TITLE: fatty acid analysis sponges 
AUTHORS: Anna de Kluijver

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol has been used to analyse fatty acid composition of sponges (Porifera). The original protocol has been developed at NIOZ Yerseke, former NIOO-CEME (Netherlands) (Boschker and Middelburg) and has been used to quantify PLFA in combination with stable isotopes for sediments, plankton, organisms (including sponges). This protocol has been adopted to the procedures of the organic geochemistry lab at Utrecht University. </div></div>

[STEPS]
?. [Total lipid extraction]
Add  Blight and Dyer mix, close with lid and shake/vortex
28.5 mL
?. [Total lipid extraction]
Weight 30-50 mg dried sponge sample in extraction tubes (Kimax®centrifuge tube).  This amount is enough to do either lipid-class or total lipid analysis,  if you want to do both, weight a double amount.
?. [Total lipid extraction]
Extract the sample in an ultrasonic bath for
?. [Total lipid extraction]
add   DCM, close lid and shake/vortex
7.5 mL
?. [Total lipid extraction]
Extract for minimal  on a roller table  - horizontal shaker. Check that lids are tightly closed!
?. [solution preparation    ]
Prepare P-buffer: dissolve   K2HPO4 in  mQ in a 1 L glass bottle, adust to pH 7-9 with  HCl, check with pH paper
8.7 g
1 L
?. [solution preparation    ]
Prepare standards: C19:0 FAME in hexane (100 x stock)   C12:0 FAME  in  hexane (100 x stock)
20 mg
2 mL
20 µl
2 mL
?. [solution preparation    ]
Prepare    methanolic NaOH by dissolving   CH3NaO in    MeOH
1.08 g
100 mL
?. [solution preparation    ]
Prepare Bligh and Dyer extraction (BDE) mix: methanol (MeOH)/ dichloromethane (DCM) / P-buffer in ratio of 2/1/0.8 (v/v/v), e.g.  MeOH,  DCM and P-buffer in a 500 mL Erlenmeyer with glass stopper
250 mL
125 mL
100 mL
?. [Total lipid extraction]
add   P-buffer, close lid and shake/vortex
7.5 mL
?. [Total lipid extraction]
Let layers separate by either leaving the tubes overnight in the freezer of by gentle centrifugation  (room temperature)
Centrifuge: 170 34, 3 min
?. [Total lipid extraction]
Collect the DCM layer (bottom) with a Pasteur pipette or a glass syringe into 50 mL glass vials. The type of vials depend on the evaporator. Take a bit of air into the syringe or pipette. Bring the tip carefully in the bottom layer. Let a few air bubbles out to clean the tip of the syringe/pipette. Collect the DCM and avoid taking the fluffy layer.      When using a glass syring, clean it in DCM: methanol (1:1) between samples.
?. [Total lipid extraction]
Add again   DCM, let layers separate (centrifugation or freezer) and collect DCM layer into the same vial
7.5 mL
?. [Total lipid extraction]
Dry samples completely under a gentle N2-stream in a turbovap ( , maximum 5 psi pressure, but start with 1-2 psi)
30 °C
?. [Total lipid extraction]
Transfer sample in (approximately) DCM:MeOH (1:1) to a pre-weighed 4 ml vial and dry in a Flexivap   under a gentle N2 stream
1 mL
26 °C
?. [Total lipid extraction]
Weigh your vials to obtain total lipid weight /sponge dry weight. Now you have your Bligh and Dyer extract (BDE)
?. [Total lipid extraction]
Optional: if your sample contains some residue, it can be cleaned over a pasteur glass pipette with pre-extracted cotton wool in DCM: MeOH (1:1)
?. [Separation into polarity classes]
Prepare columns: add a ball of pre-extracted cotton-wool into the tip of a 10 mLglass pipette. Add 1 spoon (~0.5 g) on top. The whole column is ~3 fingers width, 3 cm. The column is stored into a cylinder filled with DCM. Make sure the silica layer is soaked in DCM. Gently tap the column to get rid of air bubbles.
?. [solution preparation    ]
Activate silicic acid gel by heating it at  , for at least 2 hours and allow it to cool. Once activated, it can be kept in a closed bottle for 2 or 3 days.
120 °C
?. [solution preparation    ]
Prepare  acetic acid: add  glacial acetic acid () into  miliQ and fill to in a 100 mL vollumetric flask.
5.742 mL
25 mL
100 mL
?. [Separation into polarity classes]
Put the column in a holder, place a vial or beaker glass underneath  and wash the column with DCM
?. [Separation into polarity classes]
Dissolve and add your sample inDCM.
0.5 mL
?. [Separation into polarity classes]
Don't disturb the coyour neutral lipid fraction. Collect in a 10 mL vial if you want to analyze it or in a waste beaker/vial if it can be discarded. Don't disturb the column!
?. [Separation into polarity classes]
Elute with  acetone (picograde pestinorm), this is your glycolipid fraction. Collect in a 10 mL vial if you want to analyze it or in a waste beaker/vial if it can be discarded.
7 mL
?. [Separation into polarity classes]
Elute with  MeOH into a 20 m glass vial , this is your phospholipid fraction that will be used for further analysis.
15 mL
?. [Separation into polarity classes]
Dry the MeOH fraction in the turbovap ( max 5 psi, but start with 1-2 psi)
30 °C
?. [mild alkaline methylation]
Add to dried sample 1 ml MeOH/toluene (1:1 v/v)Add 1 ml 0.2 M methanolic NaOH (exactly with glass syringe/pipette)Add  C19:0 FAME standard
50 µl
?. [mild alkaline methylation]
Incubate at  in a oven
37 °C
?. [mild alkaline methylation]
Stop methylation by adding  hexane acetic acid (exactly, with pipette) milliQ
2 mL
0.3 mL
2 mL
?. [mild alkaline methylation]
Mix very well by shaking or vortexing. Let layers separate. The upper (hexane) layer should be clear
?. [mild alkaline methylation]
Collect the upper layer with a Pasteur pipette into a 10 ml glass vial. Avoid taking the water layer.
?. [mild alkaline methylation]
Add again  hexane, repeat 31-32, collect in the same vial
2 mL
?. [mild alkaline methylation]
Add again  hexane, repeat 31-32, collect in the same vial
2 mL
?. [mild alkaline methylation]
Evaporate hexane until complete dryness
?. [GC analysis    ]
Add   C12:0 FAME and ~  hexane or ethyl acetate Transfer samples into GC analysis vials.Store in  freezer until analysisHexane and ethyl acetate give comparable results. Make sure that C12:0 FAME is in the same solvent
50 µl
100 µl
-20 °C
?. [Total lipid extraction]
Add 1 ml of DCM and evaporate again.
1 mL