TITLE: Lipids in microalgae: The Extraction by modified Folch solvent
AUTHORS: Yingyu Hu, Zoe V Finkel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>In this protocol, lipids in miroalgae is extracted with  2 ml Folch solvent (2:1 chloroform-methanol v/v) after frozen and thawed with the addition of 100 ul water. Non-lipid substances are removed by filtration. Filtrate is then mixed with 0.88% potassium chloride solution to form a biphase system. The lower phase with extracted lipids are collected and dried under N</span><span style = "vertical-align:sub;">2 </span><span>gas flow. The residue is dissolved in 5 ml chloroform and stored under -80 ºC.</span></div></div>

[STEPS]
?. [Prepare reagent]
Folch solvent (CHCl3: MeOH=2:1 v/v)
?. [Prepare reagent]
KCl solution ( )
?. [Extraction]
Transfer sample into muffled centrifuge tube, use precombusted filter as blank.
?. [Filtration]
?. [Separation]
Calculate the volume of 0.88% KCl by multiplying (V+2) by 4/21.
In order to obtain a biphase system to separate the extract from water, the final composition of CHCl3:MeOH:H2O is 8:4:3 (v/v)
?. [Prepare reagent]
Mix two parts of chloroform and one part of methanol in a 500 ml amber bottle
?. [Prepare reagent]
Attach dispensette to the bottle
?. [Prepare reagent]
Label bottle with MSDS label.
?. [Prepare reagent]
Weigh the pyrex media bottle and press the tare button
?. [Prepare reagent]
Directly weigh  KCl in the bottle
0.44 g
?. [Prepare reagent]
Top bottle with MilliQ water to
50 g
?. [Extraction]
Add  MilliQ directly onto the sample.
100 µl
?. [Extraction]
Freeze at
-80 °C
?. [Extraction]
Vortex
?. [Extraction]
Remove vials from freezer.
?. [Extraction]
Dispense  Folch solvent into sample tube.
2 mL
?. [Extraction]
Purge the dispensette, fill the tubing with solvent before dispensing solvent into sample tube.
?. [Extraction]
Sonicate
?. [Extraction]
Keep sample in the dark at
0 Room temperature
?. [Extraction]
Centrifuge extract
Centrifuge: 32000 33, 5 min
?. [Extraction]
Remove supernatant to a clean glass centrifuge tube by pasteur pipet.Save the pasteur pipet for later.
?. [Extraction]
Add  MilliQ and  Folch solvent to the residue.
50 µl
1 mL
?. [Extraction]
Vortex
?. [Extraction]
Sonicate
?. [Extraction]
Remove supernatant  with the pasteur pipet used in #13.Combine with the supernatant obtained in #13.Save the pasteur pipet for later.
?. [Extraction]
Add  MilliQ and  Folch solvent to the residue.
50 µl
1 mL
?. [Extraction]
Vortex
?. [Extraction]
Sonicate
?. [Extraction]
Add  MilliQ and  Folch solvent to the residue.
50 µl
1 mL
?. [Extraction]
Vortex
?. [Extraction]
Sonicate
?. [Extraction]
Remove supernatant  with the pasteur pipet used in #13.Combine with the supernatant obtained in #13.Save the pasteur pipet for later.
?. [Extraction]
Remove supernatant  with the pasteur pipet used in #13.Combine with the supernatant obtained in #13.Save the pasteur pipet for later.
?. [Filtration]
Transfer extract into the funnel with the pasteur pipet used in #25.Save the pasteur pipet for later.
?. [Filtration]
Add   Folch solvent and  MilliQ into the centrifuge tube.
1 mL
50 µl
?. [Filtration]
Add   Folch solvent and  MilliQ into the centrifuge tube.
1 mL
50 µl
?. [Filtration]
Pull plunger back and push the bottom of stopper into the open top of funnel.
?. [Filtration]
In the figure:(1) 50 ml syringe (2) silicon tube (3) silicon stopper(4) filter tunnel(5) GFF filter(6) base(7) neck of the base(8) 10 ml graduated cylinder(9) clamp
?. [Filtration]
In order to avoid the loss of sample, before transfering extract into the funnel, check if the funnel is well assembled with the base by clamp.
?. [Filtration]
The neck of the base must touch the inner side of the graduated cylinder, so that the filtrate can be all collected in the cylinder. In order to maintain the balance of air pressure, leave gap between the neck and the cylider.
?. [Filtration]
Slowly and steadily push the plunger to force the filtrate into the graduated cylinder.
?. [Filtration]
Record the volume (V) of the filtrate in the cylinder to an accuracy of 0.1 ml.
?. [Filtration]
Use a clean pasteur pipet to transfer all filtrate into a clean glass centrifuge tube.
?. [Filtration]
Rinse the tube.
?. [Filtration]
Transfer the folch solvent/MilliQ mixture into the funnel with the pasteur pipet used in #27
?. [Filtration]
Rinse the tube.
?. [Filtration]
Transfer the folch solvent/MilliQ mixture into the funnel with the pasteur pipet used in #27
?. [Filtration]
Add   Folch solvent and  MilliQ into the graduated cylinder.
1 mL
50 µl
?. [Filtration]
Rinse and transfer the Folch solvent/MilliQ mixture into the same glass centrifuge tube as in #39 by the pasteur pipet used in #39.
?. [Filtration]
Add   Folch solvent and  MilliQ into the graduated cylinder.
1 mL
50 µl
?. [Filtration]
Rinse and transfer the Folch solvent/MilliQ mixture into the same glass centrifuge tube as in #39 by the pasteur pipet used in #39.
?. [Filtration]
Estimate the final volume of filtrate as (V+2)
?. [Separation]
Vortex the centrifuge tube for
?. [Separation]
Remove most of upper aqueous phase with the pasteur pipet used in #39.
?. [Separation]
Use a clean pasteur pipet to transfer the lower organic phase to a 12 ml amber vial.
?. [Separation]
Dry organic phase extract at  under a stream of N2 gas (
37 °C
?. [Separation]
Add  CHCl3 by using serological pipet to the dry residue.
5 mL
?. [Separation]
Freeze at  .
-80 °C
?. [Filtration]
Use positive pressure to force the filtrate into the same graduated cylinder.
?. [Filtration]
Use positive pressure to force the filtrate into the same graduated cylinder.
?. [Separation]
Centrifuge at  or until biphase layers separate completely.
Centrifuge: 2000 33, 5 min