Sampling Process for Carbon (δ13C) and Nitrogen (δ15N) Isotopic Analysis
- Bone, antler or dentine is sampled using a Dremmel™ with a diamond wheel attachment. Depending on thickness of cortical bone, approximately 1-2cm2 should be sampled to provide around 500mg of bone. Although yields are highly variable this should produce more than enough collagen if organic material is preserved. During sampling every effort is made to avoid diagnostic areas of bone such as epiphyses and tuberosities, with cortical bone from areas such as long bone shafts targeted where possible.
- The sampled sections of bone are abraded either by air abrasion or burring with a drill. This removes any potential contaminants.
- Bones are weighed so that collagen yields can be calculated.
- Specimens are placed in individual test tubes and demineralised by submersion in 0.5M HCL. This acid is changed approximately every 2 days until demineralisation is complete, at which point samples are soft and floppy.
- Samples are rinsed using deioinsed water and placed in PH3 H2O. They are then gelatinised by being placed in pyrex test tubes and heated for 48 hours at 70 degrees Celsius.
- Once cooled, samples are filtered using ezee filters to remove the fluid which now contains gelatinised collagen. This is poured into pierced, sealed beakers and placed in a freezer overnight.
- Samples are then lyophilised (freeze dried) to leave just solid collagen.
- Collagen is weighed into tin capsules and all samples are analysed in duplicate as a minimum.
- Analysis is undertaken using an automated elemental analyzer coupled in continuous-flow mode to an isotope-ratio-monitoring mass spectrometer and the ratio of the heavier to the lighter isotope is measured to an internationally defined scale.
- Isotopic results are reported as δ values (carbon- δ13C and nitrogen- δ15N) in parts per 1000 or ‘permil’ (‰), where:
Ancient DNA Sampling Process
- Sample preparation
(a) Bone or antler
A variable speed Dremmel™ drill is used to process both bone and antler samples. To start, all samples are surface sanded with a disposable rotary cutting disc of the Dremmel tool to ensure removal of surface contaminating DNA. The drill is then used to cut ~ 1cm³ internal segment of bone or antler. The cut segment is then powdered using a diamond cutter to yield ~ 300 mg of powder. After each sample is collected, all drill parts are soaked in 10% bleach followed by 100% ethanol and UV treated to reduce problems arising from cross contamination.
One tooth or part of a tooth (usually the root) is sufficient to collect enough DNA for sampling. The surface of tooth samples are decontaminated by soaking in 10% bleach and rinsed in sterile water. Teeth are then wrapped in UV treated foil and broken into fragments using a mortar. The pestel and mortar are then used to generate dental powder from larger fragments. Between samples, pestel and mortars are soaked in 10% bleach followed by 100% ethanol and autoclaved.
- Sample digestion and DNA extraction
All powdered samples are collected in individual sterile 1.5 ml tubes and stored at - 4˚C until digestion. For digestion, each sample is incubated at 37˚C in a solution containing 1ml extraction buffer and 20 µl proteinase K. The solution dissolves the mineral and protein content to leave a clear solution. Samples are removed from the incubator and DNA is extracted using a QIAquick purification kit™.
* Steps i. and ii are conducted in a dedicated ancient DNA laboratory at the University of Durham and strict ancient DNA protocols are adhered to.
- DNA amplification and sequencing
DNA is amplified from individual samples by polymerase chain reaction (PCR) using a Qiagen Multiplex solution (Qiagen, Basel, Switzerland). The PCR product from each sample is viewed on an agarose gel to confirm whether or not DNA has been amplified. Samples that show presence of amplifiable DNA are sent to be sequenced, a process which allows the DNA code to be read.