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{\rtf1\mac\deff2 {\fonttbl{\f0\fswiss Chicago;}{\f2\froman New York;}{\f3\fswiss Geneva;}{\f4\fmodern Monaco;}{\f16\fnil Palatino;}{\f20\froman Times;}{\f21\fswiss Helvetica;}{\f22\fmodern Courier;}{\f23\ftech Symbol;}{\f2999\fnil eWorld Tight;} {\f9840\fnil Espy Sans;}{\f9842\fnil Espy Sans Bold;}}{\colortbl\red0\green0\blue0;\red0\green0\blue255;\red0\green25 5\blue255;\red0\green255\blue0;\red255\green0\blue255;\red255\green0\b lue0;\red255\green255\blue0;\red255\green255\blue255;}{\stylesheet{ \f16 \sbasedon222\snext0 Normal;}{\s1\sl480 \f16 \sbasedon0\snext1 potoo;}}{\info{\title SilicaDNA}{\author LMS}}\margr1440\widowctrl\ftnbj \sectd \sbknone\linemod0\linex0\cols1\endnhere \pard\plain \qc\sl360 \f16 {\b\f3\fs28 DNA Extraction Protocols Using Silica}{\b\f3 \par }\pard \sl360 {\b \par }\pard \qc\sl360 Travis Glenn, Ryan Bavis, and Jon Bollback{\b \par }\pard \qc Laboratory of Molecular Systematics\par MRC 534, MSC\par Smithsonian Institution\par Washington, DC 20560\par phone: 301-238-3444\par fax: 301-238-3059\par e-mail: Glenn@onyx.si.edu{\b \par }\pard \sl360 {\b \par }\pard \sl360 \tab Below we present the protocols we have used to isolate DNA from various tissues using Silica and Guanidinium Thiocyanate. These protocols are adapted from Boom {\i et al}. (1990), H\'9ass & P\'8a\'8abo (1993), and H\'9a ss (1994). We highly recommend reading these references before using these protocols. We thank M. H\'9ass and A. Cooper for providing helpful and unpublished information.\par \pard \sl360 \par \pard \sl360 \tab Additional variations on this theme have been developed. Carter & Milton (1993) present a simpler protocol. H\'9ass et al. (1992) combined prot. K d igestions with silica extraction to get DNA from bear poop. Gerloff et al. (1995) combined the methods of Carter & Milton with the Boom et al. (1990) and H\'9ass & P\'8a\'8a bo (1993) to extract DNA from bonobo poop. Constable et al. (1995) combined silica methods with CTAB and phenol extractions to get DNA from baboon poop. So, as you can see, there are lots of variations that could be done. Note also that much of this extraction from poop has made its way into Nature.\par \pard \sl360 \par \pard \sl360 {\b Note}: Carry out all procedures in a fume hood using PCR-free chemicals and supplies.\par \pard \sl360 \par \pard \sl360 Materials needed (suggested stocks are sufficient for several hundred "extractions"):\par \pard \li360\sl360 Silica - ~100 g, Sigma # S-5631 \par GuSCN - ~500 g, Amresco (cheaper) # 0380 or Sigma # G-6639\par Triton X-100 - ~10 mL\par 0.2 M EDTA (pH 8.0) - ~100 mL\par Tris-HCl (pH 6.4) - see H\'9ass (1994), ~250 mL\par Ethanol (95% or 70%) - 500 mL\par NaCl\par 50 mL Conical Centrifuge Tubes - \par 1.5 mL centrifuge tubes\par Aluminum Foil\par 10M HCl\par \par \pard \sl360 {\b I. Preparation of solutions\par \par }\pard \li360\sl360 {\b A. Silica} - note: 2 day procedure{\i \par }\pard \fi-360\li1080\sl360 1. Add 6 g SiO{\fs18\dn4 2} to a 50 mL Falcon tube, and fill to the 50 mL mark with deionized water (dH{\fs18\dn4 2}O).\par \pard \fi-360\li1080\sl360 2. Vortex and allow to settle 24 h at room temperature.\par 3. Remove the upper 43 mL of liquid (aspirate).\par \pard \fi-360\li1080\sl360 4. Refill to 50 mL with dH{\fs18\dn4 2}O, vortex and allow to settle 5 h at room temperature.\par \pard \fi-360\li1080\sl360 5. Remove the upper 44 mL of liquid.\par 6. Add 60 \'b5L 10M HCl.\par \pard \fi-360\li1080\sl360 7. Vortex (homogenize) and aliquot for storage. H\'9ass recommends 500 \'b5L aliquots wrapped in aluminum foil (stable for 6 months at room temperature).\par \pard \sl360 \par \pard \li360\sl360 {\b B.} {\b Extraction buffer\par }\pard \fi-360\li1080\sl360 1. Combine 24 g GuSCN (guanidinium thiocyanate) and 20 mL 0.1 M Tris-HCl (pH 6.4) in a 50 mL Falcon tube.\par \pard \fi-360\li1080\sl360 2. Heat to 60 {\fs18\up10 o}C to dissolve the GuSCN.\par 3. Add 4.4 mL of 0.2 M EDTA (pH 8.0).