Feb 20, 2025
Ancient DNA Extraction from Bones and Teeth
- Michael V. Westbury1,
- Alba Rey-Iglesia1,
- Andrea A. Cabrera1,
- Vanssy Li1,
- Deon de Jager1,
- Eline D. Lorenzen1
- 1Globe Institute, University of Copenhagen
- Lorenzen Lab
Protocol Citation: Michael V. Westbury, Alba Rey-Iglesia, Andrea A. Cabrera, Vanssy Li, Deon de Jager, Eline D. Lorenzen 2025. Ancient DNA Extraction from Bones and Teeth. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9ronpv3e/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 06, 2025
Last Modified: February 20, 2025
Protocol Integer ID: 119694
Keywords: ancient dna, teeth ancient dna extraction protocol, ancient dna extraction from bone, extracting ancient dna, ancient dna from bone, ancient dna extraction, final dna extract volume, spin column protocol, using monarch spin column, extraction, monarch spin column, spin column, dna, tooth powder, bone
Abstract
Ancient DNA Extraction Protocol (spin column-based)
- This protocol outlines the procedure for extracting ancient DNA from bone or tooth powder using Monarch spin columns. The final DNA extract volume will be 35 µL.
Based on:
Protocol initially written up by Michael V. Westbury on 20-08-2018.
Annotations added by Andrea A. Cabrera and Alba Rey-Iglesia on 08-11-2019.
The procotols.io version was created by Wenxi Li and Deon de Jager.
Protocol materials
Buffer EBQiagenCatalog #19086
Tween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1379-500ml
0.5M EDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #03690
Proteinase K, recombinant, PCR GradeMerck MilliporeSigma (Sigma-Aldrich)Catalog #RPROTKSOL-RO
Buffer PBQiagenCatalog #19066
5M Sodium acetateMerck MilliporeSigma (Sigma-Aldrich)
5M Sodium chlorideFisher ScientificCatalog #10609823
Sodium acetate buffer solution 3M pH 5.2Merck MilliporeSigma (Sigma-Aldrich)Catalog #S7899
Troubleshooting
Before start
- Ensure that ~50 mg of bone or tooth powder is prepared in advance (DO NOT treat with UV).
- UV-treat aliquots (PB buffer, PE buffer, EB buffer, ddH2O) for 10 minutes before use.
- Do not UV Eppendorf Tubes/PCR TUBES
- PE buffer: Add Molecular Grade Ethanol as indicated on the bottle before use.
- Use Qiagen EB buffer directly if preferred.
- Clean all surfaces with 5% bleach solution followed by 70% ethanol before and after use.
- Clean all equipment with 70% ethanol before and after use.
Buffer Preparation
Digestion Buffer
Choose the appropriate option based on [ProK].
Prepare fresh for each use.
0.5M EDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #03690
Proteinase K, recombinant, PCR GradeMerck MilliporeSigma (Sigma-Aldrich)Catalog #RPROTKSOL-RO
Option 1: Use 10mg/mL ProK
| Reagent | Volume per sample (μL) | Final Concentration | Master Mix (14 samples) | |
| 0.5M EDTA | 900 | 0.45 | 12,600 | |
| 10 mg/mL ProK | 25 | 0.25 | 350 | |
| ddH2O | 75 | N/A | 1,050 | |
| Total | 1,000 | 14,000 |
Option 2: Use 19mg/mL ProK
| Reagent | Volume per sample (μL) | Final Concentration | Master Mix (14 samples) | |
| 0.5M EDTA | 900 | 0.45 | 12,600 | |
| 19 mg/mL ProK | 13.15 | 0.25 | 184.1 | |
| ddH2O | 86.85 | N/A | 1,215.9 | |
| Total | 1,000 | 14,000 |
Modified Binding Buffer
Prepare in bulk
Option 1 (with 5M NaOAc)
- 500 mL Buffer PBQiagenCatalog #19066
- 9 mL 5M Sodium acetateMerck MilliporeSigma (Sigma-Aldrich)
- 1.25 mL 5M Sodium chlorideFisher ScientificCatalog #10609823
- Adjust to pH 4-5 (~2 mL Hydrochloric acid)
Option 2 (with 3M NaOAc, pH 5.2)
- 500 mL Buffer PBQiagenCatalog #19066
- 15 mL Sodium acetate buffer solution 3M pH 5.2Merck MilliporeSigma (Sigma-Aldrich)Catalog #S7899
- 2.5 mL 5M Sodium chlorideFisher ScientificCatalog #10609823
- No pH adjustment necessary as the 3M sodium acetate is pH buffered at pH5.2.
