By Tara L. Fulton (auth.), Beth Shapiro, Michael Hofreiter (eds.)
Research into historical DNA started greater than 25 years in the past with the e-book of brief mitochondrial DNA series fragments from the quagga, an extinct relative of the zebra. historical DNA examine fairly received momentum following the discovery of PCR, which allowed thousands of copies to be made up of the few final DNA molecules preserved in fossils and museum specimens. In Ancient DNA: equipment and Protocols specialist researchers within the box describe a number of the protocols which are now popular to review historic DNA. those comprise directions for constructing an historic DNA laboratory, extraction protocols for quite a lot of assorted substrates, info of laboratory ideas together with PCR and NGS library education, and proposals for acceptable analytical methods to make experience of the sequences got. Written within the hugely winning Methods in Molecular Biology™ sequence structure, chapters contain introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, without difficulty reproducible laboratory protocols, and key tips about troubleshooting and heading off recognized pitfalls.
Authoritative and sensible, Ancient DNA: tools and Protocols seeks to assist scientists within the extra research of historical DNA and the methodological methods in historical research.
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Additional info for Ancient DNA: Methods and Protocols
3. Methods All steps are to be carried out at room temperature. 1. Preparing the Silica Suspension 1. 8 g of silicon dioxide into a 50-mL tube, add water to 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 mL 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).
At 4°C, the SDS will precipitate out of solution. Prior to the addition of DTT and proteinase K, the buffer should be warmed up until the SDS is fully dissolved. 2. Any bleach carryover will degrade the DNA and reagents in subsequent steps of the DNA extraction, thus it is extremely important that bleach is removed completely. 3. The volume of digestion buffer needed is sample dependent, but generally should be at least sufficient to cover the surface of the material. 4. DNA can be purified from the digestion mixture in a number of different ways.
5-mL microcentrifuge tube (see Note 11). 10. Centrifuge for 30 s and discard the supernatant. 11. If the solutions are still heavily colored, repeat steps 9–10. 12. Add 1 mL of wash buffer and resuspend the silica. 13. Centrifuge for 30 s and discard supernatant. 14. Centrifuge again briefly and remove all remaining liquid with a pipette. 15. Dry the remaining silica pellet in a heating block for 5 min at 56°C (to remove residual ethanol). 16. 05% Tween-20 and incubate with agitation for 15 min at 56°C.