Feb 25, 2022
CIDC_S16_NMR_Celegans_Extraction_Protocol
- 1University of Georgia
Protocol Citation: Brianna M Garcia, Amanda Shaver, Goncalo Gouveia 2022. CIDC_S16_NMR_Celegans_Extraction_Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.b2rbqd2n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: December 09, 2021
Last Modified: February 25, 2022
Protocol Integer ID: 55811
Abstract
A sample preparation protocol for lyophilized C. elegans samples to be analyzed by NMR spectroscopy
Materials
zirconia beadsBioSpec ProductsCatalog #11079110zx
IsopropanolFisher ScientificCatalog # A4614
MethanolFisher ScientificCatalog #A4564
WaterFisher ScientificCatalog #W64
Homogenization
Homogenization
7m 30s
7m 30s
Remove lyophilized C. elegans samples from -80 °C freezer
Add approximately 200 µL of 1.0 mm Zirconia beads (BioSpec Cat. No. 11079110zx) to each sample.
Equipment
FastPrep-96
NAME
High-throughput bead beating grinder and lysis system
TYPE
MP Biomedicals
BRAND
116010500
SKU
Equipment
QuickFlex™ Sample Holder
NAME
Adapter for 96 x 2 mL tube holder on FastPrep-96
TYPE
MP Biomedicals
BRAND
116010570
SKU
Using a FastPrep-96™ instrument (MP Biomedicals) equipped with QuickFlex™ Sample Holder (or similar equipment) homogenize samples at 420 rcf for 00:01:30
1m 30s
Place samples on dry ice for 00:01:30 to avoid overheating due and sample degradation.
1m 30s
Using a FastPrep-96™ instrument (MP Biomedicals) equipped with QuickFlex™ Sample Holder (or similar equipment) homogenize samples at 420 rcf for 00:01:30
1m 30s
Place samples on dry ice for 00:01:30 to avoid overheating due and sample degradation.
1m 30s
Using a FastPrep-96™ instrument (MP Biomedicals) equipped with QuickFlex™ Sample Holder (or similar equipment) homogenize samples at 420 rcf for 00:01:30
1m 30s
Store on dry ice and proceed with Extraction (Section 2) or store at -80 °C until ready to00:00:00 process.
Sequential non-polar (i) and polar(ii) extraction
Sequential non-polar (i) and polar(ii) extraction
7m 30s
7m 30s
Add 500 µL 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 00:00:30 to 00:01:00
1m 30s
Let sample sit at Room temperature for 00:15:00 to 00:20:00
35m
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.
Vortex each sample for 00:00:30 to 00:01:00
1m 30s
Let sample sit at Room temperature for 00:15:00 to 00:20:00
35m
Store samples Overnight (~12 hours) at -20 °C
Centrifuge samples for 00:30:00 at 20800 rcf, 4°C
30m
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
CentriVap
NAME
Benchtop Centrifugal Vacuum Concentrator
TYPE
Labconco
BRAND
7810010
SKU
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.
Shake samples at 4 °C for 00:30:00
30m
Centrifuge samples for 00:30:00 at 20800 rcf, 4°C
30m
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.