TITLE: Ancient DNA Extraction from Dental Calculus with Consolidant Removal
AUTHORS: Franziska Aron, Courtney Hofman, Zandra Fagernäs, Irina Velsko, Guido Brandt, Christina Warinner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Silica-based total DNA extraction protocol optimised for the recovery of ultra-short DNA molecules from archaeological dental calculus, modified from Dabney </span><span style = "font-style:italic;">et al.</span><span> (2013) </span><span style = "font-style:italic;">PNAS</span><span> (doi: </span><a href="https://dx.doi.org/10.1073/pnas.1314445110" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">10.1073/pnas.1314445110</span></a><span>) and adapted for dental calculus by Mann </span><span style = "font-style:italic;">et al.</span><span> (2018) </span><span style = "font-style:italic;">Scientific Reports </span><span>(doi: </span><a href="https://doi.org/10.1038/s41598-018-28091-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1038/s41598-018-28091-9</span></a><span>). </span></div><div class = "text-block">This protocol includes an optional acetone wash step to remove conservation consolidants (e.g. glue) from dental calculus that can sometimes be found on samples archived in museum collections.</div></div>

[STEPS]
?. [Day 1: Preparation of reagents (Buffer Prep Room)]
Prepare cleaned workspace with all necessary reagents and equipment.
If lab-wide large-batch pre-prepared reagent stores are used, ensure to have made personal stock-aliquot of reagents such as UV-Water, EDTA, sodium acetate, and proteinase K in amounts sufficient for this extraction.
?. [Day 1: Preparation of reagents (Buffer Prep Room)]
Prepare binding buffer calculating .  ABC1Reagent [Stock Concentration]Final ConcentrationVolume/reaction2GuHCl (1 mol=95.53 g)5 M4.77 g3UV HPLC-water6 mL4Isopropanol (100%)40%4 mL5Total10 mL
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ABC1Reagent [Stock Concentration]Final ConcentrationVolume/reaction2GuHCl (1 mol=95.53 g)5 M4.77 g3UV HPLC-water6 mL4Isopropanol (100%)40%4 mL5Total10 mL
?. [Day 1: Preparation of reagents (Buffer Prep Room)]
Weigh GuHCl and transfer into a glass bottle.
If you want to clean the area where GuHCl was used, first use water and then bleach solution. Do not use bleach directly as it reacts with GuHCl to produce toxic chlorine gas!
?. [Day 1: Preparation of reagents (Buffer Prep Room)]
Add UV-irradiated HPLC water (6 mL).
This reaction is endothermic and the tube will become very cold. Be aware of the unusual  'slushy' way of dissolving.
?. [Day 1: Preparation of reagents (Buffer Prep Room)]
Gently shake horizontally in order to get the salt dissolved. If necessary, apply short ( ) bursts in microwave (~400W) keeping the tube slightly unscrewed. Wait until bottle cools down between microwave bursts.
?. [Day 1: Preparation of reagents (Buffer Prep Room)]
Pipette isopropanol (4 mL) to reach the final reaction volume ().
10 mL
?. [Day 1: Preparation of reagents (Buffer Prep Room)]
Prepare wash buffer by pipetting  ethanol to the wash buffer from the High Pure Viral Nucleic Acid kit following manufacturer's instuctions and make an aliquot calculating .
40 mL
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?. [Day 1: Preparation of reagents (Buffer Prep Room)]
Prepare TET elution buffer by making an aliquot of TE-buffer calculating  and pipette Tween-20 accordingly to reach  concentration to make 'TET'.
[/ reaction]
Because Tween-20 is highly viscous, we dilute it 1:10 in  UV-HPLC water, and use this 10% dilution to add Tween-20 to the TE-buffer
?. [Day 1: Preparation of reagents (Buffer Prep Room)]
Irradiate all buffers with UV for   without the lids.
UV irradiation can be done together with solutions from steps 1 (binding buffer), 4 (wash buffer), and 5 (TET buffer).
?. [Day 1: Preparation of reagents (Buffer Prep Room)]
Store binding buffer in a fridge at  overnight for day 4.
4 °C
Label the bottle accordingly with the name, date and for the calculated amount of reactions. Buffer can be stored in a fridge for up to four weeks. Seal bottle with parafilm to avoid evaporation.
?. [Day 1: Preparation of reagents (Buffer Prep Room)]
Dilute proteinase K powder () in   UV HPLC-water to a working concentration of  /  .
