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}{\listlevel\levelnfc23\leveljc0\levelfollow0\levelstartat1\levelspace360\levelindent0{\leveltext\'01\u-3929 _;}{\levelnumbers;}\f14\fbias0 \fi-360\li6120\jclisttab\tx6120 }{\listname ;}\listid2096050508}{\list\listtemplateid67698689 \listsimple{\listlevel\levelnfc23\leveljc0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\'01\u-3913 _;}{\levelnumbers;}\f3\fbias0 \fi-360\li360\jclisttab\tx360 }{\listname ;}\listid2125493141}{\list\listtemplateid67698689\listsimple {\listlevel\levelnfc23\leveljc0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\'01\u-3913 _;}{\levelnumbers;}\f3\fbias0 \fi-360\li360\jclisttab\tx360 }{\listname ;}\listid2130275792}}{\*\listoverridetable{\listoverride\listid1577402864 \listoverridecount0\ls1}{\listoverride\listid1459910479\listoverridecount0\ls2}{\listoverride\listid463937144\listoverridecount0\ls3}{\listoverride\listid1723209359\listoverridecount0\ls4}{\listoverride\listid936912162\listoverridecount0\ls5} {\listoverride\listid2050105259\listoverridecount0\ls6}{\listoverride\listid733552978\listoverridecount0\ls7}{\listoverride\listid2048722723\listoverridecount0\ls8}{\listoverride\listid1231770961\listoverridecount0\ls9}{\listoverride\listid1444114457 \listoverridecount0\ls10}{\listoverride\listid1\listoverridecount0\ls11}{\listoverride\listid2\listoverridecount0\ls12}{\listoverride\listid5\listoverridecount0\ls13}{\listoverride\listid2\listoverridecount0\ls14}{\listoverride\listid3 \listoverridecount0\ls15}{\listoverride\listid805897685\listoverridecount0\ls16}{\listoverride\listid1584411579\listoverridecount0\ls17}{\listoverride\listid2096050508\listoverridecount0\ls18}{\listoverride\listid1208183804\listoverridecount0\ls19} {\listoverride\listid465322660\listoverridecount0\ls20}{\listoverride\listid1464158634\listoverridecount0\ls21}{\listoverride\listid1533572390\listoverridecount0\ls22}{\listoverride\listid2091611627\listoverridecount0\ls23}{\listoverride\listid1990673692 \listoverridecount0\ls24}{\listoverride\listid2062821488\listoverridecount0\ls25}{\listoverride\listid2125493141\listoverridecount0\ls26}{\listoverride\listid1895657098\listoverridecount0\ls27}{\listoverride\listid2130275792\listoverridecount0\ls28} {\listoverride\listid33577681\listoverridecount0\ls29}{\listoverride\listid1620837308\listoverridecount0\ls30}{\listoverride\listid1\listoverridecount0\ls31}}{\info{\title Microsatellite Isolation Protocol}{\author Rasmus}{\operator Travis Glenn} {\creatim\yr2000\mo9\dy5\hr10\min28}{\revtim\yr2000\mo9\dy21\hr19\min9}{\printim\yr2000\mo6\dy30\hr16\min45}{\version6}{\edmins45}{\nofpages24}{\nofwords7525}{\nofchars42898}{\*\company Dell Computer Corporation}{\nofcharsws52681}{\vern99}} \margl1152\margr1152\margt720\margb850 \widowctrl\ftnbj\aenddoc\noxlattoyen\expshrtn\noultrlspc\dntblnsbdb\nospaceforul\hyphcaps0\formshade\viewkind1\viewscale100\pgbrdrhead\pgbrdrfoot \fet0\sectd \linex0\endnhere\sectdefaultcl {\*\pnseclvl1 \pnucrm\pnstart1\pnindent720\pnhang{\pntxta .