{"id":141,"date":"2015-08-03T13:34:20","date_gmt":"2015-08-03T04:34:20","guid":{"rendered":"http:\/\/www-p.cc.ocha.ac.jp\/bio-shimada-lab\/?page_id=141"},"modified":"2018-05-29T14:16:56","modified_gmt":"2018-05-29T05:16:56","slug":"pr_05dna","status":"publish","type":"page","link":"https:\/\/www-p.sci.ocha.ac.jp\/bio-shimada-lab\/ex_protocol\/pr_05dna.html","title":{"rendered":"DNA\u30fbRNA\u95a2\u4fc2"},"content":{"rendered":"

<\/a>DNA\u306e\u62bd\u51fa<\/h2>\n

DNA\u62bd\u51fa\u30b5\u30f3\u30d7\u30eb\u306e\u6e96\u5099<\/h4>\n
    \n
  1. \u85fb\u4f53\u306e\u4e00\u90e8(1cm\u89d2)\u3092\u3061\u304e\u308a\u3001\u6ec5\u83cc\u6d77\u6c34\u5165\u308a\u30c7\u30a3\u30b9\u30dd\u30b7\u30e3\u30fc\u30ec\u306b\u5165\u308c,\u5b9f\u4f53\u9855\u5fae\u93e1\u3067\u78ba\u8a8d\u3057\u306a\u304c\u3089\u7b46\u3067\u6c5a\u308c\u3092\u843d\u3068\u3059\u3002(\u6c5a\u308c\u304c\u3072\u3069\u3051\u308c\u3070\u7e70\u308a\u8fd4\u3059)<\/li>\n
  2. \u30aa\u30fc\u30c8\u30af\u30ec\u30fc\u30d6\u6e08\u307f\u306e\u30a8\u30c3\u30da\u30f3\u30c1\u30e5\u30fc\u30d6(\u540d\u524d\u3092\u5fd8\u308c\u305a\u306b)<\/u>\u306b\u30af\u30ea\u30fc\u30cb\u30f3\u30b0\u3057\u305f\u85fb\u4f53\u3092\u5165\u308c,\u30ad\u30e0\u30ef\u30a4\u30d7\u3067\u6c34\u6c17\u3092\u53d6\u308b\u3002<\/li>\n
  3. \u4f7f\u3063\u305f\u30d4\u30f3\u30bb\u30c3\u30c8\u3084\u7b46\u306f70%\u30a8\u30bf\u30ce\u30fc\u30eb\u306b\u3064\u3051\u3066,\u30ad\u30e0\u30ef\u30a4\u30d7\u3067\u62ed\u3044\u3066\u30b1\u30fc\u30b9\u306b\u623b\u3059\u3002<\/li>\n
  4. \u30c7\u30b7\u30b1\u30fc\u30bf\u30fc\u306b\u5165\u308c\u771f\u7a7a\u30dd\u30f3\u30d7\u3067\u7a7a\u6c17\u3092\u629c\u304d\u306a\u304c\u3089\u30c9\u30e9\u30a4\u30a2\u30c3\u30d7(10\u5206)\u3002<\/li>\n
  5. \u8a3c\u62e0\u6a19\u672c\u3068\u3057\u3066\u62bc\u3057\u8449\u6a19\u672c\u3092\u4f5c\u6210\u3059\u308b\u3002<\/li>\n<\/ol>\n

