Feb 20, 2025

Public workspaceAncient DNA Extraction from Bones and Teeth

DOI
dx.doi.org/10.17504/protocols.io.x54v9ronpv3e/v1
  • 1Globe Institute, University of Copenhagen
Vanssy Li
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.x54v9ronpv3e/v1
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
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
ReagentBuffer EBQiagenCatalog #19086
ReagentTween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1379-500ml
Reagent0.5M EDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #03690
ReagentProteinase K, recombinant, PCR GradeMerck MilliporeSigma (Sigma-Aldrich)Catalog #RPROTKSOL-RO
ReagentBuffer EBQiagenCatalog #19086
ReagentBuffer PBQiagenCatalog #19066
Reagent5M Sodium acetateMerck MilliporeSigma (Sigma-Aldrich)
Reagent5M Sodium chlorideFisher ScientificCatalog #10609823
ReagentBuffer PBQiagenCatalog #19066
ReagentSodium acetate buffer solution 3M pH 5.2Merck MilliporeSigma (Sigma-Aldrich)Catalog #S7899
Reagent5M Sodium chlorideFisher ScientificCatalog #10609823
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
Buffer Preparation
Digestion Buffer
Choose the appropriate option based on [ProK].
Prepare fresh for each use.
Reagent0.5M EDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #03690
ReagentProteinase K, recombinant, PCR GradeMerck MilliporeSigma (Sigma-Aldrich)Catalog #RPROTKSOL-RO

Option 1: Use 10mg/mL ProK
ReagentVolume per sample (μL)Final ConcentrationMaster Mix (14 samples)
0.5M EDTA9000.4512,600
10 mg/mL ProK250.25350
ddH2O75N/A1,050
Total1,00014,000

Option 2: Use 19mg/mL ProK
ReagentVolume per sample (μL)Final ConcentrationMaster Mix (14 samples)
0.5M EDTA9000.4512,600
19 mg/mL ProK13.150.25184.1
ddH2O86.85N/A1,215.9
Total1,00014,000


Critical
Modified Binding Buffer
Prepare in bulk

Option 1 (with 5M NaOAc)
  • 500 mL ReagentBuffer PBQiagenCatalog #19066
  • 9 mL Reagent5M Sodium acetateMerck MilliporeSigma (Sigma-Aldrich)
  • 1.25 mL Reagent5M Sodium chlorideFisher ScientificCatalog #10609823
  • Adjust to pH 4-5 (~2 mL Hydrochloric acid)

Option 2 (with 3M NaOAc, pH 5.2)
  • 500 mL ReagentBuffer PBQiagenCatalog #19066
  • 15 mL ReagentSodium acetate buffer solution 3M pH 5.2Merck MilliporeSigma (Sigma-Aldrich)Catalog #S7899
  • 2.5 mL Reagent5M 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.
  • ReagentBuffer EBQiagenCatalog #19086 . Use as provided.
EBT Buffer *optional
Appropriate for when NOT using LoBind Eppendorf tubes.
  • ReagentBuffer EBQiagenCatalog #19086
  • ReagentTween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1379-500ml at a final concentration of 0.05% (v/v)
Optional
Day 1 - Sample digestion
Day 1 - Sample digestion
19h 2m
19h 2m
Optional: Pre-digestion
30m
Optional
In a 2 mL LoBind Eppendorf tube:
  • Add Amount50 mg SampleBone Powder to Amount1 mL extraction buffer.
  • Resuspend the bone powder by vortexing.
Optional
Incubate at Temperature50 °C with constant rotation for Duration00:30:00 (use Parafilm to seal the lids).
30m
Optional
Digestion

Note
  • The microcentrifuge tubes are the LoBind tubes.
  • Use a rotation machine to keep the samples well mixed during digestion.

Critical
Overnight
Remove Parafilm and centrifuge for Duration00:02:00 at maximum speed to pellet the bone powder.
2m
Remove the supernatant and add Amount1 mL fresh extraction buffer.
Resuspend the bone powder by vortexing.
Incubate at Temperature37 °C with constant rotation for ~Duration18:00:00 or overnight (use Parafilm to seal the lids).
18h
Day 2 - DNA purification
Day 2 - DNA purification
41m
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.

Critical
Remove Parafilm from eppendorf and centrifuge for Duration00:02:00 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 Duration00:10:00 min centrifugation, then adjust in Duration00:05:00 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 Duration00:01:00 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
AddAmount650 µL PE buffer to the spin column.
Centrifuge at 5,000 RPM for Duration00:01:00 min and discard the flowthrough.
1m
Centrifigation
Repeat steps 7.6-7.7.
Centrifuge at maximum speed for Duration00:01:00 min to dry the spin column.
1m
Centrifigation
Place the column in a clean 1.5 mL LoBind Eppendorf tube.
DNA Elution
10m 30s
Add Amount17.5 µL EB buffer (or EBT buffer if not using LoBind tubes) to the silica membrane and incubate for Duration00:10:00 min at room temperature.
10m
Incubation
Centrifuge at maximum speed for Duration00:00:30 seconds.
30s
Centrifigation
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