TITLE: DNA Extraction from Modern Dental Calculus
AUTHORS: Franziska Aron

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for DNA extraction from modern dental calculus samples for Illumina sequencing. This protocol uses the Qiagen PowerSoil DNA extraction kit. </div></div>

[STEPS]
?. [Sample Preparation]
Weigh out   to   of the calculus and transfer it into a 2ml-Eppendorf Safe-Lock DNA LoBind-Tube (use special accuracy scales)
3 mg
5 mg
?. [Lysis]
Crush the calculus with a sterile metal pestle within the tube.
?. [Lysis]
Add  EDTA and  Proteinase K  ( )
250 µl
12.5 µl
[/ml]
?. [Lysis]
Let it rotate in the dark at  for  (optional: over the weekend) until the calculus fully decalcifies and either disappears or becomes buoyant (floating and feathery in appearance)
0 Room temperature
[up to 48h]
?. [DNA_Clean_up ]
Remove the tubes from the rotator and briefly centrifuge the extraction tubes
This centrifugation step is just made to remove the liquid from the lid.
?. [DNA_Clean_up ]
Transfer everything with a  pipette tip (supernatant and pellet) to the Power Bead tubes
1000 µl
?. [DNA_Clean_up ]
Invert to mix.
?. [DNA_Clean_up ]
Add    C1 solution, invert several times to mix.
60 µl
?. [DNA_Clean_up ]
Secure tubes horizontally on a flat-bed vortex pad with tape, vortex at maximum speed for
?. [DNA_Clean_up ]
Centrifuge at  for  at
Centrifuge: 9400 34
0 Room temperature
Make sure the Power Bead tubes can rotate without rubbing, 10.000 x g is the maximum, otherwise the tubes may break
?. [DNA_Clean_up ]
Transfer supernatant to the 2ml collection tube (provided in the kit)
Expect   to
?. [DNA_Clean_up ]
Add  C2 Solution to the supernatant and vortex   at maximum speed
250 µl
?. [DNA_Clean_up ]
Incubate at  for  on the pre-cooled rack ( )
4 °C
4 °C
?. [DNA_Clean_up ]
Centrifuge  for  at
Centrifuge: 9400 34
0 Room temperature
?. [DNA_Clean_up ]
Transfer  of the supernatant to a clean 2ml collection tube (provided in the kit), avoid transferring the pellet.
600 µl
?. [DNA_Clean_up ]
Add   C3 solution and vortex briefly
200 µl
?. [DNA_Clean_up ]
Incubate the tubes at  for  on the pre-cooled rack ( )
4 °C
4 °C
?. [DNA_Clean_up ]
Centrifuge the tubes at  for  at
Centrifuge: 9400 34
4 Room temperature
?. [DNA_Clean_up ]
Transfer  of the supernatant to a clean 2ml collection tube (provided in the kit)
750 µl
?. [DNA_Clean_up ]
Gently shake C4 Solution to mix
?. [DNA_Clean_up ]
Add  C4 Solution to supernatant and vortex for
1.2 mL
The solution should not exceed the rim of the tube.
?. [DNA_Clean_up ]
Bind the DNA on the membrane of spin filter unit:
?. [DNA_Clean_up ]
Repeat until the entire solution has been passed through the spin filter unit (2-3 times)
?. [DNA_Clean_up ]
Load  C5 Solution into spin filter unit
500 µl
?. [DNA_Clean_up ]
Centrifuge  at
Centrifuge: 9400 34
-20 Room temperature
?. [DNA_Clean_up ]
Discard flow through
?. [DNA_Clean_up ]
Dry spin   at
Centrifuge: 9400 34
-20 Room temperature
?. [Elution ]
Place the spin filter unit into a clean 2ml collection tube (provided in the kit)
?. [Elution ]
Pipette  C6 Solution into center of the membrane in the spin filter unit and incubate for
100 µl
?. [Elution ]
Centrifuge  for  at
Centrifuge: 9400 34
-20 Room temperature
?. [Elution ]
Remove spin filter unit and quantify the DNA () with the Qubit ds BR Kit
3 µl
?. [(OPTIONAL) EDTA Wash]
Centrifuge for   at
Centrifuge: 9400 34
0 Room temperature
?. [(OPTIONAL) EDTA Wash]
Remove supernatant
Optional: Transfer the supernatant to a clean 1.5ml loBind Tube and stored it for future analysis at
?. [(OPTIONAL) EDTA Wash]
Add   of   EDTA and let it rotate on a tube rotator for  at
Note this step is optional, however it can be useful to remove exogenous fluids such as blood.
1 mL
0 Room temperature
?. [Lysis]
Clean the metal pestle after every sample by incubating for  in a  household bleach dilution , then wiping clean with HPLC-water and drying with a clean paper towel
Sterile plastic pestles can be used instead of metal pestles, but they must be changed between each sample and not re-used.
?. [DNA_Clean_up ]
Save power bead tubes at  as  backup. These can be thrown out once you know the extraction worked
-20 °C
?. [DNA_Clean_up ]
Store remaining supernatant at
-20 °C
We save this as backup until confirmation that the extraction worked. If extraction was successful, this can be discarded.
?. [DNA_Clean_up ]
Load approximately  into spin filter unit
675 µl
?. [DNA_Clean_up ]
Centrifuge   at  at
Centrifuge: 9400 34
4 Room temperature
?. [DNA_Clean_up ]
Discard flow through
?. [Elution ]
Store the eluate at
-20 °C
?. [Preparation Day 1 ]
Place a 1.5ml/2ml tube rack at  for the incubation steps on the final days
4 °C
?. [Preparation Day 1 ]
Prepare a 1:10 dilution of household bleach solution (~6% hypochlorite) in a 50ml-Falcon tube
?. [Preparation Day 1 ]
Label for each sample one 2ml-Eppendorf Safe-Lock DNA LoBind-Tube and one 1.5ml-Eppendorf Safe-Lock DNA LoBind-Tube
?. [Preparation for Final Day]
Retrieve the spin filter columns (provided in the kit) from fridge to warm them up to  for use at step 26
-20 Room temperature
"provided in the kit" refers to: DNeasy PowerSoil Kit (100) Cat No./ID: 12888-100A spin filter column is a spin filter unit sitting in a collection column. The spin filter unit contains the       membrane.
?. [Preparation for Final Day]
Label for each sample one Power Bead tube, one spin filter column and three 2ml collection tubes (all provided in the kit)