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.
| 