Stable Transfection of Adherent Cells

Modified from Qiagen Protocol for SuperFect Reagent

The following protocol is for stable transfection of adherent cells in 6-well plate.

1. Cell line to be transfected
2. High quality plasmid DNA (>0.1ug/ul in 10mM TB, pH7-8)
3. DMEM
4. Culture medium ( DMEM+10%FBS+1%PS)
5. Selective medium (culture medium + selective drug)
6. SuperFect transfection kit (Qiagen 301305)
7. 6-well tissue culture plates
8. Micripipettes and tips
9. Serrological pipettes
10. Bio-Incubator
11. Centrifuge
12. Biosafety hood

1. The day before transfection (Mon-Tue), seed 1 x 10⁵ cells per well in 2 ml culture medium in 6-well plate. The cell number seeded should produce 40-80 % confluence on the day of transfection.
2. Incubate the cells at 37°C and 5% CO₂ overnight.
3. On the day of transfection, dilute 2 µg DNA with DMEM to a total volume of 100 µl. Mix and spin down the solution for a few seconds to remove drops from the top of the tube.
4. Add 8 µl/well (ul = DNA ug x 4) SuperFect Transfection Reagent to the DNA solution. Mix by pipetting up and down 5 times, (or by vortexing for 10 sec.).
5. Incubate the samples for 5-10 min at room temperature (15-25°C) to allow transfection-complex formation.
6. While complex formation takes place, gently aspirate the growth medium from the well, and wash cells once with 4 ml PBS or medium to remove dead cells.
7. Add 600 µl cell culture medium (containing serum and antibiotics) to the reaction tube containing the transfection complexes. Mix by pipetting up and down twice, and immediately transfer the total volume to the cells in the wells.
   
8. Incubate cells with the transfection complexes for 12-16 h under their normal growth conditions.
9. Remove medium containing the remaining complexes from the cells by gentle aspiration, and wash cells 3-4 times with 2 ml growth medium.
10. Add fresh cell culture medium and incubate for 24-48 h.
11. Passage cells from each well into two 10cm dishes. After cells settling down, replace medium in one of the two dishes with the appropriate selective medium and maintain cells in selective medium under their normal growth conditions until colonies appear. The cells in regular medium can be used for observation of foci formation.

SuperFect Transfection Reagent Handbook