Technical
Information |
Preparation
of Cationic Liposomes &
Tranfection of Cells |
Equipment & Materials
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
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.
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.
Reference
- Leventis, R., & Silvius, J.R. (1990) Biochim. Biophys.
Acta 1023: 124-132.
|