\par 4. Add 0.5 mL Triton X-100.\par 5. Mix by inverting.\par \pard \fi-360\li1080\sl360 6. Add 0.5 mL of silica suspension (see above), mix, and let sit for at least 1 h at room temperature (mixing sporadically).\par \pard \fi-360\li1080\sl360 7. Spin out the silica by centrifugation. Save the supernatant wrapped in aluminum foil or transfer to a brown bottle (stable 1 month at room temperature).\par \pard \fi-360\li1080\sl360 {\b Note}: Steps 6 & 7 remove contaminating DNA from the buffer.\par \par \pard \li360\sl360 {\b C.} {\b Washing buffer }- note: make double this amount to equal extraction buffer use per sample\par \pard \fi-360\li1080\sl360 1. Combine 24 g GuSCN (guanidinium thiocyanate) and 20 mL 0.1 M Tris-HCl (pH 6.4) in a 50 mL Falcon tube.\par \pard \fi-360\li1080\sl360 2. Heat to 60 {\fs18\up10 o}C to dissolve the GuSCN.\par 3. Add 4.4 mL of 0.2 M EDTA (pH 8.0).\par 4. Mix by inverting.\par \pard \fi-360\li1080\sl360 5. Add 0.5 mL of silica suspension (see above), mix, and let sit for at least 1 h at room temperature (mixing sporadically).\par \pard \fi-360\li1080\sl360 6. Spin out the silica by centrifugation. Save the supernatant wrapped in aluminum foil or transfer to a brown bottle (stable 1 month at room temperature).\par \pard \fi-360\li1080\sl360 \par \pard \li360\sl360 {\b D.} {\b Washing Ethanol}\par \pard \fi-360\li1080\sl360 70% EtOH supplemented with 10 mM NaCl (e.g., for 500 mL add: 368.4 mL 95% EtOH, 1.25 mL 4M NaCl, fill to 500 mL with dH{\fs18\dn4 2}O).\par \pard \sl360 \par \par {\b II. Procedures}\par \par \pard \sl360 Below we present several protocols that are useful when extracting DNA from various tissues. Several parameters can be altered, however, one should keep in mind: 1) it is very difficult to break-up silica pellets when more than 100 \'b5 L of silica is used, 2) the total amount of DNA recovered depends not only on how much is initially bound to the silica, but also how well it is eluted off in TE, and 3) if the guanidine thiocyanate becomes acidic, hydrogen cyanide will be f ormed, thus it is an extremely good idea to do all manipulations in a fume hood!\par \pard \sl360 \par Set-up:\par \pard \fi-360\li720\sl360 in the hood: 3 - 500+ mL side-arm flasks, one attached to vacuum line, ready to be used for aspiration (see Sambrook et al. 1989:1.27), a vortexer, 1 mL pippeter & tips, solutions prepared above,\par \pard \fi-360\li720\sl360 in or near the hood: a microfuge.\par \par \pard \sl360 \par \pard \li360\sl360 {\b A. General Protocol for \ldblquote Soft\rdblquote Tissues (cf. Boom }{\b\i et al}{\b . 1990)\par }\pard \fi-360\li1080\sl360 1. Combine 900 \'b5L extraction buffer and 40 \'b5L silica suspension to a 1.5 mL microfuge tube and vortex.\par \pard \fi-360\li1080\sl360 2. Add tissue and vortex.\par 3. Let sit 10 minutes at room temperature.\par \pard \fi-360\li1080\sl360 4. Vortex briefly to break apart the silica, then centrifuge (15 s) at 12,000 x g. Discard supernatant by aspirating into the side-arm flask.\par \pard \fi-360\li1080\sl360 5. Wash the silica pellet twice with washing buffer by adding 1 mL buffer, vortexing (in some cases it is faster to use a pipette tip to break apart the pellet), & centrifuging (15 s at 12,000 x g). Discard (aspirate) the supernatant each time.\par \pard \fi-360\li1080\sl360 6. Wash twice with 70% ethanol as above (add 1 mL, vortex, centrifuge). Discard (aspirate) supernatants into the second side-arm flask.\par 7. Dry the final pellet in the fume hood with the cap open or in the speed-vac using low heat.\par \pard \fi-360\li1080\sl360 8. Resuspend the pellet in 75 \'b5L TE (10 mM Tris-HCl, 1 mM EDTA).\par \pard \fi-360\li1080\sl360 9. Vortex briefly and incubate at least 10 minutes (up to several hours) at 55{\fs18\up10 o}C.\par 10. Vortex. Centrifuge 2 minutes at 12,000 x g. Transfer the supernatant to a new tube.