Total Lipid Extraction from Baker's yeast (Saccharomyces cerevisiae)

Incubate plate at 30°C until colonies 1-2 mm in diameter have formed (2-3 days)

Sterilize the inoculating loop by dipping it in the ethanol and heating it for 30 second in the flame

Allow loop to cool the loop at least for 10 seconds

Streak plate from prepared glycerol stock (-80 °C) and spread on YPD agar plate

Allow plate to incubate for 2-3 days at 25-30 °C until colonies are 1-2mm in diameter

Inoculate 5 mL YPD with yeast colony from plate

Grow overnight at 30°C with shaking at 250 rpm

Check OD₆₀₀ at UV-Vis spectrophotometer

Harvest the whole cells when OD₆₀₀ is 1.5

Centrifuge at 1000 x g for 2 minutes or until clear to pellet the yeast

Pour off the supernatant without disturbing cell pellet

Wash the cells three times by resuspending pellets in 1ml PBS, centrifuge for at 500 x g for 3-5 mins.

After washing three times with PBS, resuspend in 1ml PBS and transfer this mixture to a 15 ml conical centrifuge tube.

Add 3.75 mL of chloroform/methanol (1:2) to the 1 mL of resuspended cells

Add 1.25 mL chloroform and 1.25 mL sterile water subsequently.

Vortex vigorously for 5 mins, and centrifuge at 150 x g for 5 mins at room temperature.

After centrifugation, a two-phase system is obtained: aqueous top phase and organic bottom phase which contains the lipids.

Carefully remove the top aqueous layer and the middle insoluble layers (precipitated proteins).

The organic bottom layer is dried using a speed vacuum or dried under a steady stream of nitrogen.

Record the weight of the dried sample by pre-weighing an Eppendorf tube or equivalent before or after drying the lipid.