{"id":68,"date":"2010-05-05T20:56:26","date_gmt":"2010-05-05T11:56:26","guid":{"rendered":"http:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/?p=68"},"modified":"2014-02-10T09:37:10","modified_gmt":"2014-02-10T00:37:10","slug":"ish","status":"publish","type":"post","link":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/?p=68","title":{"rendered":"\u51cd\u7d50\u5207\u7247\u306eISH"},"content":{"rendered":"

\u56fa\u5b9a<\/strong><\/p>\n

    \n
  1. 4% PFA \/ PBS\u3067\u704c\u6d41\u56fa\u5b9a<\/li>\n
  2. \u7d44\u7e54\u3092\u5207\u308a\u51fa\u3057\u3001\u6570\u6642\u9593\u6d78\u6f2c\u56fa\u5b9a<\/li>\n
  3. 30% sucrose \/ PBS\u30014\u2103\u3001O\/N<\/li>\n
  4. OCT\u30b3\u30f3\u30d1\u30a6\u30f3\u30c9\u306b\u5305\u57cb\u3057LN2\u3067\u51cd\u7d50\uff08\u30af\u30e9\u30c3\u30af\u3067\u304d\u306a\u3044\u3088\u3046\u6ce8\u610f\uff09 <\/li>\n
  5. 10\u00b5m\u3067\u30af\u30e9\u30a4\u30aa\u30b9\u30bf\u30c3\u30c8\u5207\u7247<\/li>\n<\/ol>\n


    \n\u30d7\u30ed\u30fc\u30d6<\/strong><\/p>\n

      \n
    1. \u30d7\u30e9\u30b9\u30df\u30c9\u3092Wizard\u00ae Plus SV Minipreps DNA Purification System\uff08Promega\uff09\u3067\u7cbe\u88fd\uff085ml LB\u306eO\/N\u30ab\u30eb\u30c1\u30e3\u30fc\u3001Lysate\u306f2\u56de\u9060\u5fc3\u3001Wash\u306b70% EtOH\u8ffd\u52a0\uff09<\/li>\n
    2. Miniprep\u5168\u91cf\u3092Fermentas FastDigest\u3067\u5b8c\u5168\u306b\u76f4\u9396\u5316\uff08\u30b2\u30eb\u3067\u30c1\u30a7\u30c3\u30af\uff09<\/li>\n
    3. Wizard\u00ae SV Gel and PCR Clean-Up System\u3067\u7cbe\u88fd\uff08Wash\u306b70% EtOH\u8ffd\u52a0\u3001\u6eb6\u51fa\u306f2\u00d715\u00b5l\uff09\uff08\u30b9\u30da\u30c3\u30af\u3067\u6fc3\u5ea6\u691c\u5b9a\u30011\u00b5g\u306e\u5bb9\u91cf\u3092\u7b97\u51fa\u3001PCI\u306f\u975e\u63a8\u5968\uff09<\/li>\n
    4. DIG RNA Labeling Kit (SP6\/T7)\uff08Roche\uff09\u3067\u30d7\u30ed\u30fc\u30d6\u3092\u5408\u6210<\/li>\n
    5. \u30aa\u30d7\u30b7\u30e7\u30f3\uff1a\u30a2\u30eb\u30ab\u30ea\u5206\u89e3<\/li>\n
    6. + 1\u00b5l 20mg\/ml glycogen\u3001+ 0.5vol 7.5M NH4OAc\u3001+2.5vol EtOH\u3001-20\u2103 O\/N<\/li>\n
    7. \u9060\u5fc3\u300170% EtOH\u3067\u6d17\u3044\u3001\u9060\u5fc3\uff08PCI\u306f\u4e0d\u9069\u3002\u6709\u6a5f\u76f8\u306b\u30d7\u30ed\u30fc\u30d6\u304c\u304f\u308b\uff09<\/li>\n
    8. 500\u00b5l HYB\u306b\u6eb6\u89e3<\/li>\n<\/ol>\n