\par \pard \fi-360\li1080\sl360 11. Repeat steps 8-10, combining the second aliquot of TE with the first in step 10. If significant amount of silica are carried into the new tube, then it may be worthwhile to spin again & transfer the supernatant into another new tube (see note below). \par \pard \fi-360\li1080\sl360 12. Store at 4{\fs18\up10 o}C (short term - days to weeks) or -20{\fs18\up10 o}C (long term).\par \pard \sl360 \par \pard \sl360 Note: ensure that there is no residual silica before using DNA from these protocols. If there is residual silica, then we recommend one of the following: 1) vortex the tubes briefly, centrifuge for 1-2 min. at 12,000 x g, then take out supernatant without disturbing the silica on the bottom of the tube; or 2) vortex the tubes briefly, centrifuge for 1-2 min. at 12,000xg, then transfer the supernatant to a new tube.\par \pard \fi-360\li1080\sl360 \par \pard \li360\sl360 {\b B. Protocol for Non-mammalian Vertebrate Blood. \par }\par \pard \li360\sl360 Note: This protocol is designed for maximum yield obtainable in 1.5 mL tubes. Simply increasing blood or silica has resulted in lower yields in our hands.\par \pard \li360\sl360 \par \pard \fi-360\li1080\sl360 1. Combine 900 \'b5L extraction buffer and 75 \'b5L silica suspension to a 1.5 mL microfuge tube and vortex.\par \pard \fi-360\li1080\sl360 2. Add 15 \'b5L of blood and vortex.\par \pard \fi-360\li1080\sl360 3. Let sit 10 minutes at room temperature or elevated temperature (up to 65{\fs18\up10 o}C).\par 4. Proceed as above (follow the general protocol), except, elute DNA from the silica pellet by using 125 \'b5L of TE, twice.\par \pard \fi-360\li1080\sl360 \par \pard \li360\sl360 {\b C. Protocol for \ldblquote Hard\rdblquote Tissues (cf. H\'9ass & P\'8a\'8abo 1993).}\par \pard \fi-360\li1080\sl360 1. 500 mg tissue is combined with 1 mL extraction buffer in a 1.5 ml microfuge tube.\par \pard \fi-360\li1080\sl360 2. Incubate at 60{\fs18\up10 o}C for at least 1 h with sporadic agitation.\par \pard \fi-360\li1080\sl360 3. Spin down debris (1 min. at 12,000 x g) and transfer 500 \'b5L of the supernatant to a fresh microfuge tube.\par \pard \fi-360\li1080\sl360 4. Add 500 \'b5L extraction buffer and 40 \'b5L silica suspension.\par 5. Proceed from step 3 of the general protocol.\par \par \pard \sl360 {\b Waste Disposal:\par }\par \pard \sl360 {\b The aspirated extraction and washing buffers should be placed in a brown glass bottle with} {\b a minimum of 1/20th volume 10N NaOH}. This bottle should be kept under the fume hood and should be disposed of as hazardous waste.\par \pard \sl360 \par \pard \sl360 The aspirated 70% EtOH can usually be disposed of in the same manner as any alcohol waste.\par \pard \sl360 \par {\b References}\par \par \pard \fi-360\li360\sl360 Boom, R., C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. E. Wertheim-van Dillen, and J. van der Noordaa. 1990. Rapid and simple methods for purification of nucleic acids. Journal of Clinical Microbiology 28(3):495-503. \par \pard \fi-360\li360\sl360 Carter, M. J. and I. D. Milton. 1993. An inexpensive and simple method for DNA purifications on silica particles. Nucleic Acids Research 21(4):1044.\par Constable, J. J., C. Packer, D. A. Collins, and A. E. Pusey. 1995. Nuclear DNA from primate dung. Nature 373:393.\par \pard \fi-360\li360\sl360 Gerloff, U., C. Schl\'9a tterer, K. Rassmann, I. Rambold, G. Hohmann, B. Fruth, and D. Tautz. 1995. Amplification of hypervariable simple sequence repeats (microsatellites) from excremental DNA of wild living bonobos (Pan paniscus). Molecular Ecology 515-518.\par \pard \fi-360\li360\sl360 H\'9ass, M. 1994. More about the silica method. Ancient DNA Newsletter 2(1):10-12.\par H\'9ass, M., M Kohn, S. P\'8a\'8abo, F. Knauer, and W. Schr\'9ader. 1992. Excremental analysis by PCR. Nature 359:199.\par H\'9ass, M. and S. P\'8a\'8abo. 1993. DNA extraction from Pleistocene bones by a silica-based purification method. Nucleic Acids Research 21(16):3913-3914. \par \pard \fi-360\li360\sl360 \par \par }