Drosophila Cell Transfection Protocol

Drosophila Cell Transfection Protocol

Drosophila Tissue Culture - Cell Transfection Protocol

1. Seed 5 ml of medium in a 60 mm dish with 0.2 to 0.3 ml of cell culture (5 to 8 X 10⁶ cells, i.e. a 2 to 3 day-old culture).
2. Incubate at 25°C for at least 6 hr or overnight before transfection.
3. Mix 10 μg of plasmid DNA with 0.4 ml of 0.25 M CaCl₂ and add to 0.4 ml of 2X HEBS, pH 7.1 drop-wise with swirling in a 17 x 100 mm polycarbonate test tube.
4. Incubate at room temperature for 20 to 30 min, the solution should become slightly cloudy.
5. Add 0.8 ml of the DNA precipitate to the 60 mm dish and swirl. Incubate at 25°C.
6. For selection (i.e. for transformed cell lines) after 24 hr, split the cells 1:4 into new medium. After another 24 hr, add the appropriate selective drug for the plasmid used (for example, G-418 at 1 mg/ml or hygromycin at 200 μg/ml).
7. Split cells every 7 to 10 days into selective (drug-containing) medium.
8. Grow cell lines as mixed cultures in selective medium.

Drosophila Transfection Buffers

0.25 M CaCl2
HEBS (2X)		42 mM HEPES (Free Acid) 2.8 mM Sodium Phosphate Dibasic (Na2HPO4) Sterile filter and store at -20°C 274 mM NaCl When thawing for a transfection, pH to 7.1 again, and sterile filter before use. pH to 7.1 with NaOH 9.4 mM KCl

Materials Needed for Drosophila Transfection

• Sodium Phosphate, Dibasic
• Calcium Chloride
• Sodium Hydroxide
• G-418
• HEPES, Free Acid
• Potassium Chloride
• Sodium Chloride
• Hygromycin