}}{\*\pnseclvl2\pnucltr\pnstart1\pnindent720\pnhang{\pntxta .}}{\*\pnseclvl3\pndec\pnstart1\pnindent720\pnhang{\pntxta .}}{\*\pnseclvl4\pnlcltr\pnstart1\pnindent720\pnhang{\pntxta )}}{\*\pnseclvl5 \pndec\pnstart1\pnindent720\pnhang{\pntxtb (}{\pntxta )}}{\*\pnseclvl6\pnlcltr\pnstart1\pnindent720\pnhang{\pntxtb (}{\pntxta )}}{\*\pnseclvl7\pnlcrm\pnstart1\pnindent720\pnhang{\pntxtb (}{\pntxta )}}{\*\pnseclvl8\pnlcltr\pnstart1\pnindent720\pnhang {\pntxtb (}{\pntxta )}}{\*\pnseclvl9\pnlcrm\pnstart1\pnindent720\pnhang{\pntxtb (}{\pntxta )}}\pard\plain \qc\widctlpar\adjustright \fs20\cgrid {\b\f4\fs24 Chapter 2 - Microsatellite Isolation 2000}{\b\f4\fs24\up20 \u169\'a9}{\f4\fs24 \par }\pard \widctlpar\tx2160\tx5760\adjustright {\f4\fs24 \par }\pard \qc\widctlpar\adjustright {\f4\fs24 Travis C. Glenn}{\f4\fs24\super 1,2}{\f4\fs24 , Tawnya Cary}{\f4\fs24\super 2}{\f4\fs24 , Mandy Dust}{\f4\fs24\super 1}{\f4\fs24 , Susanne Hauswaldt}{\f4\fs24\super 1,2}{\f4\fs24 , Kelly Prince}{\f4\fs24\super 2} {\f4\fs24 , Clifton Ramsdell}{\f4\fs24\super 2}{\f4\fs24 , and Irma Shute}{\f4\fs24\super 3}{\f4\fs24 \par \par }\pard\plain \s19\widctlpar\tx1440\tx5130\tx5760\adjustright \f12\cgrid {\f4 Address:\tab }{\f4\super 1}{\f4 Savannah River Ecology Lab\tab }{\f4\super 2}{\f4 Dept. Biol. Sci.; or }{\f4\super 3}{\f4 Medical School \par }\pard\plain \widctlpar\tx1440\tx5130\tx5760\adjustright \fs20\cgrid {\f4\fs24 \tab Drawer E\tab University of South Carolina \par \tab Aiken, SC 29802\tab Columbia, SC 29208 \par \par Telephone:\tab (803) 725-5746 (5881 \endash lab)\tab (803) 777-4606 \par Fax:\tab (803) 725-3309\tab (803) 777-4002 \par E-mail:\tab Glenn@srel.edu\tab Travis.Glenn@sc.edu \par }\pard \widctlpar\adjustright {\f4 \par \par \par }{\f4\fs24 Below we present explicit step by step protocols that we used in the 5-week summer class, Biology 656, at the University of South Carolina. We used these protocols to construct libraries, screen those libraries, and determi ne flanking sequences, in 3 birds, 3 reptiles, 2 mammals, 2 insects, 1 amphibian, and even one species of micro-crustacean. In theory, this protocol will work for any eukaryotic organism (i.e, anything with an appreciable number of microsatellites); or a n y other chunk of DNA you can capture with an oligonucleotide. The biggest differences among species are: 1) how the initial DNA