      RNA\u306e\u30a2\u30eb\u30ab\u30ea\u5206\u89e3<\/p>\n

        \n
      1. 50\u00b5l\u306e\u30d7\u30ed\u30fc\u30d6\u6eb6\u6db2\u306b\u300130\u00b5l\u306e200mM Na2CO3\u300120\u00b5l\u306e200mM HaHCO3\u3092\u52a0\u3048\u308b<\/li>\n
      2. 60\u2103\u3067t\u5206\u9593\uff1at = (L0 -Lf) \/ (K \u00d7 L0 \u00d7 L1)\u3001K =0.11\u3001L0=\u521d\u671f\u9577 (kb)\u3001Lf=\u76ee\u7684\u9577 (kb)<\/li>\n
      3. 500 kb\u306b<\/li>\n<\/ol>\n

        \u30cf\u30a4\u30d6\u30ea<\/p>\n

          \n
        1. PBS\u30012 min<\/li>\n
        2. PAP\u30da\u30f3\u3067\u67a0\u3092\u63cf\u304f<\/li>\n
        3. 1 \u00b5g\/ml ProK \/ PBS, 30min\u300137\u2103\uff080.1\u301c10\uff09<\/li>\n
        4. 2\u00d7SSC\u30012\u00d75 min<\/li>\n
        5. 100\u00b5l HYB\u300160 min\u300137\u2103\uff0837\u301c50\u2103\uff09<\/li>\n
        6. 100\u00b5l Probe \/ HYB (1:10), 16h\u300137\u2103\uff0837\u301c50\u2103\uff09<\/li>\n
        7. 2\u00d7SSC\u30012\u00d75 min\u300137\u2103<\/li>\n
        8. 60% formamide\u30010.2\u00d7SSC\u30013\u00d75min\u300137\u2103<\/li>\n
        9. 2\u00d7SSC\u30012\u00d75 min<\/li>\n<\/ol>\n

          \u767a\u8272<\/p>\n

            \n
          1. PBST\u30015 min<\/li>\n
          2. BLOCK\u300130 min<\/li>\n
          3. 100\u00b5l 1:200 \u03b1-DIG \/ Block\uff08\u9060\u5fc3\u3057\u3066\u6c88\u6e23\u3092\u9664\u304f\uff09\u30012h<\/li>\n
          4. PBST\u30012\u00d75 min<\/li>\n
          5. Al-P Bfr\u30015 min<\/li>\n
          6. BCIP\/NBT\uff08Roche\uff09\u3001\u6697\u6240\u3001\u6db2\u304c\u8272\u3065\u3044\u305f\u3089\u4ea4\u63db\u3001\u767a\u8272\u3059\u308b\u307e\u3067\uff08\u301cO\/N\uff09<\/li>\n<\/ol>\n

            \u6eb6\u6db2<\/strong><\/p>\n

              \n
            • PBS\uff1a\u30bf\u30ab\u30e9\u306eDPBS\u306e\u30bf\u30d6\u30ec\u30c3\u30c8<\/li>\n
            • PBST\uff1a0.1% Tween-20 \/ PBS<\/li>\n
            • PFA\uff1a20g PFA\u3001100ml\u306e\u6c34\u300165\u2103\u306e\u30d2\u30fc\u30c8\u30d6\u30ed\u30c3\u30af\u3067\u904e\u71b1\u3057\u3001200\u00b5l\u306e1N NaOH\u6dfb\u52a0\u3001\u3060\u3044\u305f\u3044\u6eb6\u89e3\u3057\u305f\u3089\u6ffe\u904e\u3059\u308b<\/li>\n
            • HYB\uff1a4\u00d7SSC\u300110%\u30c7\u30ad\u30b9\u30c8\u30e9\u30f3\u786b\u9178\u30011\u00d7\u30c7\u30f3\u30cf\u30fc\u30c8\u6db2\u30012mM EDTA\u300150% formamide\u30010.5 mg\/ml torula RNA<\/li>\n
            • BLOCK\uff1a2\uff05 NGS\u30011\uff05\u30d6\u30ed\u30c3\u30ad\u30f3\u30b0\u8a66\u85ac\uff08Roche\uff09<\/li>\n
            • Al-P Bfr\uff1a20ml 1M Tris-HCL, pH9.5\u300110ml 1M MgCl2\u30014ml 5M NaCl\u30012ml 20%Tween-20\u30012ml 20% Triton X-100\u3001240\u00b5g levamisole\u3001\u6c34\u3092\u52a0\u3048\u3066200ml<\/li>\n<\/ul>\n

