Calcium phosphate transfection of 293 cells

Procedure

1. Grow enough 293HEK cells in T150 flasks so that you have one T150 for two 15 cm dishes. 293 cells are normally grown in 10% FCS in DMEM (with Pen/Strep and Glu) but will also grow well in 10% FCS DMEM.
2. On the morning of the day of transfection, trypsinise the cells and seed 1 T150 into two 15 cm dishes in 24 ml of 10% FCS/DMEM. Transfection must be performed in DMEM not EMEM. Do this as early as possible to allow the cells to stick down and spread.
3. At about 3 pm, remove the 1× HBS and 2.5 M CaCl₂ and warm up to room temperature.
4. At about 4 pm set up the transfection as below. Set up a single 1.5 ml Eppendorf for each 15 cm dish.
   1. Add 1.3 ml of 1× HBS.
   2. Add 77 µg of plasmid and mix well by pipetting up and down.
   3. Add 38.5 µl of 2.5 M CaCl₂ and mix immediately by rapidly pipetting up and down.
   4. Add another 38.5 µl of CaCl₂ and mix immediately by rapidly pipetting up and down.
   5. Incubate at room temperature for 40 minutes.
   6. Add dropwise to each dish. Rock back and forth so you spread the transfection mix evenly.
5. The next day you should see a very fine precipitate, particularly in areas where there are no cells. You will need to use the highest magnification in TCB to see this clearly. It will almost look like a bacterial infection.
6. Remove the media and replace with cold fresh media (normally 10% FCS EMEM but DMEM will also do).
7. Expression will only be good 48 hours after transfection.

1× HEPES buffered saline (1× HBS)

• NaCl: 4 g
• KCl: 0.1775 g
• Na₂HPO₄: 0.095 g
• HEPES: 2.5 g
• pH 7.4