Neutral Lipid Methanol Chloroform Extraction_41878045

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title: "TheNessLab : Neutral Lipid Methanol-Chloroform Extraction"

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TheNessLab : Neutral Lipid Methanol-Chloroform Extraction

Created by Wen Chen on Feb 10, 2020

Use this protocol to extract neutral lipids from lyophilized samples.

Materials

• Accutherm extractor
• N2 blow down device
• at least 5.0 mg ± 0.05 of lyophilized sample x 3 replicates
• 1.5mL Safelock Eppendorf tubes
• Methanol (MeOH)
• Chloroform (CHCl₃)
• Pre-labeled and weighed 5mL glass vials
• Glass vial screw cap with Teflon lined septum

Preparation

1. Turn on the Accutherm extractor and set to 60 ⁰C
2. Turn on N₂ blow down device and set heat block to 40 ⁰C
3. Weigh out 5.0 mg ± 0.05 of each lyophilized, powdered sample into a 1.5 mL Safelock Eppendorf tube in triplicate and record the exact mass of each sample.
4. Prepare an extraction solution of MeOH:CHCl₃ (1:1). Prepare 1000 uL working solution per sample +1.
5. For each extract vial, prepare a screw cap by inserting a Teflon lined septum.
6. Pre-label two 5 mL glass screw cap vials for each extract.

Extraction

1. Add 500 mL extraction solution (MeOH:CHCl₃) to each Eppendorf tube.
2. Extract on Accuthem extractor at 60 ⁰C for 20 min with shaking at max speed (1500 rpm).
3. Spin down extracts in a microcentrifuge at max speed (16000g) for 5 min .
4. Transfer supernatant to a pre-labeled 5 mL glass vial, being careful not the touch the pellet.
5. Add 500 mL MeOH:CHCl3 to the pellet and repeat the extraction as before. Pool the supernatant with the first extract, being careful to pool extracts of the same sample.
6. Dry the pooled extracts on the nitrogen blow down device at 40 ⁰C until dryness (usually 5-10 min). Remove the samples, and weigh them. 

1. Be very careful when working with solutions containing chloroform. 

2. Check all Safelock tube seals before incubation in the Accutherm extractor. 

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