Note
- The NaOAc and NaCl can be added directly to the 500 mL of Buffer PB. Make sure to label it as Modified Buffer PB.
- Make individual aliquots in 50 mL Falcon tubes.
PE Buffer
Remember to add Molecular Grade Ethanol to PE when opening a new bottle. See instructions on
the PE buffer bottle.
EB Buffer
Appropriate for when using LoBind Eppendorf tubes.
- Buffer EBQiagenCatalog #19086 . Use as provided.
EBT Buffer *optional
Appropriate for when NOT using LoBind Eppendorf tubes.
- Buffer EBQiagenCatalog #19086
- Tween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1379-500ml at a final concentration of 0.05% (v/v)
Day 1 - Sample digestion
19h 2m
Optional: Pre-digestion
30m
In a 2 mL LoBind Eppendorf tube:
- Add 50 mg Bone Powder to 1 mL extraction buffer.
- Resuspend the bone powder by vortexing.
Incubate at 50 °C with constant rotation for 00:30:00 (use Parafilm to seal the lids).
30m
Digestion
Note
- The microcentrifuge tubes are the LoBind tubes.
- Use a rotation machine to keep the samples well mixed during digestion.
Remove Parafilm and centrifuge for 00:02:00 at maximum speed to pellet the bone powder.
2m
Remove the supernatant and add 1 mL fresh extraction buffer.
Resuspend the bone powder by vortexing.
Incubate at 37 °C with constant rotation for ~18:00:00 or overnight (use Parafilm to seal the lids).
18h
Day 2 - DNA purification
41m
Concentration of DNA with Amicon vivaspin 30kD column
Note
- Amicon® vivaspin column are also called centrifugal filter units.
- When centrifuging the Amicon columns, do not close the tubes too tight.
- MinElute spin columns can be used instead of Monarch columns, though the latter are preferable for smaller volumes, like that used in the elution step.
Remove Parafilm from eppendorf and centrifuge for 00:02:00 min at maximum speed to pellet the bone powder.
2m
Transfer the supernatant (~1 mL) to an Amicon® vivaspin 30kD column.
Centrifuge at ~4,000g until ~70 μL remains. Start with a 00:10:00 min centrifugation, then adjust in 00:05:00 min intervals as needed.
Note
- If the volume does not decrease to ~70 uL after several rounds of centrifuging, then repeat steps 7.4-7.5 until all the remaining sample is used.
15m
Mix the supernatant with 10x its volume of modified binding buffer in a Monarch column.
- Example: For 70 μL supernatant, add 700 μL of binding buffer.
- Mix by pipetting or inverting.
Centrifuge for 00:01:00 min at 8,000 RPM and discard the flowthrough.
- Tap the collection tube on a paper towel to get excess flowtrhough out before reassembling.
1m
Add650 µL PE buffer to the spin column.
Centrifuge at 5,000 RPM for 00:01:00 min and discard the flowthrough.
1m
Repeat steps 7.6-7.7.
Centrifuge at maximum speed for 00:01:00 min to dry the spin column.
1m
Place the column in a clean 1.5 mL LoBind Eppendorf tube.
DNA Elution
10m 30s
Add 17.5 µL EB buffer (or EBT buffer if not using LoBind tubes) to the silica membrane and incubate for 00:10:00 min at room temperature.
10m
Centrifuge at maximum speed for 00:00:30 seconds.
30s
Repeat steps 8.1-8.2 for a total of 35 μL DNA extract. Store DNA at -20ºC.
Qubit Quantification
- If desired, measure the DNA concentration using a Qubit. See Qubit protocol for details.
Protocol references
Allentoft ME, Sikora M, Sjögren KG, et al. Population genomics of Bronze Age Eurasia. Nature. 2015;522(7555):167-172. doi:10.1038/nature14507
Dabney J, Knapp M, Glocke I, et al. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proc Natl Acad Sci U S A. 2013;110(39):15758-15763. doi:10.1073/pnas.1314445110