Ancient DNA: Methods and Protocols by Beth Shapiro Read Free Book Online Page B

is needed) for grinding sample pieces into fi ne powder.
    4. 15-mL tubes.
    5. Rotary mixer, wheel, or similar device to keep samples constantly in motion during incubation steps.
    2.2. DNA Purifi cation
    1. Silicon dioxide (see Note 2).
    and Concentration
    2. 30% HCl.
    3. Binding buffer: 5 M Guanidinium thiocyanate, 0.3 M sodium acetate (pH 5.2). Store in the dark (see Note 3).
    4. Washing buffer: 50% Ethanol, 125 mM NaCl, 10 mM Tris–HCl, 1 mM EDTA (pH 8.0) (see Note 4)
    5. Elution buffer: 10 mM Tris–HCl, 1 mM EDTA (pH 8.0).
    6. 50-mL tubes.
    7. 50-mL disposable serological pipettes.
    8. Centrifuge capable of holding 15-mL tubes and reaching centrifugal force of 5,000 × g.
    9. Columns (e.g. MobiCol “Classic,” MobiTec, catalog number: M1003).
    10. Filter (Filter (large) 10 m m pore size, MobiTec, catalog number: M2210).
    24
    N. Rohland
    11. Filter with 1 m m pore size (e.g., glass microfi ber binder free Grade GF/B: 1 m m, Whatman, catalog number: 1821–070).
    12. Hole punch with 7 mm diameter.
    13. Forceps.
    14. Centrifuge capable of holding 2.0-mL tubes and reaching centrifugal force of 16,000 × g.
    15. Vacuum manifold and vacuum pump.
    16. Collection tubes without lids.
    17. Disposable VacConnectors (Qiagen, catalog number: 19407).
    18. 1.5-mL tubes (see Note 5).
    3. Methods
     
    All steps are to be carried out at room temperature.
    3.1. Preparing
    1. Weigh 4.8 g of silicon dioxide into a 50-mL tube, add water to the Silica Suspension
    bring the mixture to 40 mL, and vortex extensively.
    2. Let large particles settle down for 1 h.
    3. Transfer 39 mL from the top of the solution into fresh 50-mL
    tube and let the solution settle for an additional 4 h.
    4. Discard 35 mL from the top of the solution and add 48 m L of 30% HCl to the 4 mL pellet that remains.
    5. Vortex, aliquot, and store the silica suspension at room temperature in the dark (see Note 6).
    3.2. Preparing
    1. Use forceps to place a large fi lter with 10 m m pore size in the the Columns
    column. Move the fi lter to the bottom of the column using the fi lter insertion tool provided with the fi lter.
    2. Use a hole punch to make a smaller “fi ne fi lter” from the fi lter paper with 1 m m pore size.
    3. Using the forceps, place the “fi ne fi lter” in the columns, and move it on top of the larger fi lter using the insertion tool.
    3.3. Sample
    1. After removing the surface of the sample with a fresh drilling Preparation
    bit at slow speed, drill into the densest part of the bone or the and DNA Release
    tooth root. Collect the powder. If a cutting tool is used instead of a drill, remove a compact part of the bone or the tooth root (again after removing the sample surface with a single-use cutting disc or blade). Grind the pieces of sample to as a fi ne powder as possible using mortar and pestle or a freezer mill.
    Collect approximately 250 mg of powder per sample into separate 15-mL tubes (see Note 7).
    3 DNA Extraction of Ancient Animal Hard Tissue Samples…
    25
    2. Add 5 mL of extraction buffer to each sample. Seal the tubes and incubate them for 16–24 h under constant agitation in the dark (see Note 8).
    3.4. DNA Adsorption
    1. Centrifuge the samples for 2 min at 5,000 × g and transfer as to Silica, Washing
    much of the liquid as possible into new 15-mL tubes.
    Steps, and Elution
    2. Add 2.5 mL of binding buffer and 100 m L of well-mixed silica suspension to the extraction buffer in each tube. Incubate for 3 h in the dark under constant agitation (see Notes 9–11).
    3. Place a disposable VacConnector onto the luer adapter of the vacuum manifold, then place the assembled column onto the
    VacConnector (depending on the manifold used, up to 24
    columns can be handled in parallel).
    4. Centrifuge the sample for 2 min at 5,000 × g , discard the supernatant, and resuspend the silica pellet in 400 m L of binding buffer. Transfer the suspension to the column and apply the vacuum (see Notes 12–14 ).
    5. Place the column in a collection tube and