Categorized: Liposome Methods
- CHCl3:MeOH (2:1)
- 0.9% NaCl
- Obtain 2 litres of cells in late log phase.
- Centrifuge down and remove pellet to a glass container (corex tube) using a spatula.
- Add 20 fold the pellet volume of CHCl3:MeOH (2:1) and agitate vigorously.
- Centrifuge at 9000 x g for 20 minutes @ 4oC. Decant CHCl3 phase. It should be monophasic, but if it isn’t, remove the bottom layer. (Avoid sucking up any precipitate or material floating on the lower phase.)
- Add one fifth of the bottom layer volume of 0.9%NaCl. Mix vigorously. Recentrifuge as above. Discard upper phase and precipitate on interface.
- Repeat step 5.
- Reduce volume of CHCl3 to 1-2ml by evaporating under nitrogen.
Phosphlipids can be quantitated by phosphate assay and identified by thin layer chromatography. Store at -20ºC or preferably -70ºC.