Simplified calcium phosphate transfection / adherent cells

Reagents*

1) 2x HBS
Reagent Final [] grams dry
HEPES, pH 7.05 50 mM 5.0
KCl 10 mM 0.37
dextrose 12 mM 1.0
NaCl 280 mM 8.0
Na2HPO4 (FW 141.96) 1.5 mM 0.1065 g
500 ml total, pH to 7.05
2) CaCl2
CaCl2 2 M 29.4 g/ 100 ml
*stock solutions may be prepared and frozen at -20 C

Vtotal (ml) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

2xHBS (ml) 0.25 0.5 0.75 1.0 1.25 1.5 1.75 2.0 2.25
CaCl2 (µl) 31 62 93 124 155 186 217 248 279
H2O (µl) 219 438 657 876 1095 1314 1533 1752 1971

Procedure

1) 293 cells are grown in Dulbecco's minimal essential medium supplemented with 10% FCS and 1% penicillin/streptomycin. Be sure to use DME and not other media--with incorrect bicarbonate concentrations, an orange juice-like precipitate will form in 1-3 hours after addition of DNA. Cells are seeded at 5 x 106 cells/100 mm plate in a volume of 10 ml of medium, or 5 X 105 cells/well of the 6-well plate in a volume of 3 ml of medium the night before transfection.
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2) The amount of DNA per transfection varies with the intended use. For a 100 mm plate, total maxium of 10 µg DNA are precipitated in a Vtotal of 1 ml. For a single well in 6-well plate total mixium of 5 ug DNA are precipitated in a Vtotal of 0.5 ml.
a) DNA's should be clean.
b) the amount of DNA the cells will tolerate depends upon the construct; toxic genes such as bcr-abl may require smaller amounts of DNA.

3) Mix H20/DNA and CaCl2, and then add the 2x HEPES slowly with bubbling for 15 seconds using pastual pipets and automatic pipettor.

4) Add the ppt thus formed (should be slightly cloudy) immediately to tissue culture cells; add the precipitate dropwise so you do not disrupt the cells. A mild-moderate drop in pH of the medium is normal. Place cells immediately into incubator and incubate overnight.

5) Change the medium at about 12 after transfection; Transient assays or drug selection can be done at about 48 hrs after transfection.