Reagents/Supplies:

• EndoFree Plasmid Maxi Kit, 10 preps, Qiagen #12362, $218

• Effectene Transfection Kit, Qiagen #301425, $205 (contains 1 ml Effectene Transfection Reagent, 0.8 ml Enhancer, 2x 15 ml Buffer EC, sufficient for 8 transfections in 100 mm dishes, store at 2-8°C)

• 100 mm dishes, 200/case, VWR #25382-166, $99.81

• 1.5 ml Eppendorf tubes, 1000/pack, Sarstedt #72690, $16.50

• 15 ml conical tubes, 500/case, ISC #C-3317-1, $70.00

• Appropriate growth medium (e.g., cRPMI for T cell lines)

Protocol:

1. Prepare plasmids for transfection using Endofree Plasmid Kit to avoid toxicity problems.

2. Day before transfection: split cells, need 0.5-2.0 x 10⁷ cells per 100 mm dish.

3. Day of transfection: harvest cells by centrifugation, remove the medium, and wash cells once with PBS in a 15 ml tube.

4. Seed 0.5-2.0 x 10⁷ cells per 100 mm dish in 7 ml growth medium containing serum and antibiotics.

5. Dilute 4 µg DNA (dissolved in TE buffer) with the DNA-condensation bufferEC to a total volume of 600 µl.

6. Add 32 µl Enhancer and vortex for 1 second.

7. Incubate 2-5 minutes at RT, centrifuge briefly to remove drops from top of tube.

8. Add 120 µl Effectene Transfection Reagent. Mix by pipetting up and down 5 times or vortexing for 10 seconds.

9. Incubate 5-10 minutes at RT to allow transfection-complex formation.

10. Add transfection complexes to tube containing 3 ml growth medium. Mix by pipetting up and down twice then immediately add mixture drop-wise onto the cells in the 100 mm dish.

11. Gently swirl the dish to ensure uniform distribution of transfection complexes.

12. Incubate cells with transfection complexes under normal growth conditions for an appropriate time for expression of the transfected gene.

13. Assay cells to confirm gene expression (e.g. X-gal staining, FACS).