Learn to grow and transfect cells

Learn to grow and transfect cells

pTet-tTAk stably transfects fibroblasts
Culture and transfect cells
1. Cultivate adherent cells in DMEM complete culture medium. The day before transfection, the cells were changed to DMEM complete culture medium containing 0.5 μg/ml tetracycline-HCl (tetracycline). Add enough cells to each 10 cm petri dish so that on the day of transfection, the cells can have a confluence of 33%.
2. Linearize the plasmid at the appropriate restriction endonuclease sites, and adjust the concentration of the plasmid per second to ≥ 0.5 mg/ml. 10 ~ 20 μ g plasmid pTet-tTAk and 1 ~ 2 μg pSV2-His (the molar ratio of Tet plasmid to selectable marker plasmid is approximately 10: 1) and 500 μl HEPES buffer in a clean 4 ml polypropylene Mix in the vinyl tube.
3. Add 32.5 μl 2 mol/L CaCl2 to the DNA mixture. Immediately vortex gently to mix the solution. The solution is stored at room temperature and mixed from time to time. After 15 to 30 minutes, flocculent precipitation will form.
4. Aspirate all the culture fluid in the cell petri dish prepared in step 1.
5. Use a Pasteur pipette to mix CaCl2-DNA several times. Add the mixture dropwise to make it evenly cover the monolayer of cells.
6. Incubate the cells at 37°C and 5% CO2 for 30 minutes, and shake the petri dish after 15 minutes to ensure uniform coverage of the DNA sediment.
7. Add 10 ml DMEM complete culture medium containing tetracycline to each cell petri dish. Cells are cultured at 37°C and 5% CO2 for 4 to 5 hours.
8. Gently aspirate the culture medium. Avoid destroying the sediment on the cells.sterile petri dishes,types of petri dishes,disposable petri dish
9. Add 2.5 ml of HEPSE buffer containing 15% glycerol and preheated to 37°C for glycerol shock. Place the cells at room temperature for 2.5 minutes. Glycerol is added dropwise to the culture medium.
10. Suck off the glycerin solution at exactly 2.5 minutes. Operate faster because glycerin is very toxic to cells.
11. Immediately and lightly and quickly add 10 ml of DMEM containing tetracycline to wash the cells. Immediately aspirate the culture medium and repeat the washing.
12. Add 10 ml of DMEM complete culture medium containing tetracycline to the cells, and incubate overnight at 37°C.
13. About 16 to 24 hours after transfection, aspirate the culture medium and replace it with 10 ml DMEM complete culture medium containing tetracycline. After transfection, incubate at 37°C for a total of 48 hours (that is, the total incubation time of steps 12 and 13).sterile petri dishes,types of petri dishes,disposable petri dish