Jan 13, 2025

Public workspaceChelex-based DNA extraction protocol for insect museum vouchers

DOI
dx.doi.org/10.17504/protocols.io.bp2l6x54rlqe/v1
  • 1University of Illinois at Urbana-Champaign, United States of America;
  • 2Institute for Sustainable Plant Protection (IPSP), National Research Council, Italy
Valeria Trivellone
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bp2l6x54rlqe/v1
Protocol Citation: Morgan Brown, Sara Ottati, Valeria Trivellone 2025. Chelex-based DNA extraction protocol for insect museum vouchers. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6x54rlqe/v1
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: February 01, 2024
Last Modified: January 13, 2025
Protocol Integer ID: 94500
Keywords: microorganisms, voucher, museum, acid nucleic extraction, DNA, next-generation sequencing, phytoplasma, bacteria, leafhoppers, bleaching, chelex, Column-based extraction, qPCR, insect
Funders Acknowledgements:
U.S. National Science Foundation
Grant ID: DEB-2244871
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK

The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Museum biorepositories preserve precious material for scientific research in several fields such as biodiversity, phylogenetics, population genetics, toxicology, environmental monitoring and pathology.
About half of the 8.7 million described species on Earth are insects and harbor a diverse community of microorganisms representing different association types, such as parasitic, mutualistic, and commensal.
Voucher specimens may play an important role in tracking insect-microbiome associations.
Catalog records and voucher specimens are the primary source for: (1) tracking past associations and inferring evolutionary pathways; (2) documenting associations in no longer accessible collection sites; (3) revising the taxonomic status of the associates; (4) resampling the associations over time when new technologies become available.
Here we present a non-destructive, fast, inexpensive, non-toxic protocol for DNA extraction from insect museum specimens with the aim to obtain high quality nucleic acid and sufficient yield for vector-borne pathogens detection, quantification and characterization.
Image Attribution
Image source: Christopher H. Dietrich (University of Illinois at Urbana-Champaign)
Materials
ReagentUltraPure™ SDS Solution, 10%Thermo FisherCatalog #24730020 ReagentChelex-100 resinBio-Rad Laboratories
Reagent95% EtOHContributed by users
Reagent1M Tris-HCl, pH 8.0Invitrogen - Thermo FisherCatalog #15568025 ReagentEDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G

Insect specimen selection and preparation
Insect specimen selection and preparation
9m
9m
Transfer the the ethanol-preserved Sampleinsect specimen on a piece of paper towel to allow the ethanol to evaporate for a few minutes.

Note
For dry insect voucher, re-hydrate the Sampleinsect specimen in a humidifying chamberDurationOvernight


2m
Place Sampleinsect specimen in a 1.5 mL centrifuge tube containing Amount500 µL ethanol Concentration90 % (v/v) for Duration00:02:00 .

2m
Optional
Discard the ethanol and add Amount500 µL bleach Concentration2.5 % (v/v) (NaOCl - The Clorox Co., Oakland, CA, USA) for Duration00:05:00 at Temperature7.5 °C .
Note
The insect body must be fully immersed in the bleach solution.


5m
Discard bleach solution and rinse 3 X with Amount250 µL of sterile water, discard the water each time.
10m
DNA Extraction
DNA Extraction
19h 19m
19h 19m
Place the Sampleinsect specimen into a fresh 1.5 mL centrifuge tube.

5m
Pipet Amount50 µL TES buffer and Amount2 µL Proteinase K (Concentration10 mg/mL , Qiagen) into the tube.
Note
Prepare TES buffer (Tris-HCl Concentration20 millimolar (mM) pH 8.0 , EDTA Concentration10 millimolar (mM) , SDS Concentration0.5 % (v/v) )
For Amount10 mL TES add Amount0.2 mL EDTA Concentration0.5 Molarity (M) , Amount0.5 mL SDS Concentration10 % (v/v) , Amount0.2 mL Tris-HCl Concentration1 Molarity (M) , Amount9.1 mL water (MilliQ)





2m
Incubate Amount55 °C DurationOvernight ;
18h
Incubation
Overnight
Centrifugate the sample for a few seconds to spin down the liquid. With a pipet, transfer Amount50 µL of the liquid sample into a fresh tube.
Store the Sampleinsect specimen as appropriate for further morphological studies.

2m
Prepare Concentration25 Mass / % volume Chelex 100 suspension.
Note
Add 2.5 g Chelex 100 resin into Amount10 mL water (MilliQ or NANOpure)


15m
Place a flask containing a Concentration25 Mass / % volume Chelex 100 suspension on a magnetic agitator and mix.
2m
Pipet Amount40 µL of Chelex directly from the agitator into the tube with the sample.

2m
Incubate the tube at Amount55 °C for Duration00:30:00 . Do not shake the tube.

30m
Incubation
Gently invert the tube 5 times.
2m
Critical
Allow the solution to cool down to TemperatureRoom temperature .

15m
Critical
Centrifugate atCentrifigation14000 rpm for Duration00:01:00 .

2m
Centrifigation
Transfer the supernatant into a fresh 1.5 mL centrifuge tube.
Safety information
The present protocol do not include toxic chemicals

2m
Protocol references
1. Bleaching step
Arora, A. K., Pesko, K. N., Quintero-Hernández, V., Possani, L. D., Miller, T. A., & Durvasula, R. V. (2018). A paratransgenic strategy to block transmission of Xylella fastidiosa from the glassy-winged sharpshooter Homalodisca vitripennis. BMC biotechnology, 18, 1-10.
Greenstone, M. H., Weber, D. C., Coudron, T. A., Payton, M. E., & Hu, J. S. (2012). Removing external DNA contamination from arthropod predators destined for molecular gut‐content analysis. Molecular Ecology Resources, 12(3), 464-469.
2. Chelex protocol modified from
Casquet, J., Thebaud, C., & Gillespie, R. G. (2012). Chelex without boiling, a rapid and easy technique to obtain stable amplifiable DNA from small amounts of ethanol‐stored spiders. Molecular ecology resources, 12(1), 136-141.
Lienhard, A., & Schäffer, S. (2019). Extracting the invisible: obtaining high quality DNA is a challenging task in small arthropods. PeerJ, 7, e6753.