TITLE: CIDC_S16_NMR_Celegans_Extraction_Protocol
AUTHORS: Brianna M Garcia, Amanda  Shaver, Goncalo  Gouveia

[DESCRIPTION]
A sample preparation protocol for lyophilized C. elegans samples to be analyzed by NMR spectroscopy

[STEPS]
SECTION: Homogenization
1. Remove lyophilized C. elegans samples from -80 °C freezer


SECTION: Homogenization
2. Add approximately 200 µL of    Zirconia beads (BioSpec Cat. No. 11079110zx) to each sample.

SECTION: Homogenization
3. 
 

 

Using a FastPrep-96™ instrument (MP Biomedicals) equipped with QuickFlex™ Sample Holder  (or similar equipment) homogenize samples at 420 rcf  for 90 s 

SECTION: Homogenization
4. Place samples on dry ice for 90 s  to avoid overheating due and sample degradation. 

SECTION: Homogenization
5. Using a FastPrep-96™ instrument (MP Biomedicals) equipped with QuickFlex™ Sample Holder  (or similar equipment) homogenize samples at 420 rcf  for 90 s 
SECTION: Homogenization
6. Place samples on dry ice for 90 s  to avoid overheating due and sample degradation. 
SECTION: Homogenization
7. Using a FastPrep-96™ instrument (MP Biomedicals) equipped with QuickFlex™ Sample Holder  (or similar equipment) homogenize samples at 420 rcf  for 90 s 
SECTION: Homogenization
8. Store on dry ice and proceed with Extraction (Section 2) or store at -80 °C until ready to   process. 

SECTION: Sequential non-polar (i) and polar(ii) extraction
9. Add 500 µL of 100% IPA chilled to -20 °C  to each homogenized sample containing: (1) lyophilized C. elegans material and (2) zirconia beads

SECTION: Sequential non-polar (i) and polar(ii) extraction
10. Vortex each sample for 30 s to 1 min 

SECTION: Sequential non-polar (i) and polar(ii) extraction
11. Let sample sit at Room temperature for 15 min to 20 min 

SECTION: Sequential non-polar (i) and polar(ii) extraction
12. Add an additional 500 µL of 100% IPA chilled to -20 °C  to each homogenized sample containing: (1) lyophilized C. elegans material, (2) zirconia beads, and (3) 500 µL IPA. 

SECTION: Sequential non-polar (i) and polar(ii) extraction
13. Vortex each sample for 30 s to 1 min 
SECTION: Sequential non-polar (i) and polar(ii) extraction
14. Let sample sit at Room temperature for 15 min to 20 min 
SECTION: Sequential non-polar (i) and polar(ii) extraction
15. Store samples   (~12 hours) at -20 °C 

SECTION: Sequential non-polar (i) and polar(ii) extraction
16. Centrifuge samples for 30 min at 20800 rcf, 4 °C 

SECTION: Sequential non-polar (i) and polar(ii) extraction
17. Transfer via Pasteur pipette the supernatant of each centrifuged sample to a new 2 mL tube for the analysis of non-polar molecules.
SECTION: Sequential non-polar (i) and polar(ii) extraction
19. Add 1 mL of 80:20 Methanol:Water (MeOH:H2O) chilled to 4 °C to each tube containing (1) the remaining worm pellet and (2) zirconia beads. 

SECTION: Sequential non-polar (i) and polar(ii) extraction
20. Shake samples at 4 °C for 30 min 

SECTION: Sequential non-polar (i) and polar(ii) extraction
21. Centrifuge samples for 30 min at 20800 rcf, 4 °C 
SECTION: Sequential non-polar (i) and polar(ii) extraction
22. Transfer via Pasteur pipette the supernatant of each centrifuged sample to a new 2 mL tube for the analysis of polar molecules.
SECTION: Sequential non-polar (i) and polar(ii) extraction
18. Place all non-polar extracts in a CentriVap at Room temperature and monitor until completely dry.
 


SECTION: Sequential non-polar (i) and polar(ii) extraction
23. Place all polar extracts in a CentriVap at Room temperature and monitor until completely dry.
SECTION: Sequential non-polar (i) and polar(ii) extraction
24. Remaining worm pellet and zirconia beads can be stored at -80 °C for future protein and/or carbohydrate analysis or discarded appropriately.