sample is isolated, and 2) which microsatellites occur most frequently, and thus are targeted for enrichment & isolation (tho ugh this is less of problem when mixtures of oligos are used). \par \par We have built upon the approach outlined in Hamilton et al. (1999), which derives from Armour et al. (1994) and Kandpal et al. (1994). Many people have published their variations along this th eme. Using hybridization capture for enrichment of microsatellites is now in widespread use because it is faster and easier to do with multiple samples than the original enrichment described by Ostrander et al. (1992; see ftp://onyx.si.edu/protocols/Msat ManV6.rtf for step by step protocols for U-DNA enrichment). We aren\rquote t doing anything terribly unique relative to other labs, but we have brought together the best specific approaches from many protocols to reduce a lot of unnecessary time & steps from most other protocols out there. We hope you can use these protocols to help isolate microsatellites from your organism of interest. \par \par TCG 7/00 \par }\pard\plain \s15\qc\widctlpar\adjustright \b\f1\cgrid {\fs28\ul \page Microsatellite Isolation Overview \par }{\fs20\ul \par }\pard\plain \qc\widctlpar\adjustright \fs20\cgrid {\lang1024 {\shpgrp{\*\shpinst\shpleft1605\shptop9224\shpright6994\shpbottom10156\shpfhdr0\shpbxcolumn\shpbypara\shpwr3\shpwrk0\shpfblwtxt0\shpz13\shplid1026 {\sp{\sn groupLeft}{\sv 2505}}{\sp{\sn groupTop}{\sv 10990}}{\sp{\sn groupRight}{\sv 7894}}{\sp{\sn groupBottom}{\sv 11922}}{\sp{\sn fFlipH}{\sv 0}}{\sp{\sn fFlipV}{\sv 0}} {\sp{\sn rotation}{\sv 0}}{\sp{\sn lidRegroup}{\sv 26}}{\shpgrp{\*\shpinst\shplid1027{\sp{\sn groupLeft}{\sv 2505}}{\sp{\sn groupTop}{\sv 10990}}{\sp{\sn groupRight}{\sv 7894}} {\sp{\sn groupBottom}{\sv 11570}}{\sp{\sn relLeft}{\sv 2505}}{\sp{\sn relTop}{\sv 10990}}{\sp{\sn relRight}{\sv 7894}}{\sp{\sn relBottom}{\sv 11570}}{\sp{\sn fRelFlipH}{\sv 0}} {\sp{\sn fRelFlipV}{\sv 0}}{\shp{\*\shpinst\shplid1028{\sp{\sn relLeft}{\sv 2505}}{\sp{\sn relTop}{\sv 10990}}{\sp{\sn relRight}{\sv 6290}}{\sp{\sn relBottom}{\sv 11570}}{\sp{\sn fRelFlipH}{\sv 0}}{\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 202}} {\sp{\sn lTxid}{\sv 65536}}{\sp{\sn hspNext}{\sv 1028}}{\sp{\sn lidRegroup}{\sv 20}}{\shptxt \pard\plain \qc\widctlpar\adjustright \fs20\cgrid {\b\f1 X. Bacterial Transformation }{\f1 via \par electroporation or heatshock}{\b\f1 \par }}}}{\shp{\*\shpinst\shplid1029{\sp{\sn relLeft}{\sv 6321}}{\sp{\sn relTop}{\sv 11261}}{\sp{\sn relRight}{\sv 7074}}{\sp{\sn relBottom}{\sv 11261}}{\sp{\sn fRelFlipH}{\sv 0}} {\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 20}}{\sp{\sn shapePath}{\sv 4}}{\sp{\sn fFillOK}{\sv 