Apr 05, 2023

Public workspaceDecontamination of tooth roots/petrous bone cores for ancient DNA extraction

DOI
dx.doi.org/10.17504/protocols.io.eq2lynp4qvx9/v1
  • 1nstitute of Genomics, University of Tartu;
  • 2Institute of Forensic Medicine, University of Bern;
  • 3Institute of Genomics, University of Tartu;
  • 4University of Cambridge
Marcel Keller
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DOI: dx.doi.org/10.17504/protocols.io.eq2lynp4qvx9/v1
Protocol Citation: Marcel Keller, Christiana L Scheib 2023. Decontamination of tooth roots/petrous bone cores for ancient DNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lynp4qvx9/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: January 25, 2022
Last Modified: April 05, 2023
Protocol Integer ID: 57423
Keywords: ancient DNA, aDNA, archeogenetics, archaeogenetics, paleogenetics, palaeogenetics
Abstract
Protocol for decontamination of tooth roots and petrous bone cores from archaeological human remains for reduction of surface contaminations and increase of endogenous DNA prior to DNA extraction.
Guidelines
Please read the general guidelines for working in the Ancient DNA protocol collection – University of Tartu, Institute of Genomics.
Materials
Equipment and consumables:
AB
NumberEquipment and consumables
[# of samples]x250 ml tubes
3Disposable 100 ml beakers
[# of samples]Weighing boats (medium)
[# of samples]Weighing boats (small)
2 of eachToothbrushes, dental scaler, tweezers
250 ml racks
[# of samples]/6 (round up)Lid of multi-rack
[# of samples]x5 ml plus 100 ml for decontaminationNaOCl (6% v/v)
[# of samples]x5 ml plus 100 ml for decontaminationMilliQ water
[# of samples]x5 ml plus 100 ml for decontaminationEthanol (70%)
Lab equipment:
Laminar flow hood
[Scales]

Other consumables:
DNA ExitusPlus
Paper towels
Aluminum foil
Safety warnings
Reagents

NaOCl (bleach) solution (6%)
- H290 May be corrosive to metals.
- H314 Causes severe skin burns and eye damage.
- H411 Toxic to aquatic life with long lasting effects.
- EUH206 Warning! Do not use together with other products. May release dangerous gases (chlorine). Remove from surface after recommended incubation time with water-soaked tissue.


DNA ExitusPlus
H319 Causes serious eye irritation.

Ethanol
- H225 Highly flammable liquid and vapor.
- H319 Causes serious eye irritation.


Equipment
UV radiation
- UV radiation can damage eyes and can be carcinogenic in contact with skin. Do not look directly at unshielded UV radiation. Do not expose unprotected skin to UV radiation.
- UV emitters generate ozone during operation. Use only in ventilated rooms.


Before start
Previous step:
This protocol follows the sampling protocol (tooth roots and petrous bones).

Following step:
Proceed with the extraction protocol (chunk samples/high volume).

Equipment and consumables:
AB
NumberEquipment and consumables
[# of samples]x250 ml tubes
3Disposable 100 ml beakers
[# of samples]Weighing boats (medium)
[# of samples]Weighing boats (small)
2 of eachToothbrushes, dental scaler, tweezers
250 ml racks
[# of samples]/6 (round up)Lid of multi-rack
[# of samples]x5 ml plus 100 ml for decontaminationNaOCl (6% v/v)
[# of samples]x5 ml plus 100 ml for decontaminationMilliQ water
[# of samples]x5 ml plus 100 ml for decontaminationEthanol (70%)

Preparation
Preparation
Clean drill hood and table bench surfaces with DNA Exitus and rinse with water
Set up 100 ml beaker decontamination station:
1x NaOCl (bleach, 6% v/v)
1x MilliQ water
1x Ethanol
Place toothbrush and tweezers in bleach.
Prepare two 50 ml tubes per sample, label with sample ID and B for bleach and E for ethanol.
Add around Amount5 mL NaOCl (bleach, 6% v/v) to each B tube (sample needs to be submerged).

Add around Amount5 mL ethanol (70%) to each E tube (sample needs to be submerged).

Place paper towels on a drying rack (e.g. upside-down multi-rack lid) and tape in place. Label with sample IDs. Place in the hood on a piece of aluminum foil and UV the rack in the drill hood while decontaminating samples.
Decontamination
Decontamination

Note
Change gloves during the Decontamination steps whenever they get dirty or wet.


Note
Steps 10.1-10.3 only apply if the samples is visibly dirty.

Optional
Put out one medium weighing boat for each sample and label it in the corner.
Take sample to be used for extraction and place in weighing boat labeled with sample ID.
Remove surface dirt gently from the sample using the toothbrush soaked in NaOCl. Be careful with scrubbing to not spray bleach/dirt onto yourself or other samples.
Place samples in NaOCl (B tube) for Duration00:05:00 and shake while incubating.

5m
Pour excess NaOCl down the sink with normal faucet running to dilute the bleach, careful not to drop the samples.
Rinse with MilliQ water 3 times.


Shake, pour out excess water, and place sample in ethanol for Duration00:02:00 .

2m
Wash down sink with normal facet to dilute/remove remaining bleach in the drain.
Pour out excess ethanol from tubes into the sink and place back into the rack. Rinse with normal faucet water when finished.
Drying
Drying
2h
2h
In the hood, place paper towels in front of the rack for catching excess ethanol. One at a time, remove as much ethanol as possible before placing samples onto the square with the corresponding sample ID on drying rack.
Leave UV on while allowing the samples to dry at least for Duration02:00:00 or DurationOvernight .

2h

Note
Either continue on the same day with the extraction protocol or stop here:

To continue, see with protocol "ancient DNA extraction (chunk samples/high volume)".

To stop, prepare weighing boats (small), one for each sample, and label with sample ID. Tare scale with small weighing boat labeled with the sample number, place tooth/petrous core on and record weight. Place sample in a respective tube (5 ml tube for <250 mg; 15 ml tube for >250 mg).