Preparation of Cationic Liposomes & Transfection of Cells - Avanti® Polar Lipids

Equipment & Materials

  • Cationic lipid
  • Chloroform
  • Distilled water
  • Buffer
  • Glass vials with teflon liners
  • Glass syringes
  • Nitrogen or Argon gas
  • Vacuum system
  • 0.22µm filter (optional)

Procedure

  • 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.
  • 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

  1. Combine cationic lipid dispersion with DNA (1µg per 10µg lipid) in a suitable container.
  2. Incubate lipid/DNA mixture for 5 min. at room temperature.
  3. After 5 min., mixture is ready for transfection of cells.

Transfection of Cells

  1. Wash cell monolayers with HEPES-buffered saline two times.
  2. Incubate cells with lipid/DNA mixture in HEPES-buffered saline (3 ml mixture per 100 mm culture dish) at 37°C for 90 min.
  3. After the incubation period, either replace the medium or supplement as necessary.
  4. Culture cells for desired period of time and harvest.

Notes

  • 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.