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solution ( 6, 8 ) .
    4. Collect the powder and transfer it to a labelled, sterile tube (15 mL).
    16
    R. Barnett and G. Larson
    3.2. Chelation
    1. Add 15 mL of 1× chelation buffer to the powdered sample (see Note 14).
    2. Add 15 mL of 1× chelation buffer to a labelled tube that does not contain any powder. This will be the negative extraction control.
    3. Be sure that the powder and chelation buffer are well mixed.
    Place all 15-mL tubes on the rotary mixer and rotate overnight at room temperature.
    4. Concentrate the samples and negative control by centrifugation at 4,000 × g for 10 min or until all organic content has pelleted at the bottom of the tube.
    5. Remove eluate and retain pellet (see Note 15).
    3.3. Digestion
    1. Prepare 4.2 mL of the 1× digestion buffer for each sample and add to this each tube containing an organic pellet using.
    2. Add 0.6 mL of the 10× solution of SDS to each sample.
    3. Add 0.6 mL of the 10× solution of proteinase K to each sample.
    4. Add 0.6 mL of the 10× solution of DTT to each sample.
    5. Place all 15-mL tubes on the rotary mixer. Place the rotator in the oven and rotate overnight at 55°C (see Note 16).
    3.4. Phase Separation
    1. Decant each digested sample into a corresponding pre-prepared tube containing phenol.
    2. Place all 15-mL tubes on the rotary mixer and rotate at room temperature for 10 min
    3. Centrifuge at 8,000 ×
    g for 10 min. The two phases will
    separate. If phase-lock or phase-divider tubes are used, the gel should have formed a barrier between the aqueous and hydrophobic layers (see Note 17). Decant the aqueous layer into the second, pre-prepared tube containing phenol.
    4. Place all 15-mL tubes on the rotary mixer and rotate at room temperature for 10 min.
    5. Centrifuge at 8,000 × g for 10 min. As before, the two phases will separate. Decant the aqueous layer into a pre-prepared chloroform tube.
    6. Place all 15-mL tubes on the rotary mixer and rotate at room temperature for 5 min.
    7. Centrifuge at 8,000 × g for 5 min. The two phases will separate.
    3.5. Concentration
    1. Carefully transfer the aqueous layer by pipette to a micro-concentrator (30 kDa membrane) (see Note 18).
    2. Centrifuge at 8,000 × g until the sample has completely passed through the membrane. Discard the fi ltrate.
    2 A Phenol–Chloroform Protocol for Extracting DNA from Ancient Samples 17
    3. Add 5 mL of ultrapure water to each sample and centrifuge again at 8,000 × g until resolution into a fi nal solution of 100–200 m L
    (see Note 19). The eluate will now contain your DNA.
    4. Transfer the remaining solution by pipette to a sterile storage tube (e.g. 1.5 mL, see Note 20).
    5. Store the DNA extract at −20°C (see Note 21).
    4. Notes
     
    1. Shaker mills, freezer mills, and other similar devices use friction to reduce samples to powder. The increase in surface area that results from powdering the sample allows for more effi cient digestion.
    2. Prior to beginning the protocol, prepare N suffi ciently labelled tubes, where N is the number of samples plus negative controls (i.e. if 7 samples and one negative control are to be extracted, prepare N = 8 labelled tubes, 2 N = 16 phenol tubes, etc.).
    3. EDTA is a strong chelator that is able to bind metallic ions such as Ca 2+ and Mg 2+ that are released during digestion.
    4. Some experiments have shown that N -phenacylthiazone bromide may be useful in freeing DNA that has been chemically crosslinked to other biomolecules through diagenetic processes ( 9, 10 ) .
    5. SDS is a detergent that allows the solubilisation of lipids present in biological samples and denatures proteins. 10× SDS
    solution should be stored in the refrigerator. At low temperature, SDS can precipitate out of solution. If this occurs, place it in a warm oven for 5 min until the detergent has
    resolubilised.
    6. Proteinase K is a protease that cleaves proteins, reducing them to their constituent amino acids. Proteinase K should be stored in the freezer where it will