Ancient DNA extract purification (chunk samples/high volume)

Turn the hood on full power and open the glass.

Spray hood and table bench surfaces with DNA Exitus, let sit a minute and wipe down with paper towels.

Wipe down outside surfaces of reagents/tips with DNA Exitus and place in the hood.

Label the 50 ml waste tube, PB tube and PE tube as well as the 5 ml EB tube.

In the hood, make EB Buffer aliquot (100µL per sample) in a 5 ml tube.

Prepare PE (wash) buffer by adding ethanol.

Aliquot PE Buffer to 50 ml tube: [# of samples]x1000 µl plus 10%.

Aliquot PB Buffer to 50 ml tube: [# of samples]x2.4 ml plus 10%.

Check that the centrifuge rotor is the bucket and add 5 ml inserts. Wipe the rotor and inserts with DNA Exitus after taking them out of the centrifuge and before putting them into the centrifuge.

Bring in samples and on the table bench, remove parafilm and wipe with a small amount of DNA Exitus before placing in the centrifuge.

Spin down pellets at 4000rpm .

Change gloves and label your concentrators on lids and sides with sample ID numbers.

Transfer samples to the hood without disturbing the pellet

Add as much supernatant to the labeled concentrators as possible without disturbing the pellet. Chunks of collagen can interfere with the concentration process.

Change centrifuge inserts from 5 ml to 50 ml. Wipe inserts with DNA Exitus after taking them out of the centrifuge and before putting them into the centrifuge. Centrifuge at 4000rpm,0h 0m 0s for 0h 25m 0s to 0h 45m 0s or until extracts are 250 µl.

While tubes are spinning: Change gloves and label large volume columns (Roche) with Sample IDs on lids and sides (e.g. 12 on top and 12 on the side) and label Eppendorf 1.5 ml tubes:

When samples have concentrated, add 2.5mL or at least 10x [extract volume] PB (binding) buffer to the Roche columns using the 5 ml pipette. Do not let this sit too long as the buffer starts to leak through the membrane quickly.

Add concentrated extract to the PB buffer inside the Roche column and gently pipette to mix. Use 1000 µl LONG tips.

Spin Roche columns at 4000rpm.

Change gloves and place your columns back in the hood.

Add 1000µL of PE (Wash/Ethanol) buffer to each Roche column.

Spin at 4000rpm.

Change gloves and place your columns back in the hood.

Using a clean 1.5 ml tube rack, take a collection tube from the bag without touching the inside of the bag or the inside of the tube and place it in your 1.5 ml rack.

Remove the inner column from the 50 ml Roche tube and place it inside the clean collection tube. Pull the release tab on the front and twist it forward to free the column from the funnel. Discard the funnel and small plastic bits.

Close the lid on your column/tube and label immediately with the sample ID.

Change gloves and change the rotor in the centrifuge to the 2 ml rotor. Wipe the rotor and inserts with DNA Exitus after taking them out of the centrifuge and before putting them into the centrifuge.

Spin columns + collection tube at 13000rpm to dry the membrane.

Turn on the heat block 37°C.

Back in the hood, take the silica column out of the collection tube and place it into the corresponding clean, labeled 1.5 ml Eppendorf lo-bind tube.

Change gloves and add 100µL of EB (Elution) buffer to each sample.

Incubate samples in the heat block at 37°C for 0h 10m 0s.

Spin at 13000rpm. Be careful to arrange lids so that they will not break.

Back in the hood, discard the silica column, close lid, wipe tubes with DNA Exitus and wrap with Parafilm if stored for more than a few days before library prep.

Store at -20°C