By Tara L. Fulton (auth.), Beth Shapiro, Michael Hofreiter (eds.)
Research into historic DNA started greater than 25 years in the past with the book of brief mitochondrial DNA series fragments from the quagga, an extinct relative of the zebra. historic DNA examine particularly received momentum following the discovery of PCR, which allowed thousands of copies to be made up of the few ultimate DNA molecules preserved in fossils and museum specimens. In Ancient DNA: tools and Protocols specialist researchers within the box describe a number of the protocols which are now widely used to review old DNA. those comprise directions for establishing an old DNA laboratory, extraction protocols for quite a lot of various substrates, information of laboratory recommendations together with PCR and NGS library guidance, and recommendations for acceptable analytical ways to make experience of the sequences received. Written within the hugely profitable Methods in Molecular Biology™ sequence structure, chapters contain introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, with ease reproducible laboratory protocols, and key tips about troubleshooting and warding off recognized pitfalls.
Authoritative and functional, Ancient DNA: equipment and Protocols seeks to assist scientists within the additional research of old DNA and the methodological techniques in historical research.
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Extra resources for Ancient DNA: Methods and Protocols
Add 48 μL 30% hydrochloric acid (keep pH acidic <3). 7. Resuspend the pellet, and aliquot approx. 200 μL 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. 2. Paleofeces DNA Extraction 1. Add approximately 1 g of fecal material to a small weighing 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 final 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).
Nat Rev Genet 2:353–359 4. Cooper A, Poinar HN (2000) Ancient DNA: do it right or not at all. Science 289:1139 5. Leonard JA, Shanks O, Hofreiter M, Kreuz E, Hodges L, Ream W, Wayne RK, Fleischer RC (2007) Animal DNA in PCR reagents plagues ancient DNA research. J Archaeol Sci 34:1361–1366 6. Rohland N, Hofreiter M (2007) Ancient DNA extraction from bones and teeth. Nat Protoc 2:1756–1762 7. Handt O, Höss M, Krings M, Pääbo S (1994) Ancient DNA: methodological challenges. Experientia 50:524–527 8.
2. 5 mL of binding buffer and 100 mL 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 mL of binding buffer.