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

Content Overview

• Equipment & Materials
• Procedure
• Preparation of Lipid/DNA Mixtures
• Transfection of Cells
• Notes

Equipment & Materials

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

Procedure

• Dissolve each lipid component (e.g., DOTAP/DOPE) in chloroform to a convenient working concentration (1-10 mg/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., 308 mM NaCl, 40 mM Hepes, pH 7.4), and sonicate further for 2 minutes.
• Solution may be passed through a 0.22 µm 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.