Bone and tooth collagen extraction for stable isotope analysis and radiocarbon dating

Place the skeletal material to sample on a clean sheet of aluminium foil in a well-ventilated space.

With a clean mini diamond saw mounted on a rotary multifunction tool (e.g. Dremel 3000 or Proxxon), carefully cut a piece of the skeletal material aiming for the appropriate mass. The bone powder created during this step can be kept in the foil for further analysis of the material.

With a diamond drill bit, mechanically clean the bone/tooth surface by removing approximately 2mm of its outer surface.

The saws and drill bits can be cleaned by being soaked in an ultrasonic bath in DI-water, then in ethanol, then in water again (Repeat until the water is clear), 5 minutes each time.

Place the samples in a small container. Label the container with the sample ID on several surfaces, and then add DI-water. Sonicate the samples in the container for approximately 3 minutes, avoiding temperature increase. The water may become murky.

Remove the water from the container, rinse the samples 1 to 3 times and add fresh DI-water. Repeat until the water in the container remains clear. In general, two sonification steps suffice.

Let the samples dry in the container placed in a fume hood.

Weight the sample once it is dry.

Insert the bone or dentin sample in a reaction tube with a screw cap*.

In a fume hood, add approximately 10 mL of the diluted HCl solution to the reaction tube, ensuring the sample is fully submerged. For well-preserved samples, a 0.5M HCl solution at room temperature can be used.

If the sample seems fragile or highly degraded, consider working at low temperatures (e.g. 4°C ) and/or with a more dilute acid solution (e.g. 0.2M HCl solution).

Do not close the tubes tightly as CO₂forms during the demineralisation.

Every two to three days, replace the HCl solution in the tube with fresh HCl. If no bubbling is observed within 30 minutes after the addition of fresh HCl at room temperature (1h 0m 0s if HCl is stored at 4°C ), the sample can be considered as demineralised.

Once the skeletal material is demineralised (i.e. no bubbling), rinse it in the tube three times with DI-water. Rinse also the edges of the tube. If the sample is fragmented, use an Ezee filter.

If the NaOH step is not directly following, the rinsed demineralised sample can be preserved in the fridge in DI- water. If the sample is crumbly after the demineralisation, it is also possible to remove the DI-water and let the sample sit in the freezer.

Remove any DI-water from the tubes with the samples. If the sample was frozen, wait for it to thaw and remove the excess of DI-water.

Add 0.25 M NaOH solution making sure to cover the sample.

Let the sample in contact with the NaOH solution for 0h 15m 0s. Discard the NaOH solution from the tube using a Paster pipette or an Ezee filter if the sample consists of small fragments.

Rinse the sample and the edges of the tube 3 times with DI-water.

Add the diluted solution of HCl (same concentration as for the demineralisation step). Make sure the solution covers the sample.

Let the sample and the solution react for 0h 5m 0s.

Rinse the sample three times with DI-water.

Add the pH 3 HCl solution to the tubes. Make sure it largely covers the samples.

Transfer the tubes to a heat-resistant rack.

Heat the sample in pH 3 HCL solution at 70°C for 48h 0m 0s.

While waiting for the samples to solubilise, label the glass vials for each sample and weigh them. Also, prepare small pieces of parafilm to cover each glass vial.

Once the samples are solubilised, filter the liquid samples by slowly pressing the Ezee filters into the sample tubes. Then transfer the filtered liquid to the pre-weighed glass vials designated for each sample.

After the initial filtration, some of the collagen solution (pH 3 HCl solution) may remain at the bottom of the tube. To maximise recovery, add more of the pH 3 HCl solution to the tube. Then, proceed to to filter the solution again.

Add a piece of parafilm on top of each glass vial and pierce it several times with a sharp tool (e.g. a clean toothpick for each sample).

Put a cap above the parafilm and place the glass vials with the filtered samples in a freezer overnight.

Remove the caps from the vials and place the frozen samples in a freeze-dryer for at least 24 hours.

Once you obtained fully dry collagen, remove the samples from the freeze-drier.

After removing the parafilm, place the caps on the glass vials to prevent the collagen from taking up humidity.

Weight the dry collagen in the glass vials and calculate the collagen yield.

The collagen can be stored in a constantly dry environment (e.g. a freezer).