TITLE: Tooth Sampling from the inner pulp chamber for ancient DNA Extraction
AUTHORS: Gunnar Neumann, Aida Andrades Valtuena, James Fellows Yates, Raphaela Stahl, Guido Brandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to obtain powder from the inner pulp chamber of teeth for the extraction of ancient DNA. It is ideal for the simultaneous isolation of both host endogenous and microbial DNA (e.g. for pathogens). </div></div>

[STEPS]
?. [Workstation preparation]
Place a sheet of aluminium foil under the hood.Place weighing paper and drill bits on a sheet of aluminum foil on an easily accessible clean surface outside of the hood. Alternatively, you may place drill bits and weighing paper within hood but be sure to cover them with aluminum foil or a paper towel to protect them from dust produced during sampling.Install the air extraction unit, if present, to reduce airborne dust during sample cutting.You will also need:2 ml Safe-Lock Biopur tubesWeighing trays/pans (optional)Clean and labelled sample bags according to the amount of samplesPaper towelsDiluted commercial bleach solution (1:10)UV-irradiated filtered/deionised water
Everything that comes into contact with the sample needs to be decontaminated in order to avoid cross-contamination between samples. Change gloves regularly, especially between different samples.
?. [Preparation of sample]
Place a sheet of aluminium foil in the UV chamber.Place the tooth on the aluminium foil, and place the labeled sample bag next to it (to avoid mixing up samples in case you work on more than one).UV- irradiate the tooth for 15 minutes on each side.
If possible, avoid touching the tooth directly. Use precision forceps or similar to handle each tooth individually.
Tip: it is possible to UV irradiate multiple teeth at once in the same UV chamber. Ensure each tooth is on a separate bit of foil, and keep the corresponding labelled sample bag next to it to prevent sample mix up.
?. [Preparation of sample]
Take a new 2 ml tube and label it accordingly. If this is the beginning of the sampling session, tare the precision scale with the tube placed in the center. Label a new sample bag with the sample ID.
To avoid contamination, tubes should remain closed except when the tooth powder is added.
?. [Preparation of sample]
Remove the tooth from the UV chamber and tranfer it to the aluminium foil in the hood. Check the tooth for calculus. If present, sample the deposit by following this protocol (ensure thorough clean up of work space before continuing).
?. [Sampling procedure]
On a weighing tray, hold the crown of the tooth with the pliers and use the electric drill with a diamond saw blade to cut the tooth at the cementum-enamel junction (CEJ) to separate the roots from the crown.Alternatively, you can use a manual jeweler's jigsaw for cutting. In this case, fix the tooth in a foil-covered bench vice.
?. [Sampling procedure]
Remove any sawing powder that may be trapped inside the pulp chamber and root canals by gently tapping onto the weighing tray. Hold the samples with pliers.
Powder from the sawing procedure should not be used for extraction as it make contain modern surface contamination and should be set aside (clearly labeled) or discarded.
?. [Sampling procedure]
Put the weighing tray/pan to the side. Take a fresh piece of weighing paper, slightly fold it mid-way and place it under the area where you want to sample in the inner pulp chambers.
Bone powder can be statically charged and difficult to transfer to tubes. By creasing the weighing paper in advance, it makes it easier to transfer the bone powder from the weighing paper into the tube when pouring.
?. [Sampling procedure]
Replace the diamond circular saw blade of the electric drill with a diamond ball bit.
?. [Sampling procedure]
Hold the crown with the pliers above the weighing paper. With the drill at low speed (and high torque), drill from inside the pulp chamber and collect the powder on the weighing paper. In addition, you can also sample from inside the roots, if needed.
Teeth are often used as material to screen for blood-borne pathogens. For this, it is better to sample the surfaces of the pulp chamber (instead of drilling deep into the dentine) because this is where the highly vascularized pulp tissue existed during life. Deeper sampling will result in the collection of more acellular/anuclear, non-blood associated dentine tissue and will reduce possible pathogen DNA signals as well as the amount of endogenous human DNA.
Pro-Tip: If residual dirt/soil is mixed in with your collected clean powder, you can physically separate them by carefully tipping the edges of the weighing paper repeatedly. In doing so, the different weights and particle sizes will easily separate. Discard the dirt particles.
?. [Weighing of tooth powder]
Transfer the clean powder bone into a labeled 2 ml Safe-Lock Biopur tube. The folded weighing paper will help guide the powder into the tube.Close the tube, wipe it with a damp paper towel and weigh the tube.Write down the weight in mg on the cap and on the side of the tube.Store the bone powder at until further processing.
Tooth powder can become statically charged. In such case, consider using an anti-static gun and apply to the weighing paper and tube.
Ideally you wish to collect around  to   of powder for extraction (if using our protocol for ancient DNA Extraction). If you have more than   then store it as a back-up in a separate tube.
-20 °C
?. [Weighing of tooth powder]
Take photos after sampling for documentation.Put tooth parts back into a new, UV irradiated, labeled bag.
The remaining material should be stored adequately so they can be passed on to other colleagues for additional analyses (e.g. isotope studies), to minimise unnecessary additional sampling of valuable ancient specimens.
?. [Decontamination]
Carefully clean the workspace and the equipment before sampling the next sample:
Everything that came in contact with the sample needs to be cleaned/decontaminated in order to avoid cross-contamination between samples.
?. [Decontamination]
Throw away disposable material such as aluminium foil and plastic weighing tray/pan.Clean surfaces and tools such as pliers with bleach solutionUse less aggressive DNA decontamination reagents (e.g. DNA away) for sensitive material such as electronic devices (e.g. handpiece of the drill, precision balance). Wipe off bleach with water-wet paper towels afterwards.
?. [Decontamination]
Saw blades and drill bits can be reused after careful cleaning: incubate them in bleach solution (1:10 dilution) for at least 2 minutes.       You can use a UV irradiated and bleached toothbrush for brushing the saw blade. Clean with UV irradiated water to remove all bleach and let it dry.      You can use a UV irradiated and bleached toothbrush for brushing the saw blade.
Check conditions of drill bits after ~5 uses and replace with new bits if there is excessive wear.
?. [Decontamination]
(or in case of UV irradiation of multiple samples at once,  ) and repeat the sampling procedure with the next sample.