Oct 02, 2025
Proteinase K solution for ancient DNA extraction
- Anna Schmidt1,
- Sarah Nagel1
- 1Max Planck Institute for Evolutionary Anthropology
- MPI EVA Ancient DNA Core Unit
Document Citation: Anna Schmidt, Sarah Nagel 2025. Proteinase K solution for ancient DNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpm3j8gzp/v1
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: August 15, 2025
Last Modified: October 02, 2025
Document Integer ID: 224761
Keywords: ancient dna extraction from bone, ancient dna extraction, degraded dna from ancient bone, dna extraction, day of dna extraction, ultrashort dna fragment, ancient bone, degraded dna, complete mitochondrial genome sequence, dna, middle pleistocene cave bear, extraction, sediment, genome, extraction buffer stock solution, throughput sequencing
Funders Acknowledgements:
Max Planck Society
Grant ID: -
Abstract
This document describes the preparation of proteinase K 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. Proteinase K is added to the extraction buffer stock solution (see separate document) on the day of DNA 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
This protocol describes the preparation of up to 50 ml Proteinase K solution (10 mg/ml) in units of 10 ml.
Materials
| Reagent//consumable | Supplier | Catalogue number | |
| Reagents | |||
| Proteinase K, recombinant, PCR grade, lyophilised, 100 mg | Sigma Aldrich | 3115836001 | |
| TE buffer (decontaminated)*$ | self | ||
| Consumables | |||
| 50 ml Falcon tube with stand | Corning | 210261 | |
| 1.5 ml LoBind Tubes (decontaminated)** | Eppendorf | VB-0285 | |
| 25 ml serological pipette | Corning BV | 357525 | |
| 10 ml serological pipette | Corning BV | 357551 | |
| 5 ml serological pipette | Corning BV | 357543 | |
*See document in the Appendix for preparation of TE buffer.
$ See document in the Appendix for decontamination of reagents and buffers.
** See document in the Appendix for decontamination of material.
Safety information
[CAUTION: Proteinase K]
Proteinase K causes skin and eye irritation, respiratory irritation and may cause allergy or asthma symptoms or breathing difficulties if inhaled. Avoid breathing dust/fume/gas/vapours/spray and always wear protective gloves and eye protection when working with this chemical.
Always refer to the Material Safety Data Sheet attached (MSDS) for detailed information.
Equipment
- Serological pipette controller (e.g. battery-powered pipetting aid for glas pipettes, cat. no. TC16.1)
Protocol
1. Add 4.5 ml TE buffer to a vial containing 100 mg Proteinase K. Mix thoroughly by vortexing, then let the solution sit for 10 minutes to ensure the powder dissolves completely.
2. Transfer complete volume to a fresh 50 ml Falcon tube, add 5.5 ml TE buffer and mix thoroughly.
3. Prepare 1.0 ml aliquots in 1.5 ml LoBind tubes and store them in the freezer until use. Shelf life is at least 1 year from preparation.
Note
[Documentation]
Note the lot numbers and the date and initials written on the reagents used for the preparation in Labfolder (orange fields).
Note
[Labeling]
Label the tubes with "Proteinase K, 10 mg/ml", date and the initials of the person who dissolved the enzyme.
Appendix
ProtK_eng.pdf224KB