TITLE: Sampling of tooth roots for ancient DNA
AUTHORS: Marcel Keller, Christiana L Scheib

[DESCRIPTION]
Protocol for the sampling of tooth roots of archaeological human remains for the extraction of ancient DNA.

[BEFORE_START]
Previous step:
This protocol follows the introduction of samples into the ancient DNA lab (documentation and changing bags).

Following step:
Proceed with the decontamination protocol.

Equipment and consumables:
 

 

[GUIDELINES]
Please read the general guidelines for working in the Ancient DNA protocol collection – University of Tartu, Institute of Genomics.

[STEPS]
SECTION: Preparation
7. Place down sheets of aluminum foil to cover the entire surface of the drill hood.
SECTION: Preparation
8. Make an L-shaped “wall” of folded foil that runs along the back of your work area and the side without the drill to keep flying dust from expanding across the hood.
SECTION: Preparation
9. Place down a smaller piece of foil for your working surface. Fold small pieces of foil that you can wrap around the beaks of the tooth extractor. Place a big weighing boat onto the working surface. Change this setup for each sample.
SECTION: Preparation
10. Gather clean, decontaminated tools for drilling and place them on the foil. 
SECTION: Sampling
12. 
 

SECTION: Sampling
13. Turn on the drill in “FWD” mode and choose a medium speed to prevent overheating/burning of the bone or dentine.
SECTION: Sampling
14. Put on clean sleeves by pushing your thumbs through the plastic close to the cuffs to keep the sleeve in place, then gloves on top. 
SECTION: Sampling
2. Wrap foil strips around the ends of your tooth extractor (for holding the tooth).
SECTION: Sampling
3. Hold the tooth with the tooth extractor and drill carefully a few millimeters above the end of the root.
SECTION: Sampling
4. When drilling is completed, put your samples in labeled bags. Wipe these down with a small amount of DNA Exitus before adding to your sample box.
SECTION: Clean-up after each sample
5. Rinse off the drill wheel with MilliQ water first then into your 6% bleach for a few minutes or while you are drilling the next tooth. Then rinse with water and finally ethanol before drying for use again.
SECTION: Clean-up after each sample
6. Carefully remove and throw away the weighing boat, small work surface foil, dirty gloves and sleeves; put on new gloves.
SECTION: Clean-up after each sample
7. For the next sample, start from step 9.
SECTION: Final clean-up
8. When finished for the day, wipe down the tooth extractors and drill spanner with DNA Exitus and put away. 
SECTION: Final clean-up
10. Leave drill bits and scrapers to soak in bleach while you throw away all foil and other consumables and wipe down all inner hood work surfaces and the outside of the drill with DNA Exitus and wipe dry with paper towels.
SECTION: Final clean-up
9. Use a clean toothbrush with a little DNA Exitus to remove sample powder from the top/inside of the drill head.


 

SECTION: Final clean-up
10. Make sure your metal tools are rinsed and dry and no bleach remains! 
Then put them away.
SECTION: Final clean-up
11. Turn on regular faucet water and carefully pour off thebleach, water, and ethanol from the tool decontamination station. 
 

SECTION: Final clean-up
12. Clean table surface with DNA Exitus and dry with paper towels.
SECTION: Final clean-up
13. Put down the glass and turn off the hood. 
SECTION: Final clean-up
14. Turn off the drill.
SECTION: Final clean-up
15. Take any trash and personal reagents with you.
SECTION: Preparation
1. Turn the hood on full power and open the glass. 



SECTION: Clean up
45. 
SECTION: Preparation
6. Label your bags and tubes:

 


 

 

SECTION: Preparation
5. Wipe down markers and pens with DNA Exitus.
SECTION: Preparation
4. To decontaminate drill bits, drill spanner, tweezers, dental scalers and toothbrush – Soak in 6% bleach for 5 minutes, rinse with MilliQ water, rinse with 70% Ethanol. Clean the drill spanner and tooth extractors with DNA Exitus.
SECTION: Preparation
3. Set up decontamination station: 
4 disposable 100 ml beakers
6% NaOCl (bleach) aliquot [2x] 
MilliQ water [1x]  
70% ethanol aliquot [1x]
SECTION: Preparation
2. Clean drill hood and table bench surfaces with DNA Exitus and wipe clean with a paper towel. 
SECTION: Preparation
1.1. Turn the hood on full power and open the glass.
SECTION: Sampling
11. 
 

SECTION: Sampling
1. If the tooth has calculus, use the dental scaler to remove it before sampling. Put the tooth into the mini grip bag and hold the tooth outside the bag with your fingers. Now push gently with scaler and collect all calculus inside the bag. Remove the tooth and pour the calculus on weighing paper. Use weighing paper to place calculus in a labeled 1.5 ml tube after taring.
SECTION: Sampling
15. Place the bag with your sample and the clean sample bag and tube into the hood behind the foil wall.
SECTION: Sampling
16. If the tooth has calculus, use the dental scaler to remove it before sampling. Put the tooth into the mini grip bag and hold the tooth outside the bag with your fingers. Now push gently with scaler and collect all calculus inside the bag. Remove the tooth and pour the calculus on weighing paper. Use weighing paper to place calculus in a labeled 1.5 ml tube after taring.
SECTION: Sampling
17. Wrap foil strips around the ends of your tooth extractor (for holding the tooth).
SECTION: Sampling
18. Hold the tooth with the tooth extractor and drill carefully a few millimeters above the end of the root.
SECTION: Sampling
19. When drilling is completed, put your samples in labeled bags. Wipe these down with a small amount of DNA Exitus before adding to your sample box.
SECTION: Clean-up after each sample
20. Rinse off the drill wheel with MilliQ water first then into your 6% bleach for a few minutes or while you are drilling the next tooth. Then rinse with water and finally ethanol before drying for use again.
SECTION: Clean-up after each sample
21. Carefully remove and throw away the weighing boat, small work surface foil, dirty gloves, and sleeves. Put on new gloves.
SECTION: Final clean-up
23. When finished for the day, wipe down the tooth extractors and drill spanner with DNA Exitus and put away.
SECTION: Clean-up after each sample
22. For the next sample, start from step 9.
SECTION: Final clean-up
24. Use a clean toothbrush with a little DNA Exitus to remove sample powder from the top/inside of the drill head.

 

SECTION: Final clean-up
25. Leave drill bits and scalers to soak in bleach while you throw away all foil and other consumables and wipe down all inner hood work surfaces and the outside of the drill with DNA Exitus and wipe dry with paper towels.