Read Ancient DNA: Methods and Protocols for Free Online

sample. Further processing sequesters the nucleic acid fraction from other components by repeated separation into hydrophobic and aqueous phases. Final passing through a membrane fi lter concentrates the DNA in the sample. The fi nal solution of total DNA can be used for PCR, next-generation sequencing, and other applications.
    2. Materials
     
    All reagents, consumables, and equipment used throughout this protocol should be sterilised, either through purchase or through processing with UV irradiation or cleaning with bleach solution and ethanol ( 5– 7 ) . Plasticware should be sterile, single-use, and preferably designed to prevent cross-contamination (e.g. pipette tips with aerosol barrier). All solutions should be prepared using ultrapure water (18.2 M W at 25°C).
    2.1. Sample
    1. Hand-held drill with disposable abrasive discs.
    Preparation
    2. Sodium hypochlorite (bleach, 10–20% solution).
    3. Ethanol (95–100%).
    4. Freezer mill, shaker mill, or other device for grinding samples into powder (see Note 1).
    5. Aluminium foil.
    6. N prepared sterile plastic tubes (15 mL) (see Note 2).
    2.2. Chelation
    1. EDTA chelation buffer: Ethylenediaminetetraacetic acid (EDTA) 1 M solution pH 8.0 (see Note 3).
    2. Rotary mixer, wheel or similar device to keep samples constantly in motion during incubation steps, suitable for use with 15-mL tubes.
    3. Centrifuge suitable for use with 15-mL tubes.
    2.3. Digestion
    1. 1× Buffer:15 mM Tris–HCl (pH 8.0), 2.5 mM
    N -phenacylthiazone bromide (see Note 4).
    2. 10×: Sodium dodecyl sulphate (Fisher) 10%w/v (see Note 5).
    3. 10×: 25 mg mL −1 Proteinase K (see Note 6).
    4. 10×: 500 mM Dithiothreitol (DTT) (see Note 7).
    5. Laboratory incubator large enough to accommodate rotator.
    6. Rotary mixer, wheel or similar device to keep samples constantly in motion during incubation steps, suitable for use with 15-mL tubes.
    2 A Phenol–Chloroform Protocol for Extracting DNA from Ancient Samples 15
    2.4. Phase Separation
    1. 2 N pre-prepared 15-mL phase separating tubes (light gel see Note 8) containing 6 mL of saturated pH 6.6 Phenol (see
    Note 9).
    2. N pre-prepared 15-mL sterile plastic tubes containing 6 mL of chloroform (see Note 10).
    3. Rotary mixer, wheel or similar device to keep samples constantly in motion during incubation steps, suitable for use with 15-mL tubes.
    4. Centrifuge suitable for use with 15-mL tubes.
    2.5. Concentration
    1. N labelled micro-concentrators with a nominal molecular weight limit of 30 kDa and able to process 6 mL of solution (see Note 11).
    2. Centrifuge suitable for use with 15-mL tubes.
    3. N labelled sterile plastic tubes (1.5 mL).
    3. Methods
     
    As the method described below attempts to extract degraded, damaged DNA from samples that may be anywhere up to several hundred thousands of years old, it is necessary to work in an isolated aDNA workspace, ideally one that is isolated from normal molecular biology (especially PCR) work ( 4 ) .
    3.1. Sample
    1. Prepare the work area by sterilising surfaces. First wash all sur-
    Preparation
    faces with bleach and then rinse with ethanol. Wait until the surfaces dry completely before proceeding. A fume hood with integrated extraction fans is an ideal location for this protocol to be performed. Prepare several layers of aluminium foil to collect powdered sample as it is produced.
    2. Thoroughly abrade the external surface of the bone/tooth sample using a hand-held drill with disposable cutting discs or equivalent (see Note 12). Discard the resulting powder, for example by collecting it in the upper layer of aluminium foil and discarding the foil and powder.
    3. Reduce the bone/tooth section to powder using a shaker mill, freezer mill or similar device (see Note 13). The speed and other conditions of the powdering device should be adjusted to suit the mineralisation state of the sample. Generate as fi ne a powder as possible to maximise the surface area of the sample that will eventually contact the chelation