Mar 04, 2026

Public workspaceARISE Insect DNA Extraction

DOI
https://dx.doi.org/10.17504/protocols.io.eq2lyw1dqvx9/v1
  • Kevin Beentjes1,
  • Nafiesa Ibrahim1,
  • Tara van der Plas1,
  • Cas Lekkerkerker1,
  • Thirza van de Wetering1,
  • Emma Wubben1
  • 1Naturalis Biodiversity Center
Kevin Beentjes
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2lyw1dqvx9/v1
Protocol Citation: Kevin Beentjes, Nafiesa Ibrahim, Tara van der Plas, Cas Lekkerkerker, Thirza van de Wetering, Emma Wubben 2026. ARISE Insect DNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyw1dqvx9/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: July 30, 2024
Last Modified: March 04, 2026
Protocol Integer ID: 104295
Keywords: DNA extraction, magnetic bead, DNA purification, insect, arise insect dna extraction magnetic, dna extraction, dna extraction with home, tissue dna extraction, throughput dna extraction, standard for tissue dna extraction, arise program at naturalis biodiversity center, suitable for insect, insect, naturalis biodiversity center, more information about arise, arise program, other taxonomic group, bead, arise
Funders Acknowledgements:
NWO Roadmap
Grant ID: 2020/ENW/00901156
Abstract
Magnetic bead DNA extraction with home-made chemicals, suitable for insects (both destructive and non-destructive sampling) and other taxonomic groups. This protocol is designed as a low-cost, high-quality, high-throughput DNA extraction (ca € 0,35 per sample).

This protocol is the standard for tissue DNA extractions within the ARISE program at Naturalis Biodiversity Center.
For more information about ARISE, visit https://www.arise-biodiversity.nl/
Materials
Chemicals:

  • Milli-Q water
  • Tris hydrochloride (1 M, pH 8)
  • EDTA (0.5 M, pH 8)
  • Guanidine thiocyanate
  • Trisodium phosphate dodecahydrate
  • Sodium chloride
  • Tween 20
  • Guanidine hydrochloride
  • 100% absolute ethanol
  • 96.2% technical grade ethanol
  • Isopropanol
  • Proteinase K (36 mg/ml)
  • NaOH / 5M HCl to adjust pH
  • SeraSil-Mag 400 magnetic beads (Sigma-Aldrich, GE29357371)
Troubleshooting
Safety warnings
When preparing or handling chemicals, wear protective clothing: lab coats, gloves and safety glasses. Be aware of the dangers of each chemical, more information can be found in the relevant Safety Data Sheets (SDS).

Chemicals used in this protocol must be disposed of as hazardous chemical waste through an approved facility, complying with local and national regulations. Do not pour solutions down the drain, as it is harmful to aquatic life.

Do not add bleach or acidic solutions directly to the sample-preparation waste. Buffers containing guanidine thiocyanate or guanidine hydrochloride can form toxic gasses when combined with bleach.
Buffer Preparation
Lysis Buffer A (per 100 ml)
  • Add 13.4 mL of 1 M tris hydrochloride (pH 8) to 50 ml MilliQ.
  • Add 10.6 mL of 0.5 M EDTA (pH 8).
  • Stir the mixture until all solids are dissolved.
  • Adjust to pH 9.0 with NaOH.
  • Bring to a final volume of 100 ml with MilliQ.
Lysis Buffer B (per 100 ml)
  • Add 3.4 g of guanidine thiocyanate to 50 ml MilliQ.
  • Add 17.4 g of trisodium phosphate dodecahydrate.
  • Add 0.4 g of sodium chloride.
  • Stir on a heated magnetic stirrer until all solids are dissolved.
  • Adjust to pH 9.0 with 5M HCl.
  • Bring to a final volume of 100 ml with MilliQ.
Tissue Lysis Additive (15% Tween 20) (per 100 ml)
  • Add 15 mL of Tween 20 to 85 ml MilliQ.
  • Stir the mixture gently, don't create bubbles.
Tissue Binding Buffer (per 100 ml)
  • Dissolve 26.3 g guanidine hydrochloride in 20 ml milliQ.
  • Stir until dissolved.
  • Adjust the volume to 50 ml with milliQ.
  • Add 50 mL 100% ethanol.
Elution Buffer (per 100 mL)
  • Add 1 mL 1 M Tris HCl (pH 8) to 75 ml milliQ .
  • Add 0.2 mL 0.5 M EDTA (pH 8).
  • Bring to a final volume of 100 ml with milliQ.
  • Vortex/stir briefly to mix.
Calibration of Tissue Binding Buffer
  • Prepare a lysis mixture by mixing 2.17 ml Lysis Buffer A, 2.17 ml Lysis Buffer B, 330 µl milliQ and 330 µl 15% Tween.
  • Make a calibration solution by adding 10 ml Tissue Binding Buffer to the 5 ml prepared lysis mixture in a 50 mL tube.
  • Adjust to pH 4.0 - 4.5 with 5M HCl, while making sure the mixture is at room temperature and that it has been thoroughly mixed.
  • Take note of the volume 5M HCl added (this will be approximately 350 µl 5M HCl).
  • Dispose of the used calibration solution.

