Methods

1. Plate cells in a 35-mm tissue culture dish in 2 mL of appropriate culture medium. Incubate cells at 37°C until they are 90-95% confluent.

2. For each 35-mm dish, dilute separately 0.6-12 pig plasmid DNA to 50 |iL in OptiMEM I (reduced serum medium) and 18-30 |Jg lipofectin reagent to 50 |iL in OptiMEM. The DNA and lipofectin reagent must be diluted separately to avoid precipitation.

3. Combine 50 pL of diluted DNA and 50 jjL of diluted Lipofectin Reagent in a polystyrene tube (e.g., Falcon 2024, see Note 2) to obtain 100 |iL complex solution per tissue culture dish. Mix gently (do not vortex) and let stand for 5 min at room temperature.

4. Wash the cells twice with OptiMEM I to remove serum that may inhibit transfection (see Note 3).

5 Add 2 mL of OptiMEM I to the cells and swirl plates gently to ensure all cells are covered.

6 Add 100 pL of lipofectin reagent DNA complex to the cells dropwise as uniformly as possible. Swirl plates gently to mix.

7. Incubate the cells for 5-24 h at 37°C in ahumidified 5-10% C02 environment

8. Remove medium.

9. Add 2 mL of appropriate culture medium to the cells and incubate at 37°C in a humidified 5-10% C02 environment for 48-72 h.

10. Harvest the cells by aspirating the medium and wash the monolayers twice with PBS.

11. Add 0.5 mL of PBS and harvest cells by scraping with rubber plunger. Transfer the cell suspension to an Eppendorf tube. Harvest the remaining cells with a second 0.5-mL aliquot of PBS.

12. Pellet cells by centrifugation for 5 min, 5000 rpm in a microcentrifuge at room temperature. Discard the supernatant (cell pellet may now be frozen if desired).

13 Resuspend the pellet in appropriate buffer for reporter enzyme assay

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