CIDC_S16_NMR_Celegans_Extraction_Protocol
Brianna M Garcia, Amanda Shaver, Goncalo Gouveia
Abstract
A sample preparation protocol for lyophilized C. elegans samples to be analyzed by NMR spectroscopy
Steps
Homogenization
Remove lyophilized C. elegans samples from -80°C freezer
Add approximately 200µL of 1.0 mm Zirconia beads (BioSpec Cat. No. 11079110zx) to each sample.
Equipment
| Value | Label |
|---|---|
| FastPrep-96 | NAME |
| High-throughput bead beating grinder and lysis system | TYPE |
| MP Biomedicals | BRAND |
| 116010500 | SKU |
| http://www.mpbio.com | LINK |
Equipment
| Value | Label |
|---|---|
| QuickFlex™ Sample Holder | NAME |
| Adapter for 96 x 2 mL tube holder on FastPrep-96 | TYPE |
| MP Biomedicals | BRAND |
| 116010570 | SKU |
| http://www.mpbio.com | LINK |
Using a FastPrep-96™ instrument (MP Biomedicals) equipped with QuickFlex™ Sample Holder (or similar equipment) homogenize samples at 420rcf,0h 0m 0s for 0h 1m 30s
Place samples on dry ice for 0h 1m 30s to avoid overheating due and sample degradation.
Using a FastPrep-96™ instrument (MP Biomedicals) equipped with QuickFlex™ Sample Holder (or similar equipment) homogenize samples at 420rcf,0h 0m 0s for 0h 1m 30s
Place samples on dry ice for 0h 1m 30s to avoid overheating due and sample degradation.
Using a FastPrep-96™ instrument (MP Biomedicals) equipped with QuickFlex™ Sample Holder (or similar equipment) homogenize samples at 420rcf,0h 0m 0s for 0h 1m 30s
Store on dry ice and proceed with Extraction (Section 2) or store at -80°C until ready to process.
Sequential non-polar (i) and polar(ii) extraction
Add 500µL of 100% IPA chilled to -20°C to each homogenized sample containing: (1) lyophilized C. elegans material and (2) zirconia beads
Vortex each sample for 0h 0m 30s to 0h 1m 0s
Let sample sit at Room temperature for 0h 15m 0s to 0h 20m 0s
Add an additional 500µL of 100% IPA chilled to -20°C to each homogenized sample containing: (1) lyophilized C. elegans material, (2) zirconia beads, and (3) 500µL IPA.
Vortex each sample for 0h 0m 30s to 0h 1m 0s
Let sample sit at Room temperature for 0h 15m 0s to 0h 20m 0s
Store samples (~12 hours) at -20°C
Centrifuge samples for 0h 30m 0s at 20800rcf,4°C,0h 0m 0s
Transfer via Pasteur pipette the supernatant of each centrifuged sample to a new 2 mL tube for the analysis of non-polar molecules.
Place all non-polar extracts in a CentriVap at Room temperature and monitor until completely dry.
Equipment
| Value | Label |
|---|---|
| CentriVap | NAME |
| Benchtop Centrifugal Vacuum Concentrator | TYPE |
| Labconco | BRAND |
| 7810010 | SKU |
| https://www.labconco.com/category/centrivap-cold-traps | LINK |
Add 1mL of 80:20 Methanol:Water (MeOH:H2O) chilled to 4°C to each tube containing (1) the remaining worm pellet and (2) zirconia beads.
Shake samples at 4°C for 0h 30m 0s
Centrifuge samples for 0h 30m 0s at 20800rcf,4°C,0h 0m 0s
Transfer via Pasteur pipette the supernatant of each centrifuged sample to a new 2 mL tube for the analysis of polar molecules.
Place all polar extracts in a CentriVap at Room temperature and monitor until completely dry.
Remaining worm pellet and zirconia beads can be stored at -80°C for future protein and/or carbohydrate analysis or discarded appropriately.
