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remain stable for several months.
    7. DTT is a reducing agent that can cleave cystine–cystine bridges and disrupt the tertiary structure of some proteins, prior to digestion. DTT should be stored in the freezer where it will remain stable for several months.
    8. Phase-lock or phase-dividing gels are useful in this capacity as they allow easy decanting of the aqueous phase. The inert gel can be added to tubes, and upon centrifugation, forms a barrier between the two phases. In the absence of gel, the aqueous phase can be transferred by careful manual pipetting.
    9. Phenol is dangerous. Extreme caution must be exercised when aliquotting and transferring phenolic samples. Familiarise yourself with the appropriate safety information and correct disposal 18
    R. Barnett and G. Larson
    methods before use. Polyethylene glycol should be kept to
    hand wherever phenol is used and those performing the technique should acquaint themselves with emergency procedures in case of phenol spills.
    10. Chloroform is hazardous and should be handled with caution.
    11. Concentrators use a membrane barrier of pre-determined pore size to prevent the passage of molecules larger than a certain molecular weight. A pore size with a molecular weight cut-off of 30,000 Da should prevent the loss of any nucleic acid larger than about 50 bp in length, but will allow removal of almost all other digested biomolecules.
    12. This step removes any preservative coatings and potentially adsorbed environmental contaminants. Recent work suggests
    that the speed setting of the drill and the amount of friction produced can have a negative effect on DNA recover y ( 11 ) .
    Generally, use the lowest speed setting possible for abrasion and cutting of bone and tooth.
    13. Some of the sample should be retained for further extractions.
    Replication, either internal or external, may be necessary for ancient samples.
    14. To adapt the protocol for tissue/hair/nail, the overnight chelation stage can be omitted. Begin at the digestion stage and add 0.5 mL of 1× Chelation buffer to the digestion buffer, SDS, proteinase K, and DTT ( 12 ) . Follow the remainder of the protocol as described.
    15. The eluate is also likely to contain DNA and can be retained for processing, either in parallel or at a later date ( 8 ) .
    16. The temperature setting for digestion can be modifi ed depending on the sample. Recent work suggests that lower temperatures may have a benefi cial effect on DNA recovery. If the temperature is lowered, increase the length of time the samples are left to rotate until complete digestion is achieved ( 8 ) .
    17. Very occasionally, the phase-lock tubes may not separate properly between aqueous and hydrophobic phases. In this case, care should be taken when manually removing the aqueous
    phase by pipette.
    18. Adding a small volume of ultrapure water to the fi lters prior to adding the extract may aid in absorption of DNA to the membrane. Depending on the volume of extract to be processed, this step may have to be repeated multiple times until the entire sample has passed through the membrane.
    19. Flushing water through the membrane after the entire sample has been passed through may help to further remove any
    potential inhibitors from the fi nal extract.
    20. After extraction, DNA can be roughly quantifi ed by measurement on a spectrophotometric platform. Note that this does 2 A Phenol–Chloroform Protocol for Extracting DNA from Ancient Samples 19
    not give an indication of how much of the DNA in the extract is derived from the sample vs. from coextracted environmental contaminants.
    21. It may be useful to subdivide the fi nal extracts into aliquots of 20–50 m L and to use these as necessary. DNA is susceptible to damage from repeat fr eeze–thaw cycles ( 13 ) and should be defrosted as infrequently as possible.
    Acknowledgments
    Thanks to Beth Shapiro for the opportunity to contribute this chapter.
    References
    1. Hagelberg E, Clegg JB (1991) Isolation and 8.