May 21, 2026
Step-By-Step Procedure for Non-destructive DNA Extractions from Pinned or Preserved Arthropod Specimens
- Axel David Gonzalez Murillo1,
- Jackson H. Turner1,
- William Klingeman III1,
- John Moulton1
- 1University of Tennessee
- Axel Gonzalez Murillo
Protocol Citation: Axel David Gonzalez Murillo, Jackson H. Turner, William Klingeman III, John Moulton 2026. Step-By-Step Procedure for Non-destructive DNA Extractions from Pinned or Preserved Arthropod Specimens. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3mr8pl25/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: May 20, 2026
Last Modified: May 21, 2026
Protocol Integer ID: 317620
Keywords: dna extraction, extracting genomic dna, molecular genetic analysis of valuable museum specimen dna, valuable museum specimen dna, arthropod specimens this protocol, preserved arthropod specimen, dna quantification, arthropod specimen, dsdna high sensitivity assay, dna, availability of specimen, extracted fluid, genomic dna, using silica spin column chromatography, molecular genetic analysis, specimen
Abstract
This protocol describes a non-destructive method for extracting genomic DNA from pinned or preserved arthropod specimens that retain physical integrity as a voucher specimen for use in future morphological studies. Specimens used with this protocol were hand collected, netted, and pinned (from 1 to 90 years old), or recovered from adhesive traps and cleaned (see adhesive removal description at Protocol.io: https://dx.doi.org/10.17504/protocols.io.4r3l2rd6gq1y/v1). For DNA extraction, the sample is subjected to partial relaxation and minimal integumental manipulation, after which, internal soft tissues are digested for a minimum of 8 hours with Proteinase K in a custom lysis buffer containing Tris-HCl, EDTA, SDS, NaCl, and Triton X-100 at 52-55°C. DNA from extracted fluids is purified using silica spin column chromatography with a hydrogen bond weakening binding buffer, washed with ethanol-based buffer, and eluted in 50 μL of pre-warmed Tris buffer. DNA quantification is performed, (e.g., by using a Qubit 2.0 fluorometer with dsDNA High Sensitivity assay). This approach enables molecular genetic analysis of valuable museum specimen DNA, as well as associated eDNA, without compromising the availability of specimens for future taxonomic research.
Materials
- (hydroxymethyl) aminomethane hydrochloride (Tris) (pH 8.0): 50mM (2.5 ml)
- Ethylenediaminetetraacetic acid (EDTA) (pH 8.0): 50mM (5 ml)
- Sodium dodecyl sulfate (SDS) solution: 2% (5 ml)
- NaCl: 100mM (6.5 ml)
- Triton X-100: 0.50% (31.0 μl)
- Nucleus free water
- Sucrose (Optional): 50mM (2.5 ml: if used; reduce Triton X-100 volume, accordingly)
- Proteinase K (20 mg/ml)
- 1N HCl
- Binding buffer solution (containing guanidine thiocyanate, sodium perchlorate monohydrate, and absolute isopropanol)
- Wash buffer (10mM Tris, pH 8.0, 80% molecular grade or absolute ethanol)
Before start
Before beginning the extraction, prepare the lysis buffer with the following working concentrations for the desired lysis buffer volume (50 ml volume in the example given below):
- Complete the 50 ml volume with nucleus free water
- Note: addition of sucrose can act as a cushion to reduce DNA shearing and can increase osmotic pressure that will facilitate cell wall lysis. Addition of sucrose to the lysis buffer changes solution storage conditions. A filtered, sterile sucrose addition to lysis buffer solution can be stored at 4 C for 3 months; if left at lab ambient temperature, sucrose-supplemented lysis buffer should be used within about one week.
Reagent Preparation
Before beginning the extraction, prepare the lysis buffer with the following working concentrations for the desired lysis buffer volume (50 ml volume in the example given below):
- (hydroxymethyl) aminomethane hydrochloride (Tris) (pH 8.0): 50mM (2.5 ml)
- Ethylenediaminetetraacetic acid (EDTA) (pH 8.0): 50mM (5 ml)
- Sodium dodecyl sulfate (SDS) solution: 2% (5 ml)
- NaCl: 100mM (6.5 ml)
- Triton X-100: 0.50% (31.0 µl)
- Complete the 50 ml volume with nucleosfree water
- Sucrose (Optional): 50mM (2.5 ml: if used; reduce Triton X-100 volume, accordingly)
o Note: addition of sucrose can act as a cushion to reduce DNA shearing and can increase osmotic pressure that will facilitate cell wall lysis. Addition of sucrose to the lysis buffer changes solution storage conditions. A filtered, sterile sucrose addition to lysis buffer solution can be stored at 4 C for 3 months; if left at lab ambient temperature, sucrose-supplemented lysis buffer should be used within about one week.
