaCl2 (fridge door) at room temperature in the TC room.
3. Use between 2 and 5 µg DNA per well of a six-well dish, or 10-20 µg per 100 mm dish. Prepare the DNA and DNA- Calcium solutions in sterile 1.5 ml microfuge tubes.
(Optional: Ethanol precipitate the amount of DNA required for each set of transfections. For convenience dilute DNA first to 100 µl before precipitating. Add 1/10 3M NaOAc pH 5.2 (sterile), mix and 3 volumes of 100% Ethanol (-20°C). Place at -20°C for 20 min (minimum, or overnight in fridge) then spin in the coldroom at max speed. Remove the supernatant carefully without disturbing the pellet. Add 200 ul 70% ethanol (-20°C) without disturbing the pellet, spin for another 5 min (can be at RT), remove the ethanol as completely as possible, and air dry (RT or 37°C) until all the ethanol has evaporated. Resuspend DNA by adding water and flicking, the resuspending.)
4. In the hood: Dilute DNA with sterile water (work out volume with reference to final target volume), mix using a vortex mixer, and then add 37µl of 1 M CaCl2 to give 150 µl DNA-solution per well of a 6 well plate (for 10 cm dish, add 124 µl per dish to give 500 µl final volume), mix again (vortex).
5. Add 150 µl (500 µl for 10 cm dish) of 2x HBS (vortex) to sterile 5ml universal. Adjust the vortex so the HBS can be gently vortexed. Add the DNA-calcium solution dropwise (ca. 1 drop/sec). The solution will be slightly opaque. Leave for 30 min at RT.
6. Vortex the transfection solution again and add the solution dropwise to the plates along the periphery of the dish, swirl to distribute the precipitate and return the cells to incubator.
7. The next day (after 12-16 hrs) wash the cells twice with PBS and add fresh culture medium.
8. Cells may be harvested after 16-72 hours. This time has to be optimized for each new construct and depends on the promoter and the toxicity of the protein.
Solutions: 2xHBS (Hepes buffered saline)
50 mM Hepes pH 7.1
280 mM NaCl
1.5 mM Na2HPO4
pH to 7.1 exact with NaOH
filter sterilize and freeze in 5 ml aliquots
1M CaCl
2