0}}{\sp{\sn fFilled}{\sv 0}}{\sp{\sn lineEndArrowhead}{\sv 1}}{\sp{\sn fArrowheadsOK}{\sv 1}}{\sp{\sn lidRegroup}{\sv 20}}}} {\shp{\*\shpinst\shplid1030{\sp{\sn relLeft}{\sv 7014}}{\sp{\sn relTop}{\sv 11033}}{\sp{\sn relRight}{\sv 7894}}{\sp{\sn relBottom}{\sv 11433}}{\sp{\sn fRelFlipH}{\sv 0}}{\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 202}}{\sp{\sn lTxid}{\sv 131072}} {\sp{\sn dxTextLeft}{\sv 54864}}{\sp{\sn dyTextTop}{\sv 54864}}{\sp{\sn dxTextRight}{\sv 54864}}{\sp{\sn dyTextBottom}{\sv 54864}}{\sp{\sn hspNext}{\sv 1030}}{\sp{\sn lidRegroup}{\sv 20}}{\shptxt \pard\plain \qc\widctlpar\adjustright \fs20\cgrid {\f1 3 hrs}{ \par }}}}}}{\shp{\*\shpinst\shplid1031{\sp{\sn relLeft}{\sv 4320}}{\sp{\sn relTop}{\sv 11582}}{\sp{\sn relRight}{\sv 4320}}{\sp{\sn relBottom}{\sv 11922}}{\sp{\sn fRelFlipH}{\sv 0}} {\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 20}}{\sp{\sn shapePath}{\sv 4}}{\sp{\sn fFillOK}{\sv 0}}{\sp{\sn fFilled}{\sv 0}}{\sp{\sn lineWidth}{\sv 12700}}{\sp{\sn lineEndArrowhead}{\sv 1}}{\sp{\sn fArrowheadsOK}{\sv 1}}{\sp{\sn fLine}{\sv 1}}}} }{\shprslt{\*\do\dobxcolumn\dobypara\dodhgt8205\dppolygon\dppolycount4\dpptx0\dppty0\dpptx5389\dppty0\dpptx5389\dppty932\dpptx0\dppty932\dpx1605\dpy9224\dpxsize5389\dpysize932 \dpfillfgcr255\dpfillfgcg255\dpfillfgcb255\dpfillbgcr255\dpfillbgcg255\dpfillbgcb255\dpfillpat1\dplinew15\dplinecor0\dplinecog0\dplinecob0}}} {\shpgrp{\*\shpinst\shpleft2125\shptop8260\shpright10038\shpbottom9199\shpfhdr0\shpbxcolumn\shpbypara\shpwr3\shpwrk0\shpfblwtxt0\shpz12\shplid1032{\sp{\sn groupLeft}{\sv 3025}}{\sp{\sn groupTop}{\sv 9906}}{\sp{\sn groupRight}{\sv 10938}} {\sp{\sn groupBottom}{\sv 10845}}{\sp{\sn fFlipH}{\sv 0}}{\sp{\sn fFlipV}{\sv 0}}{\sp{\sn rotation}{\sv 0}}{\sp{\sn lidRegroup}{\sv 26}}{\shpgrp{\*\shpinst\shplid1033 {\sp{\sn groupLeft}{\sv 3025}}{\sp{\sn groupTop}{\sv 9906}}{\sp{\sn groupRight}{\sv 10938}}{\sp{\sn groupBottom}{\sv 10566}}{\sp{\sn relLeft}{\sv 3025}}{\sp{\sn relTop}{\sv 9906}} {\sp{\sn relRight}{\sv 10938}}{\sp{\sn relBottom}{\sv 10566}}{\sp{\sn fRelFlipH}{\sv 0}}{\sp{\sn fRelFlipV}{\sv 0}}{\shp{\*\shpinst\shplid1034{\sp{\sn relLeft}{\sv 3025}} {\sp{\sn relTop}{\sv 9914}}{\sp{\sn relRight}{\sv 5835}}{\sp{\sn relBottom}{\sv 10505}}{\sp{\sn fRelFlipH}{\sv 0}}{\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 202}} {\sp{\sn lTxid}{\sv 196608}}{\sp{\sn hspNext}{\sv 1034}}{\sp{\sn lidRegroup}{\sv 20}}{\shptxt \pard\plain \s3\qc\keepn\widctlpar\outlinelevel2\adjustright \b\f1\fs22\cgrid {\fs20 IX.A. 