This protocol uses the DOTAP Liposomal Transfection Kit from Roche.
1. Seed cells overnight before the day of transfection.
2. For each 35 mm plate or each well of a 6-well plate, dilute
2.5 mg DNA to 0.1 mg/ml
in HBS (HEPES-buffer saline: 20 mM HEPES, 150 mM NaCl, pH7.4).
3. In a separate sterile tube, dilute 15 ml
DOTAP in HBS to a final volume of 50 ml.
4. Transfer the DNA/HBS to DOTAP/HBS and mix by pipetting.
5. Incubate at room temperature for 10-15 minutes.
6. Mix the DOTAP/DNA with 1-2 ml of culture medium.
7. Remove medium from cell plate and replace with the culture medium
8. Return cells to incubator, incubate for 3-6 hours.
9. Replace medium with fresh culture medium (without DOATP/DNA).