Johannes A. Schmid

 

Calcium-Phosphate Transfection (optimised for 293 cells)

 

Buffers

· HeBS-Buffer (Hepes buffered saline)

400 ml A.dest.

final conc.

8 g NaCl

280 mM

0.2 g Na2HPO4.7H2O (or 0.107 g anhydrous)

1.5 mM

6.5 g Hepes (Sigma H-7006) (or 5.96 g of free acid)

50 mM

 

Adjust the pH to exactly 7.05 (calibrate pH-meter with pH 4.01 and pH 7.00 buffers before).

Add A.dest. to 500 ml, filter through 0.2 µm filters and store in aliquots at -20°C

(not longer than 6 months). Thawed aliquots shouldn't be frozen again.

· CaCl2: 29.4 g CaCl2.2H2O (MW=147) in 100 ml A.dest (final conc.: 2 M)

Filter through 0.2 µm filters and store aliquoted at -20°C.

· Chloroquine (optional): chloroquine. 2H2O (Sigma C-6628): 12.9 mg/ml in PBS

(conc.: 25 mM). Filter through 0.2 µm filters and store at -20°C.

 

Procedure (amounts are given for 6-wells):

1. Seed cells (about 500 000 cells per 6-well = per 10 cm2 ) one day before the transfection (in

DMEM/10% FCS)

2. (Optional: 1 h before transfection, exchange the medium for medium containing

25µM chloroquine)

3. Thaw HeBS and CaCl2 at room temperature

4. For each transfection prepare aliquots of 71 µl HeBS

5. Prepare the DNA/ CaCl2-Mix: 4 µg DNA (total) in 62 µl A.dest. + 9 µl CaCl2

6. Add the DNA/ CaCl2-Mix drop-wise to the HeBS aliquots (by screwing the Gilson

pipette) and slightly mix after each drop. Incubate for 2 - 3 min at R.T. to form the

DNA-precipitate (not longer).

7. Add the DNA-precipitate drop-wise to the cells (by screwing the Gilson pipette

and moving it to cover the whole surface of the cell culture; don't swirl the dish).

8. Carefully transfer the dish back to the incubator. Incubate for 24 h (or in the

presence of chloroquine: for 10 h) and exchange the medium afterwards.

(The transfection is in the presence of FCS!). The efficiency of transfection is in

the range of 70-90%. Harvest the cells after 48 h.

The protocol is adapted from Neil Perkins who adapted it from Gary Nolan in 1995

(See web site: http://www.stanford.edu/group/nolan/

or CP in Mol.Biol. 9.1 and 9.11.2-3)