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

(to remove residual ethanol).
    16. Add 60 μ L of 0.1× TE pH 8.0 plus 0.05% Tween-20 and incubate with agitation for 15 min at 56°C.
    17. Centrifuge for 3 min at maximum speed and transfer supernatant to a 0.5-mL tube.
    18. Centrifuge again for 3 min at maximum speed and transfer supernatant to a clean 1.5-mL tube (see Note 12).
    19. Freeze at −20°C.
    20. Although counterintuitive, thaw the extract after completely freezing and make 10 μ L aliquots and refreeze and store at −80 or −20°C (see Note 13).
    21. Prepare a 1:10 dilution from the extract for PCR.
    4. Notes
     
    1. If less than 1 g of material is available, it is possible to scale the entire procedure down to 100 or 50 mg of sample
    using 1.75 mL of the GuSCN extraction-buffer. Volumes for
    L6-buffer + silica and all wash buffers remain the same.
    5 Extraction of DNA from Paleofeces
    41
    2. Cut the feces into smaller pieces to allow for more coverage of the surface area of the feces with the extraction-buffer.
    3. Add the PTB directly to the extraction buffer just before using it.
    4. While it may seem odd, it appears (although the evidence for this is not statistically signifi cant) to be better to add the fecal remains to the solution rather than add the solution to the fecal remains. We suspect this has something to do with the molecular availability of embedded nucleic acids and their accessibility to the salts in the buffer.
    5. It appears to be benefi cial to avoid the transfer of any solid material into the binding buffer (L6).
    6. We have noticed a signifi cant improvement in DNA recovery when silica and L6-buffer are incubated together prior to adding the supernatant from the GuSCN extraction-buffer/sample
    mix. Pre-incubate on a rotating wheel to insure proper mixing of silica and buffer.
    7. We have attempted various compositions of extraction-buffer, for example, phosphate-based buffers and other chaotropic salts such as sodium periodate; none appear to be as successful in achieving the balance between DNA recovery and inhibitor
    removal as the buffer presented here.
    8. Make up exactly enough fresh buffer for each use, and do not store the buffer, as guanidinium is light and temperature—
    sensitive and thus loses effi cacy as it ages.
    9. Keeping the buffers acidic is important ( 15, 16 ) . Measure the pH of the solution after the sample has been added and adjust accordingly. The guanidinium must remain protonated to be
    an effective bridge between the phosphates on the DNA and
    the silica hydroxyl groups. In addition, alkaline conditions are far more conducive to DNA backbone degradation.
    10. In our experience, extending the incubation time has not signifi cantly altered total DNA yield.
    11. It is wise to resuspend the silica pellet by placing the tip of the pipette at the edge of the pellet in the base of the tube and pipetting up and down slowly. Be careful not to allow the solution to bleed over the edge of the tube. While 50 μ L of silica is suffi cient for more than 10 μ g of DNA, the silica clearly gets “clogged” with other polar molecules. Using less than 50 μ L
    is therefore not advised. However, in our experience, increasing the amount of silica above 50 μ L has not been shown to yield quantitatively more DNA.
    12. Any silica remaining in the DNA solution may inhibit downstream reactions. As a precaution, spin down the extract before taking an aliquot for subsequent PCR. See ref. ( 17 ) for other helpful tips.
    42
    M. Kuch and H. Poinar
    13. Storage : It is wise to freeze and thaw the extract post purifi cation and prior to PCR. It appears that many of the inhibitors are precipitated out of solution during this step.
    References
    1. Martin P (1975) Sloth droppings. Nat Hist
    Bryant VM, Cooper A, Pääbo S (2001) A
    74–78
    molecular analysis of dietary diversity for three
    2. Sobolik K (2003) Archaeobiology. AltaMira,
    archaic Native Americans. Proc Natl Acad Sci
    Walnut Creek
    U S A 98:4317–4322
    3. Hofreiter M, Mead