Take the desired volume of Tissue Binding Buffer and add the same volume 5M HCl added to the calibration solution for every 10 ml of tissue binding buffer (e.g. if 350 µl HCl was added to the 10 ml calibration solution, adjusting the pH of 100 ml of Tissue Binding Buffer would require the addition of 3.5 ml HCl).

Verify you got the right pH by adding 10 ml of binding buffer to 5 ml of fresh lysis mixture and measure the pH. Continue measuring and adjusting the pH as needed, the final combination of Lysis Buffer A and B should be between pH 4.0 and 4.5. If a new Lysis Buffer B is made, the last verification step must be performed again.
Tissue Sampling and Lysis
Tissue Sampling

Note: For high throughput, DNA extraction can be performed in plates rather than individual tubes. Within ARISE animal tissues are processed in Nunc 96-wells plates (Avantor cat no. 732-2661), which are used for photography, tissue lysis and sample storage.

  • Add 50 μl of Lysis Buffer A into a plate or 1.5 ml eppendorf tube.
  • Place up to 40 mg of tissue (e.g. a single insect leg) into the plate / tube, small insects can be added to the buffer in their entirety for a non-destructive DNA extraction. This is at your own risk! Only applicable for hard-bodied insects, there is no guarantee that specimens are not damaged or destroyed during lysis.
  • Optional: Grind tissue with a tube pestle or cut the tissue with a scalpel. For non-destructive DNA extractions, skip this step.

Possible stop, store samples at 4 °C. At this step, photography of specimens is possible without the risk of buffers crystallizing (since Lysis Buffer B has not been added yet).
Imaging
Pause
Chemical Lysis

Important: The salts present in the Lysis Buffer B and the Tissue Binding Buffer can crystallize, heat the buffers at ca. 55°C for 10 minutes, or until the salts are dissolved. The entire process is scalable depending on initial sample weight or transferred lysate amount. Where relevant, scalable volumes are shown in brackets next to reagent volumes used (e.g. 2x volume), e.g. when using the extraction method on larger bulk samples.

Add a mix of the following buffers per sample:
  • 50 μl of Lysis Buffer B
  • 7.7 μl Tissue Lysis Additive
  • 7.7 μl ProtK Solution (36 mg/ml)

  • Incubate at 55°C, 400 RPM on a shaking incubator or with occasional vortexing, at least 3 hours or overnight.

Possible stop, store samples at 4 °C.
Pause
Overnight
DNA Purification
Note: For high throughput, DNA extraction in plates can be automated using a purification instrument (e.g. a ThermoFisher Scientific KingFisher Flex or a MolGen PurePrep 96) or a liquid handling platform.

Binding

  • Centrifuge the incubated samples down for 1 minute at max centrifuge speed at room temperature to pellet solid particles at the bottom of the plate / tube.
  • Transfer 75 µl of the supernatant to a deep well plate / tube.
  • Add 20 µl SeraSil-Mag 400 beads to each sample (make sure beads are properly shaken into suspension).
  • Add 150 μl (2x the lysate volume) of Tissue Binding Buffer to each sample.
  • Incubate 5 minutes at room temperature on a shaker.

When a non-destructive DNA extraction was done, for example for small insects that were added to the buffer in their entirety, the plate in which the lysis was done can be filled with ethanol and sealed to preserve the remaining specimen. For destructive extractions, the plate can be discarded.
Washing

  • Settle the beads on a magnetic rack and discard the supernatant.
  • Remove the plate / tube from the magnet and add 500 µl isopropanol.
  • Shake at room temperature at 500 rpm for 2 minutes.
  • Settle the beads on the magnet and discard the supernatant.
  • Remove the plate / tube from the magnet and add 400 µl 96% ethanol.
  • Settle the beads on the magnet and discard the supernatant.
  • Repeat last two steps to wash twice with 400 µl 96% ethanol.
  • Remove all ethanol and dry the beads for approximately 5-10 minutes at room temperature (or 50 °C).
Elution

  • Add 50-70 µl Elution Buffer (or MilliQ) to each sample and shake at 500 rpm for at least 5 minutes.
  • Settle the beads on the magnet and transfer the supernatant to a new plate / tube.
Protocol references
Based on the Mu-DNA protocol:
Sellers GS, Di Muri C, Gómez A, Hänfling B (2018) Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types. Metabarcoding and Metagenomics 2:24556.
https://doi.org/10.3897/mbmg.2.24556

We acknowledge support from the Dutch Research Council (NIEBA-ARISE project NWO Roadmap grant number 2020/ENW/00901156).