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)