Calcium phosphate transfection

Edited by Chang Zhu
1. Seed ~1 x 106 cells in 100 mm culture dish one day before transfection. On the day of transfection, cells should be 50-60% confluent.

2. On the day of transfection, change to fresh warm media and put cells back to the incubator.

3. Meanwhile, add 0.5 ml 2X HEBS to 15 ml tube or 35 mm tissue culture plate. Prepare 1-20 mg DNA in 0.5M CaCl2 (by dilution 2M CaCl2 with ddH2O).

2X HEBS Buffer
NaCl 274 mM 8.0 g
KCl 10 mM 0.38 g
Na2HPO4*7H2O 1.4 mM 8.0 g
Glucose 15 mM 1 g
Hepes 42 mM 5 g
ddH2O add to 450 ml, and to 500 ml after adjusting pH

Adjust 2X HEBES buffer's pH to 7.05 ± 0.05 with NaOH. Filter sterilize, aliquot, and store frozen or at 4 C.

4. Add dropwise DNA/CaCl2 to 2X HEBES. Mix by blowing bubbles with a 2 ml pipette or by gently swirling of the tissue culture plate.

5. Stand at room temperature for 20-30 minutes. An opalescent precipitate should form within 10-20 minutes.

6. Add the DNA/CaPO4 precipitate dropwise to the cells. Swirl gently to mix the DNA/CaPO4 precipitate and medium.

7. After 4-16 hours, remove the medium containing DNA/CaPO4 precipitate. Add new growth media.

8. Havest cells 48-72 hours after transfection.