TITLE: Ancient DNA Extraction from Skeletal Material
AUTHORS: Irina Velsko, Eirini Skourtanioti, Guido Brandt

[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 skeletal material powder (e.g. bone, teeth), 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>). </span></div></div>

[STEPS]
?. [Day 1: Binding buffer preparation (Buffer Prep Room)]
Prepare binding buffer calculating .  ABC1Reagent [Stock Concentration]Final ConcentrationVolume/reaction2GuHCl (1 mol=95.53 g)5 M4.77 g3UV HPLC-water up to6 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-water up to6 ml4Isopropanol (100%)40%4 ml5Total10 ml
?. [Day 1: Binding buffer preparation (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: Binding buffer preparation (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: Decalcification and Protein Denaturation (DNA Extraction Room)]
Seal tubes with Parafilm, rotate   with low overhead rotation speed (e.g., 12-16 rpm)  at  in the incubator.
[(12-18h)]
37 °C
Post-incubation lysate and pellet can safely be stored in a freezer (  ) before isolation and clean-up.
After starting the incubation, we recommend beginning preparations for day 2, such as pre-labelling the falcon tubes that will be used in step 12.
?. [Day 2: 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 2: 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 2: DNA isolation and clean-up (DNA Extraction Room)]
Remove parafilm from extraction tubes, then spin the tubes for  at  to pellet bone powder.
Centrifuge: 18500 34
If pellet is not solid, repeat centrifugation.
?. [Day 2: DNA isolation and clean-up (DNA Extraction Room)]
Pipette supernatant to matching  Falcon tube, mix contents by inversion. If pellet is too fragile, repeat centrifugation before transferring supernatant. Store the bone pellet at .
50 mL
-20 °C
?. [Day 2: DNA isolation and clean-up (DNA Extraction Room)]
Pipette binding buffer/extract mix to High Pure Extender Assembly.
?. [Day 2: 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 2: 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 collection tube and take off the funnel.
?. [Day 2: DNA isolation and clean-up (DNA Extraction Room)]
Dry spin the column in the collection tube for  at .
Centrifuge: 18500 34
?. [Day 2: DNA isolation and clean-up (DNA Extraction Room)]
Pipette  wash buffer from the High Pure Viral Nucleic Acid kit and spin at  for .
450 µl
Centrifuge: 8000 34
?. [Day 2: 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 collection tube.
?. [Day 2: 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 2: 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 2: Elution (DNA Extraction Room)]
Transfer column into   LoBind Tube, pipette  of TET to the center of column to elute DNA, incubate for  and spin  at .
1.5 mL
50 µl
Centrifuge: 18500 34
Eluted DNA will be stored in this tube. Label tube on top and side accordingly.
?. [Day 2: Elution (DNA Extraction Room)]
Repeat elution step for a total elution volume of  TET.
100 µl
?. [Day 2: Elution (DNA Extraction Room)]
Store the DNA extracts at  until use.
-20 °C
?. [Day 1: Binding buffer preparation (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'.
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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: Binding buffer preparation (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 gases!
?. [Day 1: Binding buffer preparation (Buffer Prep Room)]
Fill up with UV-irradiated HPLC water to final volume.
This reaction is endothermic and the tube will become very cold. Be aware of the unusual  'slushy' way of dissolving.
?. [Day 1: Binding buffer preparation (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: Binding buffer preparation (Buffer Prep Room)]
Pipette isopropanol to reach the final reaction volume ().
10 mL
?. [Day 1: Extraction Buffer Preparation (DNA Extraction Room)]
Prepare extraction buffer calculating to a total of  .  ABC1Reagent [Stock Concentration]Final ConcentrationVolume/reaction2EDTA [0.5 M]0.45 M0.9 ml3UV HPLC water0.075 ml4Proteinase K [10 mg/ml]0.25 mg/ml0.025 ml5Total1 ml
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ABC1Reagent [Stock Concentration]Final ConcentrationVolume/reaction2EDTA [0.5 M]0.45 M0.9 ml3UV HPLC water0.075 ml4Proteinase K [10 mg/ml]0.25 mg/ml0.025 ml5Total1 ml
?. [Day 1: Extraction Buffer Preparation (DNA Extraction Room)]
Prepare extraction buffer by following the table ( ).
?. [Day 1: Extraction Buffer Preparation (DNA Extraction Room)]
Make an aliquot of EDTA (, ) calculating   and irradiate with UV for .
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UV irradiation can be done together with solutions from steps 1 (binding buffer), 4 (wash buffer), and 5 (TET buffer).
?. [Day 1: Binding buffer preparation (Buffer Prep Room)]
Store binding buffer in a fridge at  overnight for day 2.
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: Decalcification and Protein Denaturation (DNA Extraction Room)]
Pipette of from the extraction buffer aliquot to a   -  aliquot of bone powder.
1 mL
30 mg
50 mg
Preparation of the bone or tooth powder ideally should be done prior to beginning of the extraction protocol in a dedicated sampling room, and stored in a labelled   Safe-Lock tube.
?. [Day 1: Binding buffer preparation (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 2: DNA isolation and clean-up (DNA Extraction Room)]
Prepare cleaned workspace with all necessary reagents and equipment.
?. [Day 1: Binding buffer preparation (Buffer Prep Room)]
Dilute proteinase K powder () to a concentration of  /  .
100 mg
10 mg
1 mL
Remaining proteinase K solution should be stored in