100 mg
10 mL
10 mg
1 mL
Store proteinase K solution at   for use on day 2.
?. [Day 1: Sample DNA decontamination (DNA extraction room)]
UV sterilize calculus. Irradiate samples for  , then shake the samples and irradiate for another  .
Aim for 2-5 mg starting material.
If samples were treated with acetone, just UV sterilize the calculus for 30sec.
If the sample is in powder-form, irradiate in the original tube with the lid open. If the sample is in large pieces, place calculus in an aluminium foil boat in the crosslinker to maximize exposure.
?. [Day 1: Sample DNA decontamination (DNA extraction room)]
If the calculus was in a boat for irradiation place it back in a 1.5 mL or 2 mL tube. Add  of 0.5 M EDTA to each sample, to remove surface contaminants.
1 mL
This step should be skipped if the purpose of the study is to detect infectuous pathogens, as these are more likely to be located on the outer layer of the calculus deposit (however, this may lead to a higher proportion of environmental contaminants in the data).
?. [Day 1: Sample DNA decontamination (DNA extraction room)]
Vortex and incubate on rotator at room temperature for  .
The incubation step can lead to some sample loss, as the calculus will start dissolving and releasing DNA. If the calculus is very powdery, the supernatant can be removed directly after vortexing, without any incubation, because the calculus solubilizes within the 15 min and you lose DNA while removing the EDTA.
?. [Day 1: Sample DNA decontamination (DNA extraction room)]
Spin tubes   at .
Centrifuge: 18500 34
?. [Day 1: Sample DNA decontamination (DNA extraction room)]
Transfer supernatant to a separate tube, labelled "EDTA-wash". Store the "EDTA-wash" tube at  for potential future analyses.
-20 °C
?. [Day 1: Decalcification (DNA Extraction Room)]
Add  of fresh 0.5 M EDTA to each sample pellet.
1 mL
?. [Day 1: Decalcification (DNA Extraction Room)]
Seal tubes with Parafilm, rotate   with low overhead rotation speed (e.g., 12-16 rpm)  at  room temperature. If the tubes will be exposed to UV irridation, such as whole-room irridation for sterilization, be sure to cover them with foil or otherwise shield them.
[(12-18h)]
Post-incubation sample suspended in EDTA can safely be stored in a freezer (  ) before isolation and clean-up.
?. [Day 2-3: Protein digestion (DNA Extraction Room)]
Remove Parafilm and make sure that the tubes have not leaked (if they have leaked, clean them).
The appearance of the calculus will vary depending on its initial state. Larger pieces will have barely changed at all, while powder will be almost gone.
?. [Day 2-3: Protein digestion (DNA Extraction Room)]
Briefly centrifuge tubes to remove any liquid from the lids.
?. [Day 2-3: Protein digestion (DNA Extraction Room)]
Add  of 10 mg/ml proteinase K to each tube.
50 µl
?. [Day 2-3: Protein digestion (DNA Extraction Room)]
Vortex tubes, seal with Parafilm, and continue decalcification by rotating at low overhead rotation speed (e.g., 12-16 rpm)  at  room temperature for 24-48 hours.
After starting the incubation, we recommend beginning preparations for day 4, such as pre-labelling the falcon tubes that will be used in step 18.
Decalcification time can be adapted, so that the sample is completely decalcified before starting DNA isolation. After complete decalcification, the sample should appear buoyant and feathery, or disappear altogether.
Post-incubation suspension can safely be stored in a freezer (  ) before isolation and clean-up.
?. [Day 4: DNA isolation and clean-up (DNA Extraction Room)]
Prepare cleaned workspace with all necessary reagents and equipment.
?. [Day 4: DNA isolation and clean-up (DNA Extraction Room)]
For each reaction prepare one   Falcon tube, one High Pure Extender Assembly (i.e. Falcon tube from kit containing funnel and purification column), two collection tubes from the kit, and one LoBind tube for final elution step.
50 mL
1.5 mL
?. [Day 4: DNA isolation and clean-up (DNA Extraction Room)]
In every  Falcon tube pipette  binding buffer and  sodium acetate (UV-irradiated). Mix by inversion and measure pH (should be 5-6).
50 mL
10 mL
400 µl
Add more sodium acetate if the pH is too high. If the pH is too low you can add sodium hydroxide.
