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Star Republic: Guide for Biologists

Transfection --- liposomal

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 containing DOTAP/DNA.

8. Return cells to incubator, incubate for 3-6 hours.

9. Replace medium with fresh culture medium (without DOATP/DNA).