TITLE: Ancient DNA extract purification (chunk samples/high volume)
AUTHORS: Marcel Keller, Christiana L Scheib

[DESCRIPTION]
Protocol for the purification of extracts, modified from Dabney et al. (2013) PNAS (doi: 10.1073/pnas.1314445110). 

[BEFORE_START]
Previous step:
This protocol follows the extraction protocol and is to be performed on day 3 after the extraction (after 72 h).

Following step:
Proceed with one of the library preparation protocols.

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
2. Turn the hood on full power and open the glass.
SECTION: Preparation
3. Spray hood and table bench surfaces with DNA Exitus, let sit a minute and wipe down with paper towels.
SECTION: Preparation
4. Wipe down outside surfaces of reagents/tips with DNA Exitus and place in the hood. 
SECTION: Preparation
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.
SECTION: Purification
11. Bring in samples and on the table bench, remove parafilm and wipe with a small amount of DNA Exitus before placing in the centrifuge.
SECTION: Purification
12. Spin down pellets at 4000 rpm, 5 min .

SECTION: Purification
13. Change gloves and label your concentrators on lids and sides with sample ID numbers.
SECTION: Preparation
6. In the hood, make EB Buffer aliquot (100 µL per sample) in a 5 ml tube.

SECTION: Purification
14. Transfer samples to the hood without disturbing the pellet
SECTION: Purification
15. Add as much supernatant to the labeled concentrators as possible without disturbing the pellet. Chunks of collagen can interfere with the concentration process.
SECTION: Purification
16. 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 4000 rpm  for 25 min to 45 min  or until extracts are 250 µl. 

SECTION: Purification
17. 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:
 
 

 
 
SECTION: Purification
18. When samples have concentrated, add 2.5 mL 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.
SECTION: Purification
19. Add concentrated extract to the PB buffer inside the Roche column and gently pipette to mix. Use 1000 µl LONG tips.
 

SECTION: Purification
20. Spin Roche columns at 4000 rpm, 1 min.
SECTION: Purification
21. Change gloves and place your columns back in the hood. 
SECTION: Purification
22. Add 1000 µL of PE (Wash/Ethanol) buffer to each Roche column.
 


SECTION: Purification
23. Spin at 4000 rpm, 1 min.

SECTION: Purification
24. Change gloves and place your columns back in the hood. 
SECTION: Purification
25. 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. 
SECTION: Purification
26. 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. 
 

SECTION: Purification
27. Close the lid on your column/tube and label immediately with the sample ID.
SECTION: Purification
28. 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.
SECTION: Purification
29. Spin columns + collection tube at 13000 rpm, 1 min  to dry the membrane.

SECTION: Purification
30. Turn on the heat block 37 °C.

SECTION: Purification
31. 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.
SECTION: Purification
32. Change gloves and add 100 µL of EB (Elution) buffer to each sample. 
 



SECTION: Purification
33. Incubate samples in the heat block at  37 °C  for 10 min.

SECTION: Purification
34. Spin at 13000 rpm, 2 min. Be careful to arrange lids so that they will not break.

SECTION: Purification
35. 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. 
SECTION: Purification
36. Store at -20 °C 

SECTION: Prerequirements
1. 
 

SECTION: Preparation
7. Prepare PE (wash) buffer by adding ethanol.
SECTION: Preparation
8. Aliquot PE Buffer to 50 ml tube: [# of samples]x1000 µl plus 10%.
SECTION: Preparation
9. Aliquot PB Buffer to 50 ml tube: [# of samples]x2.4 ml plus 10%.
SECTION: Preparation
5. Label the 50 ml waste tube, PB tube and PE tube as well as the 5 ml EB tube.