Specimen Pre-treatment and Soft Tissue Digestion
- Relaxation: Pinned specimens should be relaxed in a humidity chamber before processing.
- Integument Opening: Use sterile forceps to gently open the genital chamber and pierce the integumental membrane in the terminal abdominal segment of the adult specimen. This step is helpful for enabling better lysis buffer access into internal soft tissues.
- Centrifuge Tube Setup: Place each specimen into an individual new 2.0 ml Eppendorf centrifuge tube.
- Addition of Lysis Buffer Components: Add 300 µL of lysis buffer and then 15 µL of Proteinase K (at a concentration of 20 mg/ml) to each tube.
- Incubation: Incubate the tubes in a dry heat block set between 52 °C and 55 °C for a minimum of 8 hours.
o Note: improved results are observed when, during the incubation step, the specimen(s) are inverting/reverting manually in a constant cycle for at least 1 minute; this step can be repeated every 2 hrs.
if possible.
Silica Column Reconditioning
(Optional Step: Reconditioning the silica column can be helpful if the columns have been in storage for more than 6 months.)
To ensure maximal binding affinity for the DNA, the PuroSPIN MICRO Silica Spin Columns can be reconditioned by treating with a strong acid solution (e.g., 1N (normal) HCl), particularly if columns have been stored for more than 6 months.
- Acid Treatment: Add 125 µL of 1N HCl to the silica column.
- Centrifugation: Centrifuge immediately for 1 minute at 13,000 × g and then discard the eluent.
- Buffer Treatment: Add 125 µL of lysis buffer to the column.
- Centrifugation: Centrifuge again for 1 minute at 13,000 × g and then discard the eluent.
DNA Binding
- Binding Buffer: Add 500 µL of binding buffer solution, (containing guanidine thiocyanate, sodium perchlorate monohydrate, and absolute isopropanol) to the extraction tube containing the lysate.
o guanidine thiocyanate (GITC): 5M (135 ml)
o sodium perchlorate monohydrate (NaClO 4): 5M (135 ml)
o isopropanol: absolute (300 ml)
- Mixing: Invert the tube several times to ensure the solution is thoroughly mixed.
- Loading the Column: Transfer the entire volume of the mixture into a new or reconditioned silica spin column that has been inserted into a 2 mL collection tube.
- Centrifugation: Centrifuge for 30 seconds at 8,000 x g and then discard the eluent.
o Note: Sample DNA is now contained within the silica spin column.
Washing and Drying
- Wash Steps: Perform two washes by adding 200-300 µL of wash buffer (10mM Tris, pH 8.0, 80% molecular grade or absolute ethanol) to the column.
- Centrifugation (Wash): Centrifuge for 30 seconds at 8,000 x g for each wash and discard the eluent after each spin.
- Drying: Dry the column membrane by centrifuging at 13,000 x g for 1 minute.
DNA Elution
- Transfer: Place the dried, DNA impregnated column into a fresh, clean 2 ml microcentrifuge collection tube.
- Elution Buffer: Add 50 µL of elution buffer (10 mM Tris; pH 8.0-8.5) directly to the center of the silica membrane.
o Note: For highest recovery, the elution buffer should be pre-warmed to 55 °C
- Incubation: Let the columns stand undisturbed for at least 1 minute.
- Final Centrifugation: Centrifuge at 13,000 × g for 30 seconds to collect the purified DNA.
o Note: Sample DNA is now contained within the collection tube: DO NOT DISCARD THIS ELUENT VOLUME.
Quantification
- Measurement: Quantify the resulting DNA using a Qubit 2.0 fluorometer, using the dsDNA High Sensitivity assay, following the manufacturer’s specific protocol for accurate results.