?. [Day 4: DNA isolation and clean-up (DNA Extraction Room)]
Remove parafilm from extraction tubes, then spin the tubes for  at  to pellet calculus.
Centrifuge: 18500 34
If pellet is not solid, repeat centrifugation.
?. [Day 4: DNA isolation and clean-up (DNA Extraction Room)]
Pipette supernatant to respective  Falcon tube, mix contents by inversion. If pellet is too fragile, repeat centrifugation before transferring supernatant. Store the calculus pellet at .
50 mL
-20 °C
?. [Day 4: DNA isolation and clean-up (DNA Extraction Room)]
Pipette binding buffer/extract mix to High Pure Extender Assembly.
?. [Day 4: DNA isolation and clean-up (DNA Extraction Room)]
Spin at a maximum of  for
Centrifuge: 1500 33
This RPM is specific to a 50 mL Thermo Scientific TX-400 Swinging Bucket Rotor. As this is a swing rotor, the rpm value maybe inconsistent for other models. Therefore this value must be adjusted on a per-machine basis. Convert the rpm to rcf (g) and determine the appropriate rpm for your instrument.
You can also turn the tube 180o after   to ensure the liquid does not get stuck on the inner rim of the funnel
During this centrifugation step, we recommend preparing downstream steps, such as labelling of final elution tubes.
?. [Day 4: DNA isolation and clean-up (DNA Extraction Room)]
Pipette any liquids remaining in the funnel onto the column. Remove funnel from column and insert the column into a fresh 2 mL collection tube.
The flow-through stays in the collection tube of the 50mL High Pure Extender Assembly and can be discarded.
?. [Day 4: DNA isolation and clean-up (DNA Extraction Room)]
Dry spin the column in the collection tube for  at .
Centrifuge: 18500 34
?. [Day 4: DNA isolation and clean-up (DNA Extraction Room)]
Pipette  wash buffer from the High Pure Viral Nucleic Acid kit to the column, and spin at  for .
450 µl
Centrifuge: 8000 34
?. [Day 4: DNA isolation and clean-up (DNA Extraction Room)]
Remove column from the collection tube, discard the flow-through and the old collection tube, and put the column into a fresh 2 mL collection tube.
?. [Day 4: DNA isolation and clean-up (DNA Extraction Room)]
Repeat washing step once () reusing the collection tube, and discard flow-through.
Discard flow-through in one of two following ways: Remove all liquid in the collection tube with a pipette,  orPour off the liquid into a fresh waste tube, and pat the rim of the collection tube dry on a paper tissue or towl. Use just one spot on the paper tissue per sample. Be careful not to touch the rim of the tube on the waste container. Be sure to clean the surface with DNA Away or bleach after discarding the paper.
?. [Day 4: DNA isolation and clean-up (DNA Extraction Room)]
Dry spin at  for
Centrifuge: 18500 34
To ensure the liquid does not get stuck on the inner rim of the funnel, you can optionally spin for   , turn the tube 180o, and dry spin for another
?. [Day 4: Elution (DNA Extraction Room)]
Transfer column to a fresh   LoBind Tube, pipette  of Tris-EDTA-tween (TET) to the center of column, incubate for  on the benchtop, and spin  at  to elute the DNA.
1.5 mL
50 µl
Centrifuge: 18500 34
Eluted DNA will be stored in this tube. Label tube on top and side accordingly.
?. [Day 4: Elution (DNA Extraction Room)]
Repeat elution step for a total elution volume of  TET.
100 µl
?. [Day 4: Elution (DNA Extraction Room)]
Store the DNA extracts at  .
-20 °C
?. [Day 1: OPTIONAL Sample Consolidant Removal (Laboratory fume hood)]
Add  of acetone per sample
500 µl
Acetone wash is optional, and should only be used if the presence of consolidants (synthetic glues, conservation chemicals etc.) is observed on samples.
?. [Day 1: OPTIONAL Sample Consolidant Removal (Laboratory fume hood)]
Soak for  with an open lid under the fume hood
?. [Day 1: OPTIONAL Sample Consolidant Removal (Laboratory fume hood)]
Centrifuge the samples for
Centrifuge: 15800 1324, 1 min, 22 10
?. [Day 1: OPTIONAL Sample Consolidant Removal (Laboratory fume hood)]
Remove acetone with a pipette and discard. Leave the tube open so the remaining acetone is removed via evaporation until the calculus is dry.