              RNA\u306e\u6271\u3044<\/strong><\/p>\n

                \n
              • \u624b\u888b\u7740\u7528\u3002<\/li>\n
              • \u5bb9\u5668\u306f\u6ec5\u83cc\u6e08\u307f\u306e\u88fd\u54c1\u307e\u305f\u306f\u30aa\u30fc\u30c8\u30af\u30ec\u30fc\u30d6\u3057\u305f\u3082\u306e\u3002<\/li>\n
              • \u624b\u888b\u3092\u3057\u305f\u624b\u3084\u30de\u30a4\u30af\u30ed\u30d4\u30da\u30c3\u30c8\u306a\u3069\u306fRNase Away\u3067\u51e6\u7406\u3002<\/li>\n
              • \u6c34\u306f\u5206\u5b50\u751f\u7269\u5b66\u7528\u306e1ml\u5165\u308a\u88fd\u54c1\u3001\u307e\u305f\u306fDEPC\u51e6\u7406\u3057\u305f\u6c34\u3002<\/li>\n
              • \u96fb\u6c17\u6cf3\u52d5\u88c5\u7f6e\u306f\u6d17\u5264\u3067\u3088\u304f\u6d17\u3044\u3001\u30b2\u30eb\u306f\u4f7f\u3046\u3068\u304d\u306b\u4f5c\u308b\u3002DNA\u306fSYBR Safe\u3092\u3001RNA\u306e\u53cd\u5fdc\u6db2\u306fEtBr\u3092\u30d0\u30c3\u30d5\u30a1\u306b\u52a0\u3048\u3066\u6cf3\u52d5<\/li>\n<\/ul>\n","protected":false},"excerpt":{"rendered":"

                \u56fa\u5b9a 4% PFA \/ PBS\u3067\u704c\u6d41\u56fa\u5b9a \u7d44\u7e54\u3092\u5207\u308a\u51fa\u3057\u3001\u6570\u6642\u9593\u6d78\u6f2c\u56fa\u5b9a 30% sucrose \/ PBS\u30014\u2103\u3001O\/N OCT\u30b3\u30f3\u30d1\u30a6\u30f3\u30c9\u306b\u5305\u57cb\u3057LN2\u3067\u51cd\u7d50\uff08\u30af\u30e9\u30c3\u30af\u3067\u304d\u306a\u3044\u3088\u3046\u6ce8\u610f\uff09 10\u00b5m\u3067\u30af\u30e9\u30a4\u30aa\u30b9\u30bf\u30c3\u30c8\u5207<\/p>\n","protected":false},"author":3,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"inline_featured_image":false,"_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":"","jetpack_publicize_message":"","jetpack_publicize_feature_enabled":true,"jetpack_social_post_already_shared":false,"jetpack_social_options":{"image_generator_settings":{"template":"highway","default_image_id":0,"font":"","enabled":false},"version":2},"jetpack_post_was_ever_published":false},"categories":[3,5,4],"tags":[],"class_list":["post-68","post","type-post","status-publish","format-standard","hentry","category-buffers-media","category-microscopy","category-molecular-biology"],"jetpack_publicize_connections":[],"jetpack_featured_media_url":"","jetpack_sharing_enabled":true,"jetpack_shortlink":"https:\/\/wp.me\/s4fFyn-ish","jetpack-related-posts":[],"_links":{"self":[{"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=\/wp\/v2\/posts\/68","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=68"}],"version-history":[{"count":1,"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=\/wp\/v2\/posts\/68\/revisions"}],"predecessor-version":[{"id":187,"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=\/wp\/v2\/posts\/68\/revisions\/187"}],"wp:attachment":[{"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=68"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=68"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=68"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}