Transfection of Mouse N2a Cells using Lipofectamine/plus

1.  Quick thaw the cells in 37 C water bath and add to 10 ml of growth medium. Spin 5 min at 800 rpm to remove DMSO from freezing medium.  DMSO is toxic to growing cells.  Resuspend in 10 ml growth medium in 75 cm2 TC flask and place in 5% CO2/37 C incubator.

2. Plate approximately 5 x 105 cells into 60mm petri dishes. Wait until the cells reach 80% confluent.

3.  Dilute 2-4 ug DNA of each construct to be transfected into 250 ul of OptiMEM or DMEM medium. Add 8 ul of Plus Reagent (Invitrogen # 11514-015) to the DNA/MEM mix.  Let sit 15 min at room temperature.

4. Separately, mix 12 ul of Lipofectamine (Invitrogen # 18324-111) with 250 ul of OptiMEM or DMEM.

5. Mix DNA/MEM/Plus solution with Lipofectamine/MEM.  Let sit 15 min at room temperature.

6. Remove growth medium from 60mm cell cultures.  Rinse once with 2 ml OptiMEM or DMEM, replace with another 2 ml MEM to remove serum from the plate.

7. Add preincubated DNA/Plus/Lipofectamine/MEM solution to the cells.  Mix gently.

8. Grow cells 3 hr – overnight.  Add 2.5 ml N2a medium and continue culture.

If developing stable transfectants, add G418 (Gibco/Life Tech # 1181-031) to the growth medium  to select for positive cells.  Use at 400-800 ug/ml for selection of clones, and 200-400 ug/ml for maintenance.

9. 48 hours post transfection, harvest the cells for the future assay.

Growth medium:

DMEM, high glucose, w/L-gluatmine       Gibco # 11965-092     250ml

Fetal bovine serum, qualified           Gibco # 26140-079      50ml

OptiMEM 1 medium                    Gibco # 31985-070          250ml

Penicillin-Streptomycin            Gibco # 15140-122             5ml

Filter to 0.2 um.  Stable for several months at 4 C.

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