Calcium Phosphate transfection of 293T cells

 

 

Materials

Complete medium: DMEM(high glucose) supplemented w/ 10% BCS and Gln (Pen/Streptmycin: optional)

Transfection reagents:

i) 2M CaCl2: 2M in H2O, filter sterile, store at 4C

ii) 2xHBS: 8.0g NaCl, 0.37g KCl, 201mg Na2HPO47H2O, (1.0g glucose), 5.0g HEPES/500ml (adjust pH to 7.05 with NaOH and filter sterile, store at 4C)

DNA

Chloroquine stock, 25 mM in PBS (freeze in vials)

(Optimem supplemented w/ ITS: optional)

 

 

Procedure

The following protocol is for 10 cm dish (medium: 10ml). If you use 6-well or 12-well plates, total volume of the medium should be 3ml and 1.5ml, respectively, and decrease the amount of each reagent accordingly.

 

1. Plate cells the night before to give 60-80% confluence at the day of transfection.

 

2. One hour prior to the transfection, change to medium containing 25M chloroquine (from x1000 stock in PBS, stored at -20C). Volume should be 10 ml per dish. (Chloroquine can be omitted, but increases efficiency about x2)

 

3. Add 10g DNA and ddH2O to 438 l in 15-ml sterile tube, then add 62 l 2M CaCl2 to a final volume of 500 l. When you are ready, add 500 l of 2xHBS dropwise while gently mixing. Add this mixture directly to the cells dropwise through the medium. Do this within 1-2 min after adding 2xHBS. You will notice that the medium turns to orange. Make sure you evenly sprinkle the droplet over the entire area.

 

4. Incubate for 7-11 h. Very fine, dust-like precipitate visible. After incubation, rinse once and change to medium without chloroquine (or to Optimem with ITS).

 

5. Culture supernatant, 2-6 days after transfection. For secreted protein, you can change media once (at day 3 or 4, save the collected sup) and give it another 3-4 days of culture. As long as the cells are alive, they produce proteins. However, the time when the secretion level reaches maximum varies among proteins.