Equipment & Materials
- #850725: 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE) Avanti Cat.
- Cationic lipid
- Distilled water
- Glass vials with teflon liners
- Glass syringes
- Nitrogen or Argon gas
- Vacuum system
- 0.22µm filter (optional)
- Dissolve each lipid component (e.g., DOTAP/DOPE) in chloroform to a convenient working concentration (1-10mg/mL).
- Aliquot the desired amount of each component into a glass vial using a glass syringe. More Information On Handling Organic Solutions Of Lipids.
- Thoroughly mix the components in the glass vial.
- Carefully evaporate the chloroform (with adequate ventilation) using a nitrogen or argon stream.
- Place the lipid residue on a vacuum pump for 10-15 minutes to remove any residual organic solvent.
- Remove the vial from the vacuum pump and immediately suspend in distilled water at twice the final lipid concentration.
- Bath sonicate the lipid dispersion to clarity (2-5 min).
- Add an equal volume of buffer (e.g., 308mM NaCl, 40mM Hepes, pH7.4), and sonicate further for 2 minutes.
- Solution may be passed through a 0.22µm filter to sterilize.
Preparation of Lipid/DNA Mixtures
- Combine cationic lipid dispersion with DNA (1µg per 10µg lipid) in a suitable container.
- Incubate lipid/DNA mixture for 5 min. at room temperature.
- After 5 min., mixture is ready for transfection of cells.
Transfection of Cells
- Wash cell monolayers with HEPES-buffered saline two times.
- Incubate cells with lipid/DNA mixture in HEPES-buffered saline (3 ml mixture per 100 mm culture dish) at 37°C for 90 min.
- After the incubation period, either replace the medium or supplement as necessary.
- Culture cells for desired period of time and harvest.
- Common molar ratios of DOPE:cationic lipid are 3:1 and 1:1. In some cases, a lipid system composed entirely of cationic lipid has been used to transfect cells.
- Buffer system cited is intended for in vitro use and does not suggest that this system may be suitable for in vivo use.
- Leventis, R., & Silvius, J.R. (1990). Biochim. Biophys. Acta 1023: 124-132.