Free Ancient DNA: Methods and Protocols by Beth Shapiro Page A

weights, sizes and charges to DNA. The extraction of DNA from feces therefore requires achieving a balance between minimizing DNA loss during extraction and removing coeluates that would otherwise inhibit downstream applications. We use a chaotropic salt/silica-based procedure that is well known for its ability to remove inhibitors ( 14 ) .
    2. Materials
     
    2.1. Laboratory
     
    Supplies (per Sample)
    Forceps
    1×
    Small weight boat
    1×
    Scalpel blade
    1×
    15-mL tube
    3×
    1.5-mL tube
    2×
    0.5-mL tube
    1×
    pH paper
    1+
    2.2. Laboratory
    1. Pipettes 1–1,000 μ L.
    Equipment
    2. Vortex.
    3. Incubator with rotating wheel, or rotary mixer or similar device capable of being placed in an incubator.
    4. Microcentrifuge as well as a large swing bucket centrifuge for 15 mL.
    5. Shaking/heating block.
    5 Extraction of DNA from Paleofeces
    39
    2.3. Solutions and
    1. GuSCN extraction-buffer (14 mL per sample): 6-M guanidine Buffers (per Sample)
    thiocyanate. (GuSCN); 20 mM Tris-HCl, pH 8.0; 0.5% sodium
    lauroyl sarcosinate (Sarcosyl); 8 mM Dithiothreitol (DTT); 4%
    Polyvinylpyrrolidone (PVP); 10 mM
    N -phenacyl thiazolium
    bromide (PTB).
    2. L6-buffer (4 mL per sample): 5 M GuSCN; 0.05 M Tris-HCl, pH 8.0; 0.0225 M Natrium chloride (NaCl); 0.02 M
    Ethylenediaminetetraacetic acid (EDTA), pH 8.0; 1.25%
    Triton X-100; (add 200 μ L Silica, vortex, let sit).
    3. Silica solution (50 μ L per sample, see Subheading 3.1 ).
    4. L2-buffer (1–2 mL per sample): 5 M GuSCN; 0.05 M Tris-HCl, pH 8.0; 0.0225 M NaCl (add 200 μ L Silica, vortex, let sit).
    5. New Wash substitute (1–2 mL per sample): make an 80% ethanol solution using 1× TE (10 mM Tris-HCl and 1 mM EDTA (pH 7.5)).
    6. 0.1× TE pH 8.0 plus 0.05% Tween-20 (60 μ L per sample).
    7. Glacial Acetic acid (~15 μ L per sample).
    3. Methods
     
    3.1. Preparing the
    1. Weigh out 4.8 g silica and place it into a 50-mL gamma-
    Silica Suspension
    sterilized tube.
    2. Add double-distilled (dd) H O to the tube containing the silica 2
    to 40 mL, vortex for 2 min, then let sit for 24 h at room temperature in the dark.
    3. Carefully remove 35 ml of supernatant (without distrurbing the pellet) and discard.
    4. Add ddH O to 40 mL, vortex for 2 min, let sit for 6 h at room 2
    temperature in the dark.
    5. Carefully remove 36 ml of supernatant (without disturbing the pellet) and discard.
    6. Add 48 μ L 30% hydrochloric acid (keep pH acidic <3).
    7. Resuspend the pellet, and aliquot approx. 200 μ L of solution into separate tubes for later use.
    8. Store in the dark at +4°C.
    9. Prior to use, vortex to resuspend any pelleted material.
    3.2. Paleofeces DNA
    1. Add approximately 1 g of fecal material to a small weighing Extraction
    boat (see Note 1).
    2. Cut the fecal remains into small pieces using scalpel blades (see Note 2).
    40
    M. Kuch and H. Poinar
    3. Add fecal material to a fi nal volume of 14 mL of the GuSCN
    extraction-buffer in a 15-mL tube and incubate, rotating
    overnight at 37°C in the dark (see Notes 1, 3 and 4).
    4. Centrifuge at maximum speed for 5 min and transfer supernatant to a clean 15-mL tube.
    5. Centrifuge again at maximum speed for 5 min and transfer supernatant to 4 mL of room temperature
    pre-incubated
    L6-buffer and 50 μ L of Silica for at least 15 min (see Notes 5–8).
    6. Adjust the pH to ~5 by adding glacial acetic acid (see Note 9).
    7. Incubate while rotating at room temperature for 3 h in the dark (see Note 10).
    8. Centrifuge for 5 min at maximum speed and discard the supernatant.
    9. Add 1 mL of L2-buffer, resuspend, and transfer solution to a 1.5-mL microcentrifuge tube (see Note 11).
    10. Centrifuge for 30 s and discard the supernatant.
    11. If the solutions are still heavily colored, repeat steps 9–10.
    12. Add 1 mL of wash buffer and resuspend the silica.
    13. Centrifuge for 30 s and discard supernatant.
    14. Centrifuge again briefl y and remove all remaining liquid with a pipette.
    15. Dry the remaining silica pellet in a heating block for 5 min at 56°C