HEK 293 EXPRESSION SYSTEM

Transfection of HEK 293 Cells

(using Lipofectamine 2000 system)

Reagents:

Lipofectamine (LFA) - Gibco, cat. # 18324-012

OptiMEM (OM) - Gibco, cat. # 31985-070

Media:

500 ml Dulbecco's Modified Eagle (DME-HG)

50 ml Fetal Bovine Serum (FBS) - 10 %

and

2) DME-HG/ FBS/ Pn/ Str, which contains:

500 ml Dulbecco's Modified Eagle (DME-HG)

50 ml Fetal Bovine Serum (FBS) - 10 %

5 ml penicillin/streptomycin (10 kU/ml Pn and 10 mg/ml Str)

Phosphate Saline Buffer (PBS), pH 7.0-7.2

Trypsin (0.05% solution with 0.5 mM EDTA)

Procedures:

Split HEK 293 cells, grown in DME-HG/ FBS/ Pn/ Str into 6-well Poly-D-lysine with 2 ml medium. You can get ~18 wells (~90% confluent the following day) from a 15 cm confluent dish.
When cells reach 70-90% confluence or so, wash the cells with PBS and change the media (DME-HG/ FBS/ Pn/ Str ) with media without antibiotics (DME-HG/ FBS). Antibiotics are much more toxic to cells after treatment with Lipofectamine.
Dilute 8 ul Lipofectamine into 150 ul prewarmed (37°C) OptiMEM
Dilute 2.5 ug plasmid DNA, containing your gene of interest, into 150 ul OM without serum
Incubate 5 min. at room temperature
Mix both DNA-Opti Mem and LFA-Opti Mem solutions and let them sit 20 min. at RT
Add nucleolipid complexes directly onto the cells and incubate for 24 h at 37°C
Change the media after 24h (carefully aspirate the old and pipette new) with DME-HG/ FBS/ Pn/ Str and incubate for an additional 24 h. If the cells are too confluent-you may split the cells to 15 cm dishes. You must, however, wait an additional 24 h before adding hygro.
Split the cells into 150 mm plates with DME-HG/ FBS/ Pn/ Str, containing 150 ug/ml Hygromycin B. Incubate until foci appear (appr. 8-9 days). Change media every 3-5 days. When most of the "non-stable" cells start dying as a result of the drug selection (app. on day 5) the media has to be changed.