Feb 25, 2022

Public workspaceCIDC_S16_NMR_Celegans_Extraction_Protocol

DOI
https://dx.doi.org/10.17504/protocols.io.b2rbqd2n
  • Brianna M. Garcia1,
  • Amanda Shaver1,
  • Goncalo Gouveia1
  • 1University of Georgia
Brianna M Garcia
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DOI: https://dx.doi.org/10.17504/protocols.io.b2rbqd2n
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
Keywords: nmr, sample preparation protocol
Abstract
A sample preparation protocol for lyophilized C. elegans samples to be analyzed by NMR spectroscopy
Materials
Reagentzirconia beadsBioSpec ProductsCatalog #11079110zx
ReagentIsopropanolFisher ScientificCatalog # A4614
ReagentMethanolFisher ScientificCatalog #A4564
ReagentWaterFisher ScientificCatalog #W64

Troubleshooting
Homogenization
7m 30s
Remove lyophilized C. elegans samples from Temperature-80 °C freezer


Add approximately Amount200 µL of Thikness1.0 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 Centrifigation420 rcf for Duration00:01:30

1m 30s
Place samples on dry ice for Duration00:01:30 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 Centrifigation420 rcf for Duration00:01:30
1m 30s
Place samples on dry ice for Duration00:01:30 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 Centrifigation420 rcf for Duration00:01:30
1m 30s
Store on dry ice and proceed with Extraction (Section 2) or store at Temperature-80 °C until ready toDuration00:00:00 process.

Sequential non-polar (i) and polar(ii) extraction
7m 30s
Add Amount500 µL of 100% IPA chilled to Temperature-20 °C to each homogenized sample containing: (1) lyophilized C. elegans material and (2) zirconia beads

Vortex each sample for Duration00:00:30 to Duration00:01:00

1m 30s
Let sample sit at TemperatureRoom temperature for Duration00:15:00 to Duration00:20:00

35m
Add an additional Amount500 µL of 100% IPA chilled to Temperature-20 °C to each homogenized sample containing: (1) lyophilized C. elegans material, (2) zirconia beads, and (3) Amount500 µL IPA.

Vortex each sample for Duration00:00:30 to Duration00:01:00
1m 30s
Let sample sit at TemperatureRoom temperature for Duration00:15:00 to Duration00:20:00
35m
Store samples DurationOvernight (~12 hours) at Temperature-20 °C

Centrifuge samples for Duration00:30:00 at Centrifigation20800 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 TemperatureRoom temperature and monitor until completely dry.
Equipment
CentriVap
NAME
Benchtop Centrifugal Vacuum Concentrator
TYPE
Labconco
BRAND
7810010
SKU


Add Amount1 mL of 80:20 Methanol:Water (MeOH:H2O) chilled to Temperature4 °C to each tube containing (1) the remaining worm pellet and (2) zirconia beads.

Shake samples at Temperature4 °C for Duration00:30:00

30m
Centrifuge samples for Duration00:30:00 at Centrifigation20800 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 TemperatureRoom temperature and monitor until completely dry.
Remaining worm pellet and zirconia beads can be stored at Temperature-80 °C for future protein and/or carbohydrate analysis or discarded appropriately.