Ancient DNA extraction (chunk samples/high volume)
Marcel Keller, Christiana L Scheib
Abstract
Extraction protocol for ultra-short ancient DNA molecules from skeletal chunk samples modified from Dabney et al. (2013) PNAS (doi: 10.1073/pnas.1314445110).
Before start
Previous steps:
This protocol follows the sampling of chunk samples (tooth roots, petrous cores, other bones) and decontamination. Step 2 assumes that the samples have been dried under UV light in the laminar flow hood (see decontamination). Alternatively, the samples can be transferred into tubes.
Following step:
This protocol ends with a 72 h incubation step. Proceed with the purification (chunk samples/high volume) after the incubation.
Equipment and consumables:
| A | B |
|---|---|
| Number | Equipment and consumables |
| 4 | disposable 100 ml beakers |
| 2x100 ml | NaOCl (6% v/v) |
| 100 ml | MilliQ water |
| 100 ml | 70% ethanol |
| 2 | tweezers |
| depending on sample weight | 5 ml tubes (LoBind) |
| depending on sample weight | 15 ml tubes (LoBind) |
| [# of samples] | weighing boats (small) |
| 1 | tweezers |
| Parafilm | |
| 1 | 5 ml rack |
| 200 µl filter tips | |
| 1000 µl filter tips | |
| 5000 µl filter tips |
Steps
Prerequirements
Extraction
Set up decontamination station:
4 disposable 100 ml beakers
6% NaOCl (bleach) aliquot [2x]
MilliQ water [1x]
70% ethanol aliquot [1x]
To decontaminate tweezers, soak in 6% bleach for 5 minutes, rinse with MilliQ water, rinse with 70% Ethanol.
Clean table bench surface with DNA Exitus and wipe dry. Turn the hood on full power and open the glass.
Clean hood surface with DNA Exitus and put down a new sheet of aluminum foil. Wipe down reagents with DNA Exitus.
Place all reagents (except proteinase K) and consumables in the hood and UV while weighing.
Make EDTA 0.5Molarity (M)``8.0, Proteinase K 18.18mg/mL, and tube calculations:
| A | B | C | D | E |
|---|---|---|---|---|
| Sample ID | Weight (mg) | EDTA (ml) | PK (µl) | Tube |
| [Sample ID] | x | 2 ml per 100 mg (x/50) | 50 µl per 100 mg (x/2) | 5 ml < 250 mg < 15 ml |
| ABC001A | 100 | 2 | 50 | 5 |
| ABC001A | 300 | 6 | 150 | 15 |
Label your tubes for the extractions:
| A | B | C |
|---|---|---|
| Top | Side | |
| Project ID | PROJ | PROJ |
| Sample ID | ABC001A | ABC001A |
| Extraction # | 1 | 1 |
| Initials | YZ | |
| Date | DD/MM/YYYY | |
| Content | P | Pellet |
If necessary: Make Proteinase K solution by adding 5.5mL PCR clean water to 100mg of powder. Shake to dissolve. Let sit before using. Store in the freezer at -20°C.
Add calculated amount of EDTA to each tube. If the tubes are empty you can use the same pipette tip as long as you do not get liquid into the filter or touch any surfaces with the tip.
Add the calculated amount of Proteinase K to each tube and pipette-mix. Change your tips each time.
Add samples to their individual extraction tube.
Make sure that the lids are tightly sealed to avoid leakage.
Wipe tubes with DNA Exitus, wrap tubes with Parafilm.
Place on the nutating mixer for 24rpm.