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Linker ligation \par }\pard\plain \s2\qc\keepn\widctlpar\outlinelevel1\adjustright \f1\cgrid {\fs20 SNX linker for }{\i\fs20 Rsa}{\fs20 and }{\i\fs20 Hae}{\fs20 III digest \par }}}}{\shp{\*\shpinst\shplid1063{\sp{\sn relLeft}{\sv 6427}}{\sp{\sn relTop}{\sv 3839}}{\sp{\sn relRight}{\sv 7266}}{\sp{\sn relBottom}{\sv 3839}}{\sp{\sn fRelFlipH}{\sv 0}}{\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 20}}{\sp{\sn shapePath}{\sv 4}} {\sp{\sn fFillOK}{\sv 0}}{\sp{\sn fFilled}{\sv 0}}{\sp{\sn lineEndArrowhead}{\sv 1}}{\sp{\sn fArrowheadsOK}{\sv 1}}{\sp{\sn lidRegroup}{\sv 34}}}}{\shp{\*\shpinst\shplid1064{\sp{\sn relLeft}{\sv 7293}}{\sp{\sn relTop}{\sv 3648}} {\sp{\sn relRight}{\sv 8173}}{\sp{\sn relBottom}{\sv 4048}}{\sp{\sn fRelFlipH}{\sv 0}}{\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 202}}{\sp{\sn lTxid}{\sv 983040}} {\sp{\sn dxTextLeft}{\sv 54864}}{\sp{\sn dyTextTop}{\sv 54864}}{\sp{\sn dxTextRight}{\sv 54864}}{\sp{\sn dyTextBottom}{\sv 54864}}{\sp{\sn hspNext}{\sv 1064}}{\sp{\sn lidRegroup}{\sv 34}}{\shptxt \pard\plain \qc\widctlpar\adjustright \fs20\cgrid {\f1 10 hrs \par }{ \par }}}}{\shp{\*\shpinst\shplid1065{\sp{\sn relLeft}{\sv 8706}}{\sp{\sn relTop}{\sv 3512}}{\sp{\sn relRight}{\sv 10726}}{\sp{\sn relBottom}{\sv 4192}}{\sp{\sn fRelFlipH}{\sv 0}} {\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 202}}{\sp{\sn lTxid}{\sv 1048576}}{\sp{\sn dxTextLeft}{\sv 54864}}{\sp{\sn dyTextTop}{\sv 54864}}{\sp{\sn dxTextRight}{\sv 54864}} {\sp{\sn dyTextBottom}{\sv 54864}}{\sp{\sn hspNext}{\sv 1065}}{\sp{\sn lineDashing}{\sv 2}}{\sp{\sn lineEndCapStyle}{\sv 0}}{\sp{\sn fLine}{\sv 1}}{\sp{\sn lidRegroup}{\sv 34}}{\shptxt \pard\plain \s18\qc\widctlpar\adjustright \i\f1\fs22\cgrid {\fs20 PCR: check for successful ligation \par }}}}{\shp{\*\shpinst\shplid1066{\sp{\sn relLeft}{\sv 8177}}{\sp{\sn relTop}{\sv 3848}}{\sp{\sn relRight}{\sv 8712}}{\sp{\sn relBottom}{\sv 3848}}{\sp{\sn fRelFlipH}{\sv 0}}{\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 20}}{\sp{\sn shapePath}{\sv 4}} {\sp{\sn fFillOK}{\sv 0}}{\sp{\sn fFilled}{\sv 0}}{\sp{\sn lineEndArrowhead}{\sv 1}}{\sp{\sn fArrowheadsOK}{\sv 1}}{\sp{\sn lidRegroup}{\sv 34}}}}}}{\shp{\*\shpinst\shplid1067{\sp{\sn relLeft}{\sv 4572}}{\sp{\sn relTop}{\sv 4182}} {\sp{\sn relRight}{\sv 4572}}{\sp{\sn relBottom}{\sv 4602}}{\sp{\sn fRelFlipH}{\sv 0}}{\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 20}}{\sp{\sn shapePath}{\sv 4}}{\sp{\sn fFillOK}{\sv 0}}{\sp{\sn fFilled}{\sv 0}}{\sp{\sn lineWidth}{\sv 12700}} {\sp{\sn lineEndArrowhead}{\sv 1}}{\sp{\sn fArrowheadsOK}{\sv 1}}{\sp{\sn fLine}{\sv 1}}{\sp{\sn lidRegroup}{\sv 33}}}}}{\shprslt{\*\do\dobxcolumn\dobypara\dodhgt8199\dppolygon\dppolycount4\dpptx0\dppty0\dpptx7949\dppty0\dpptx7949\dppty1168 \dpptx0\dppty1168\dpx1625\dpy92\dpxsize7949\dpysize1168\dpfillfgcr255\dpfillfgcg255\dpfillfgcb255\dpfillbgcr255\dpfillbgcg255\dpfillbgcb255\dpfillpat1\dplinew15\dplinecor0\dplinecog0\dplinecob0}}}}{ \par }\pard \qc\widctlpar\adjustright { \par }\pard \widctlpar\adjustright { \par \par \par }{\lang1024 {\shpgrp{\*\shpinst\shpleft1584\shptop130\shpright6994\shpbottom930\shpfhdr0\shpbxcolumn\shpbypara\shpwr3\shpwrk0\shpfblwtxt0\shpz6\shplid1068{\sp{\sn groupLeft}{\sv 2481}}{\sp{\sn groupTop}{\sv 4486}}{\sp{\sn groupRight}{\sv 7894}} {\sp{\sn groupBottom}{\sv 5286}}{\sp{\sn fFlipH}{\sv 0}}{\sp{\sn fFlipV}{\sv 0}}{\sp{\sn rotation}{\sv 0}}{\sp{\sn lidRegroup}{\sv 26}}{\shpgrp{\*\shpinst\shplid1069{\sp{\sn groupLeft}{\sv 2481}}{\sp{\sn groupTop}{\sv 4486}}{\sp{\sn groupRight}{\sv 7894}} {\sp{\sn groupBottom}{\sv 4942}}{\sp{\sn relLeft}{\sv 2481}}{\sp{\sn relTop}{\sv 4486}}{\sp{\sn relRight}{\sv 7894}}{\sp{\sn relBottom}{\sv 4942}}{\sp{\sn fRelFlipH}{\sv 0}}{\sp{\sn fRelFlipV}{\sv 0}}{\shp{\*\shpinst\shplid1070{\sp{\sn relLeft}{\sv 2481}} {\sp{\sn relTop}{\sv 4486}}{\sp{\sn relRight}{\sv 6333}}{\sp{\sn relBottom}{\sv 4942}}{\sp{\sn fRelFlipH}{\sv 0}}{\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 202}} {\sp{\sn lTxid}{\sv 1114112}}{\sp{\sn dxTextLeft}{\sv 54864}}{\sp{\sn dyTextTop}{\sv 54864}}{\sp{\sn dxTextRight}{\sv 54864}}{\sp{\sn dyTextBottom}{\sv 54864}}{\sp{\sn hspNext}{\sv 1070}}{\sp{\sn lidRegroup}{\sv 20}}{\shptxt \pard\plain \qc\widctlpar\adjustright \fs20\cgrid {\b\f1 IV. 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Recovery PCR}{\f1 of enriched elution \par }}}}{\shp{\*\shpinst\shplid1087{\sp{\sn relLeft}{\sv 7262}}{\sp{\sn relTop}{\sv 6273}}{\sp{\sn relRight}{\sv 8143}}{\sp{\sn relBottom}{\sv 6673}}{\sp{\sn fRelFlipH}{\sv 0}} {\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 202}}{\sp{\sn lTxid}{\sv 1507328}}{\sp{\sn dxTextLeft}{\sv 54864}}{\sp{\sn dyTextTop}{\sv 54864}}{\sp{\sn dxTextRight}{\sv 54864}} {\sp{\sn dyTextBottom}{\sv 54864}}{\sp{\sn hspNext}{\sv 1087}}{\sp{\sn lidRegroup}{\sv 31}}{\shptxt \pard\plain \qc\widctlpar\adjustright \fs20\cgrid {\f1 2 hrs \par }{ \par }}}}{\shp{\*\shpinst\shplid1088{\sp{\sn relLeft}{\sv 6646}}{\sp{\sn relTop}{\sv 6480}}{\sp{\sn relRight}{\sv 7280}}{\sp{\sn relBottom}{\sv 6480}}{\sp{\sn fRelFlipH}{\sv 0}}{\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 20}}{\sp{\sn shapePath}{\sv 4}} {\sp{\sn fFillOK}{\sv 0}}{\sp{\sn fFilled}{\sv 0}}{\sp{\sn lineEndArrowhead}{\sv 1}}{\sp{\sn fArrowheadsOK}{\sv 1}}}}}}}{\shprslt{\*\do\dobxcolumn\dobypara\dodhgt8212\dppolygon\dppolycount4\dpptx0\dppty0\dpptx8229\dppty0\dpptx8229\dppty936\dpptx0\dppty936 \dpx1584\dpy205\dpxsize8229\dpysize936\dpfillfgcr255\dpfillfgcg255\dpfillfgcb255\dpfillbgcr255\dpfillbgcg255\dpfillbgcb255\dpfillpat1\dplinew15\dplinecor0\dplinecog0\dplinecob0}}}}{ \par \par }{\lang1024 {\shp{\*\shpinst\shpleft678\shptop148\shpright1584\shpbottom1836\shpfhdr0\shpbxcolumn\shpbypara\shpwr3\shpwrk0\shpfblwtxt0\shpz17\shplid1089{\sp{\sn shapeType}{\sv 0}}{\sp{\sn fFlipH}{\sv 0}}{\sp{\sn fFlipV}{\sv 0}} {\sp{\sn geoRight}{\sv 1192}}{\sp{\sn geoBottom}{\sv 1735}}{\sp{\sn shapePath}{\sv 4}}{\sp{\sn pVerticies}{\sv 8;3;(1192,0);(0,0);(0,1735)}}{\sp{\sn pSegmentInfo}{\sv 2;7 ;16384;44032;1;44032;1;44032;32768}}{\sp{\sn fFillOK}{\sv 1}}{\sp{\sn fFilled}{\sv 0}}{\sp{\sn lineWidth}{\sv 12700}}{\sp{\sn lineStyle}{\sv 0}}{\sp{\sn lineStartArrowhead}{\sv 0}}{\sp{\sn lineEndArrowhead}{\sv 5}}{\sp{\sn lineStartArrowWidth}{\sv 1}} {\sp{\sn lineStartArrowLength}{\sv 1}}{\sp{\sn lineEndArrowWidth}{\sv 1}}{\sp{\sn lineEndArrowLength}{\sv 1}}{\sp{\sn fArrowheadsOK}{\sv 1}}{\sp{\sn fLine}{\sv 1}}}{\shprslt{\*\do\dobxcolumn\dobypara\dodhgt8209\dppolygon\dppolycount3\dpptx906\dppty0 \dpptx0\dppty0\dpptx0\dppty1688\dpx678\dpy148\dpxsize906\dpysize1688\dpfillfgcr255\dpfillfgcg255\dpfillfgcb255\dpfillbgcr255\dpfillbgcg255\dpfillbgcb255\dpfillpat0\dplinew20\dplinecor0\dplinecog0\dplinecob0}}}}{ \par }\pard \qc\widctlpar\adjustright {\lang1024 {\shp{\*\shpinst\shpleft1320\shptop54\shpright1320\shpbottom54\shpfhdr0\shpbxcolumn\shpbypara\shpwr3\shpwrk0\shpfblwtxt0\shpz0\shplid1090 {\sp{\sn shapeType}{\sv 20}}{\sp{\sn fFlipH}{\sv 0}}{\sp{\sn fFlipV}{\sv 0}}{\sp{\sn shapePath}{\sv 4}}{\sp{\sn fFillOK}{\sv 0}}{\sp{\sn fFilled}{\sv 0}}{\sp{\sn fArrowheadsOK}{\sv 1}}}{\shprslt{\*\do\dobxcolumn\dobypara\dodhgt8192 \dpline\dpptx0\dppty0\dpptx0\dppty0\dpx1320\dpy54\dpxsize0\dpysize0\dplinew15\dplinecor0\dplinecog0\dplinecob0}}}}{ \par }\pard \widctlpar\adjustright { \par }{\lang1024 {\shpgrp{\*\shpinst\shpleft2448\shptop3\shpright6993\shpbottom750\shpfhdr0\shpbxcolumn\shpbypara\shpwr3\shpwrk0\shpfblwtxt0\shpz14\shplid1091{\sp{\sn groupLeft}{\sv 3600}}{\sp{\sn groupTop}{\sv 7024}}{\sp{\sn groupRight}{\sv 8145}} {\sp{\sn groupBottom}{\sv 7771}}{\sp{\sn fFlipH}{\sv 0}}{\sp{\sn fFlipV}{\sv 0}}{\sp{\sn lidRegroup}{\sv 26}}{\shp{\*\shpinst\shplid1092{\sp{\sn relLeft}{\sv 3600}}{\sp{\sn relTop}{\sv 7024}}{\sp{\sn relRight}{\sv 5472}}{\sp{\sn relBottom}{\sv 7415}} {\sp{\sn fRelFlipH}{\sv 0}}{\sp{\sn fRelFlipV}{\sv 0}}{\sp{\sn shapeType}{\sv 202}}{\sp{\sn lTxid}{\sv 1572864}}{\sp{\sn dxTextLeft}{\sv 54864}}{\sp{\sn dyTextTop}{\sv 54864}}{\sp{\sn dxTextRight}{\sv 54864}}{\sp{\sn dyTextBottom}{\sv 54864}} {\sp{\sn hspNext}{\sv 1092}}{\sp{\sn lidRegroup}{\sv 30}}{\shptxt \pard\plain \s16\widctlpar\adjustright \f1\cgrid {\b\fs20 VII. 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