Calcium phosphate transfection
1. Cell plate: Collect cells by trypsinization and plate cells on a 60 mm tissue culture dish at a density of 1 × 105 to 4 × 105 cells/cm² with appropriate complete medium (choose the culture dish according to the experimental needs, The area occupied by the cells after adherence reaches 40-70% of the total area of the culture dish). The cells were incubated in a 37 ℃ incubator with 5% CO2 for 8-24 h, and the transfection could be started when the cells adhered completely. Change the medium 2 h before transfection (replace the old medium with 4 ml of fresh complete medium).
Note: In order to obtain higher transfection efficiency, try to use exponentially growing cells, and the density of cells during transfection should not exceed 80%.
2. Preparation of calcium phosphate-DNA precipitation: Take a 60 mm tissue culture dish with a total reaction volume of 500 μl as an example. Add plasmid DNA (preferably total 4-10 μg) in sterilized water, and then add 31 μl 2 M CaCl2 to make the total volume of the three to 250 μl, and mix well. Add an equal volume of 2×HBS salt solution dropwise, while flicking the tube wall to mix in time after each drop is added. After standing for 2 min, the 500 μl calcium phosphate-DNA suspension was immediately added dropwise to the cell culture medium of the above monolayer cells, and the plate was gently shaken to mix.
Note: It can be observed that the medium at the dripping site will appear turbid orange instantly. It should be mixed as soon as possible to avoid the formation of excessively large particles and affect the transfection efficiency.
3. If the transfected cells are not treated with a transfection accelerator, they are incubated in a 37°C incubator with 5% CO2. After 8 h, the culture medium and DNA precipitation were aspirated, 5 ml of pre-warmed complete medium at 37°C was added, and the cells were incubated in an incubator for 16-40 h to observe the transfection efficiency.
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Post time: Jan-28-2022