Transient Expression Transfection Kit Protocol

Application

DEAE dextran mediated transfection (Millipore cat. no. S-100) is used for high efficiency, transient expression of cloned genes. This method is most efficient in COS-7, COS-1 and CV-1 cells. There are, however, a number of other cells that can be transiently transfected by this method. These include L cells, GH-1, Vero, CHO and NIH-3T3. Transfection of these other cell types required modification of this protocol with respect to the time of incubation of cells with DEAE dextran-DNA-media and chloroquine.

Kit Components

All reagents are supplied sterile. One kit will transfect up to 100 x 100 mm plates of cells.

10X Tris 1 bottle 50 mL

100X DEAE Dextran 3 vials 1.7 mL/vial

1000X chloroquine 1 vial 0.5 mL

10X Tris	1 bottle	50 mL
100X DEAE Dextran	3 vials	1.7 mL/vial
1000X chloroquine	1 vial	0.5 mL

Specification

Storage: Store 10X Tris and 100X DEAE dextran at 4°C. Store 1000X chloroquine at –20°C (light sensitive).

Protocol

Plate cells 24 hours before transfection. Split 90% confluent plates of cells 1:5 in complete growth medium.
Remove medium and wash cells twice with 10 mL of growth medium without serum per 100 mm plate.
Add 4 mL of DEAE-DNA-medium per 100 mm plate.
Formula for 10 mL of DEAE-DNA-medium (Mix Gently):

Growth medium w/L-glutamine, penicillin and streptomycin, w/o serum 8.9 mL

100X DEAE Dextran 0.1 mL

10X Tris 1.0 mL

DNA nto be transfected, 5 µg per 100 mm plate 50 µL

Growth medium w/L-glutamine, penicillin and streptomycin, w/o serum	8.9 mL
100X DEAE Dextran	0.1 mL
10X Tris	1.0 mL
DNA nto be transfected, 5 µg per 100 mm plate	50 µL

Incubate cells with DEAE-DNA-medium at 37°C in 5% carbon dioxide and 90% to 95% air atmosphere for 12 to 14 hours.
Remove DEAE-DNA-medium and wash cells with 10 mL of growth medium without serum per 100 mL plate.
Add 5 mL of growth medium with serum per 100 mm plate. Add to this 5 µL of 1000X chloroquine while gently swirling the medium in the plate. Incubate at 37°C in a 5% carbon dioxide, 90% to 95% air atmosphere for exactly 2.5 hours.
Immediately remove medium and wash cells with growth medium without serum, add 10 mL growth medium with serum per 100 mm plate and incubate cells at 37°C in a 5% carbon dioxide, 90%–95% air atmosphere for 24 hours.
Remove medium and feed fresh medium for harvest of medium and/or cells 26 to 72 hours later.

References

Vaheri, A. and Pagano, J.S. Infectious poliovirus RNA: A sensitive method of assay. Virology 1965; 27:434.
McCutchan, J.H. and Pagano, J.S. Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethyl aminoethyl-dextran. J. Natl. Cancer Inst. 1968; 41:351.
Warden, K. and Thorne, H.V. Infectivity of polyoma virus DNA for mouse embryo cells in presence of diethylaminoethly-dextran. J. Gen. Virol. 1968; 3:371.
Luthman, H. and Magnusson, G. High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Research 1983; 11:1295.

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