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 m filters and store in aliquots at -20C

(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 m filters and store aliquoted at -20C.

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

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

 

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

25M chloroquine)

3. Thaw HeBS and CaCl2 at room temperature

4. For each transfection prepare aliquots of 71 l HeBS

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