Dec 16, 2025

Public workspaceExtraction buffer stock solution (w/o Proteinase K)

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld1br7l5b/v1
Extraction buffer stock solution (w/o Proteinase K)
  • Anna Schmidt1,
  • Sarah Nagel1
  • 1Max Planck Institute for Evolutionary Anthropology
  • MPI EVA Ancient DNA Core Unit
Ancient DNA Core Unit
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ld1br7l5b/v1
External link: https://doi.org/10.1073/pnas.1314445110
Document Citation: Anna Schmidt, Sarah Nagel 2025. Extraction buffer stock solution (w/o Proteinase K). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld1br7l5b/v1
Manuscript citation:
Dabney, J., Knapp, M., Glocke, I., Gansauge, M., Weihmann, A., Nickel, B., Valdiosera, C.E., García, N., Pääbo, S., Arsuaga, J.L., & Meyer, M. (2013). Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proceedings of the National Academy of Sciences, 110, 15758 - 15763.
License: This is an open access document 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
Created: December 16, 2025
Last Modified: December 16, 2025
Document Integer ID: 235211
Keywords: Extraction Buffer, Ancient DNA extraction, ancient dna extraction from bone, ancient dna extraction, dna extraction, degraded dna from ancient bone, final extraction buffer, ultrashort dna fragment, complete mitochondrial genome sequence, extraction, extraction buffer stock solution, preparation of extraction buffer stock solution, degraded dna, middle pleistocene cave bear, day of extraction, ancient bone, dna, sediment, genome, throughput sequencing, bone
Funders Acknowledgements:
Max Planck Society
Grant ID: -
Abstract
This document describes the preparation of extraction buffer stock solution for ancient DNA extraction from bones, teeth, sediments and other materials, following the method described by Dabney et al. 2013 and refined by Rohland et al. 2018. To obtain the final extraction buffer, proteinase K solution (see separate document) must be added on the day of extraction.

References

Dabney, J., Knapp, M., Glocke, I., Gansauge, M.-T., Weihmann, A., Nickel, B., Valdiosera, C., García, N., Pääbo, S., Arsuaga, J.-L., & Meyer, M. (2013). Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proceedings of the National Academy of Sciences of the United States of America, 110(39), 15758-15763.

Rohland, N., Glocke, I., Ayinuer-Petri, A., & Meyer, M. (2018). Extraction of highly degraded DNA from ancient bones, teeth and sediments for high-throughput sequencing. Nature Protocols, 13, 2447-2461.
Troubleshooting
Note

The protocol provided here yields 243.75 ml stock solution (corresponding to 250 ml ready-to-use extraction buffer after addition of proteinase K), which suffices for 250 samples if 1 ml extraction buffer is used per sample.

Materials

Reagent/consumableSupplierCatalogue number
Reagents
Water Sigma Aldrich/Merck1153332500
0.5 M EDTA, pH 8.0AppliChemA4577, 1000
Tween-20Thermo Fisher Scientific11417160
Consumables
Square media bottle, sterile 250 mlVWR391-0629
50 ml serological pipetteCorning BV357550

Equipment

  • serological pipette controller (e.g. battery-powered pipetting aid ROTILABO, cat. no. TC16.1)


Protocol

1. Prepare the buffer in a 250 ml square media bottle by adding the following reagents. Use the glass pipette for transfer of large volumes (> 1 ml). Mix reagents by shaking the bottle.

ReagentVolume (ml)Final Concentration
0.5M EDTA, pH 8.0 2250.462 M
Water18.625
100% Tween-200.1250.051%

2. The buffer must be decontaminated using UV treatment. Instructions for UV-decontamination are provided in the Appendix.

Note
[Labeling]
Label the bottle with the buffer name, batch ID, date and the initials of the person who prepared the buffer.

Attention: Every single bottle prepared at the same day gets a new batch ID. Name the batches with Roman numerals (e.g. batch I, batch II, etc.)

3. Store the buffer at room temperature until used. Shelf life is at least one year from preparation.

Note
[Documentation] Note the lot numbers and the date and initials of the reagents used for the preparation in Labfolder (orange fields).


Appendix
Document
NAME
UV decontamination of reagents/buffers
CREATED BY
Elena Essel