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and a matching nut. Prior to use, the threaded end of the boltswere ground flat to provide an even grinding surface to maximizethe pulverization of the drill shavings (Thomas and Moore 1997).The nuts and bolts were then wrapped in aluminum foil and sterilizedin an autoclave. The nut was partially threaded on to one boltcreating a small cup that was then filled with approximately 0.1 gof drill shavings. The second bolt was added to make a sealedchamber encapsulating the drill shavings (Thomas and Moore1997). The bolt assembly was placed in liquid nitrogen for 30seconds to one minute (Thomas and Moore 1997). Upon removalfrom the liquid nitrogen, the sample was pulverized by simultaneouslytwisting both bolts together. The bolt assembly was tightenedand loosened several times and tapped on the hard surface toassure the sample was uniformly pulverized (Thomas and Moore1997). The bolt assembly was then carefully dismantled over apiece of filter paper to collect the powder and facilitate transfer toa 1.5 ml microcentrifuge tube.The powdered drill shavings were decalcified in 500 µl of 0.5M pH 8.0 EDTA at 37ºC while shaking at 225 rpm until the pelletshad broken down, (approximately three to five days) following amodified protocol for the Qiagen DNeasy Blood and Tissue kit(Qiagen, Valencia, California) (Qiagen 2006). The decalcificationstep is critical since the cells containing DNA must be freed fromthe calcified matrix in order for the extraction to be successful.The samples were centrifuged at 13,000 rpm to pellet and washedthree times with sterile deionized water to remove ions that hadaccumulated during the decalcification process. Decalcifiedsamples were then processed using a Qiagen DNeasy Blood andTissue kit following the modified protocol above.Results.—A total of 0.1 g of drill shavings yielded enough DNAfor multiple genetic analyses. Total DNA yields ranged from 110to 4650 ng. The high molecular weight DNA produced with thismethod has been verified by ultraviolet spectrophotometry andPCR. Ratios of A260/A280 measurements ranged from 1.6 to 2.0.PCR amplifications with seven species-specific microsatelliteprimers produced strong, clear bands in >91% of the samples tested.Discussion.—Our results show that DNA can be obtained froma standard method of marking turtles. By refining and combiningexisting techniques, we have developed a protocol that requireslesser amounts of shell material and minimizes the invasive procedureswhen compared to currently published techniques. Thedrilling or filing of the marginal scutes has been observed to showno signs of physical pain in the animals (Gibbons 1968). Pike etal. (2005) also noted that handling and drilling stress wore offquickly enough to have no discernable effect on recapture rates,demonstrating that drill marking has little detrimental effect ontortoises. Mockford et al. (1999) described a similar protocol forDNA extraction from freshwater turtle hatchlings; however it isimportant to note that the shell material was not ossified at thetime of removal making the DNA extraction process less problematic.Our technique provides the basis to easily gather and processgenetic material from ossified shell to examine chelonian geneticdiversity and long-term viability. A better understanding of thepopulation structure and effective populations of these long-livedand slow reproducing animals is necessary to properly formulatemanagement strategies for these animals (Gibbons et al. 2000; Scottand Seigel 1992). Our protocol uses a waste product of a commonmarking technique to obtain high-quality DNA allowing for theaddition of genetic analysis to ongoing mark-recapture studies.Our technique enables non-invasive sampling of a population toget DNA samples of comparable quality to those obtained withmore invasive methods.Acknowledgments.—Special thanks go to R. Bolt for her constant andgracious help with this project. Much of what we have done could nothave been accomplished without her assistance. Thanks also go to S. Weissand P. Cain for their help in locating tortoises for this study. We wouldalso like to thank T. Crabill for her advice on bucket trap placement and J.Sinclair for his valuable comments on this manuscript. We are also gratefulto R. Hinkle of Dynamac and J. Stiner of the National Park Service forfinancial support. We thank personnel from the Merritt Island NationalWildlife Refuge (especially M. Epstein and M. Legare) for providingpermits to conduct this study. We also thank the Canaveral National Seashore(J. Stiner) for logistical support and special use permits. All procedureswere approved by the NASA Animal Care and Use Permit #GRD-06-042 with R. Bolt as the point of contact.LITERATURE CITEDAVERY, H. R., AND L. J. VITT. 1984. How to get blood from a turtle. Copeia1984:209–210.CAGLE, F. R. 1939. A system of marking turtles for future identification.Copeia 1939:170–173.ERNST, C. H., R. W. BARBOUR, AND M. F. HERSHEY. 1974. A new codingsystem for hard shelled turtles. Trans. Kentucky Acad. Sci. 35:27–28.GIBBONS, J. W. 1968. Population structure and survivorship in the paintedturtle, Chrysemys picta. Copeia 1968:360–368.––––––, D. E. SCOTT, T. J. RYAN, K. A. BUHLMANN, T. D. TUBERVILLE, B. S.METTS, J. L. GREEN, T. MILLS, Y. LEIDEN, S. POPPY, AND C. T. WINNE.2000. The global decline of reptiles, dèjá vu amphibians. Bioscience50:653–666.HEDRICK, P. W., AND S. T. KALINOWSKI. 2000. Inbreeding depression inconservation biology. Annu. Rev. Ecol. Syst. 31:139–162.HSIEH, H.-M., L.-H. HUANG, L.-C. TSAI, C.-L. LIU, Y.-C. KUO, C.-T. HSIAO,A. LINACRE, AND J. C.-I. LEE. 2006. Species identification of Kachugatecta using the cytochrome b gene. J. Forensic Sci. 51:52–56.JACOBSON, E. R., J. SCHUMACHER, AND M. GREEN. 1992. Field and clinicaltechniques for sampling and handling blood for hematologic and selectedbiochemical determinations in the desert tortoise Xerobatesagassizii. Copeia 1992:237–241.JEHLE, R., AND J. W. ARNTZEN. 2002. Microsatellite markers in amphibianconservation genetics. Herpetol. J. 12:1–9.MOCKFORD, S. W., J. M. WRIGHT, M. SNYDER, AND T. B. HERMAN. 1999. Anon-destructive source of DNA from hatchling freshwater turtles foruse in PCR base assays. Herpetol. Rev. 30:148–149.PIKE, D. A., A. DINSMORE, T. CRABILL, R. B. SMITH, AND R. A. SEIGEL.2005. Short-term effects of handling and permanently marking gophertortoises (Gopherus polyphemus) on recapture rates and behavior.Appl. Herpetol. 2:139–147.QIAGEN. 2006. Purification of total DNA from compact animal bone usingDNeasy blood & tissue kit. http://www1.qiagen.com/literature/protocols/pdf/DY01.pdf.ROSSKOPF, W. J. 1982. Normal hemogram and blood chemistry values forCalifornia desert tortoises. Vet. Med./Small Anim. Clin. 77:85–87.SCOTT JR., N. J., AND R. A. SEIGEL. 1992. The management of amphibianand reptile populations: species priorities and methodological and theoreticalconstraints. In D. R. McCullough, and R. H. Barrett (eds.), Wildlife2001: Populations, pp. 343–368. Elsevier Applied Science, London.THOMAS, M. G., AND L. J. MOORE. 1997. Preparation of bone samples forDNA extraction: a nuts and bolts approach. BioTechniques 22:402.Herpetological Review 39(2), 2008 191