     <\/p>\n

    DNA\u62bd\u51fa(QIAGEN DNeasy Plant Mini Kit)<\/h4>\n
      \n
    1. \u30a8\u30c3\u30da\u30f3\u30c1\u30e5\u30fc\u30d6(\u30a8\u30c3\u30da\u30f3)\u306b\u30da\u30c3\u30b9\u30eb\u3092\u3055\u3059\u3002<\/li>\n
    2. \u6db2\u4f53\u7a92\u7d20\u3092\u30b3\u30c3\u30d7\u306b\u5165\u308c\u308b\u3002\u30b5\u30f3\u30d7\u30eb\u306e\u5165\u3063\u305f\u30a8\u30c3\u30da\u30f3\u3092\u30c8\u30ec\u30fc\u306b\u7acb\u3066,\u6db2\u4f53\u7a92\u7d20\u3092\u3086\u3063\u304f\u308a\u6ce8\u304e,\u8efd\u304f\u6cb8\u9a30\u3057\u305f\u3089,\u518d\u5ea6\u3044\u3063\u3071\u3044\u5165\u308c\u308b\u3002\u6db2\u4f53\u7a92\u7d20\u304c\u534a\u5206\u3050\u3089\u3044\u307e\u3067\u84b8\u767a\u3057\u305f\u3089,\u30da\u30c3\u30b9\u30eb\u3092\u3044\u308c,\u51b7\u3084\u3059\u3002\u6db2\u4f53\u7a92\u7d20\u304c\u7121\u304f\u306a\u308b\u5bf8\u524d\u3067,\u30da\u30c3\u30b9\u30eb\u3092\u30b0\u30ea\u30b0\u30ea\u3057\u3066\u30b5\u30f3\u30d7\u30eb\u3092\u78e8\u308a\u3064\u3076\u3059(\u30da\u30c3\u30b9\u30eb\u306f\u51fa\u3055\u306a\u3044)\u3002<\/li>\n
    3. \u7834\u7815\u3057\u305f\u30b5\u30f3\u30d7\u30eb\u306bAP1\u3092300\u03bcl\u3044\u308c,\u30da\u30c3\u30b9\u30eb\u3092\u56de\u3057\u306a\u304c\u3089\u629c\u304d,\u30d5\u30bf\u3092\u3057\u3063\u304b\u308a\u3057\u3066,\u30dc\u30eb\u30c6\u30c3\u30af\u30b9\u3002<\/li>\n
    4. 65\u2103\u306710\u5206\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30c8\u30022-3\u5206\u6bce\u306b\u30c1\u30e5\u30fc\u30d6\u3092\u8efd\u304f\u64b9\u62cc\u3002\u5404\u7a2e\u30c1\u30e5\u30fc\u30d6\u7528\u610f\u3002 (\u3053\u306e\u6642AE\u3092sample\u6570\u00d7100\u03bcl\u3092\u30a8\u30c3\u30da\u30f3\u306b\u53d6\u308a\u540c\u3058\u304f65\u2103)<\/li>\n
    5. \u8efd\u304f\u30c1\u30d3\u30bf\u30f3\u3002\u3086\u3063\u304f\u308a\u30d5\u30bf\u3092\u958b\u3051\u3066,P3 (AP2)\u3092130\u03bcl\u52a0\u3048\u3001\u30dc\u30eb\u30c6\u30c3\u30af\u30b9\u3001\u6c37\u4e0a5\u5206\u3001 14000rpm\u9060\u5fc35\u5206<\/li>\n
    6. \u4e0a\u6f84\u307f300\u03bcl\u3092QIA\u30ab\u30e9\u30e0(\u7d2b\u8272)\u306b\u79fb\u3057\u300114000rpm\u9060\u5fc32\u5206<\/li>\n
    7. 450\u03bcl\u306eAW1(AP3\/E)\u3092\u65b0\u3057\u3044\u30a8\u30c3\u30da\u30f3\u306b\u5165\u308c\u3066\u304a\u304f\u3002\u9752\u30d4\u30da\u30c3\u30c8\u3092850\u03bc\u306b\u3057,\u7d2b\u30ab\u30e9\u30e0\u3092\u6368\u3066,\u843d\u6db2\u3092\u5168\u91cf\u5438\u3044,AP3\/E\u5165\u308a\u306e\u30a8\u30c3\u30da\u30f3\u306b\u79fb\u3057,\u3084\u3055\u3057\u304f\u30d4\u30da\u30c3\u30c6\u30a3\u30f3\u30b05\u56de,\u767d\u30ab\u30e9\u30e0\u306b\u5168\u91cf\u79fb\u3059\u300210000rpm\u9060\u5fc31\u5206<\/li>\n
    8. \u843d\u6db2\u30fbcollection tube\u3092\u6368\u3066\u3066,\u30ab\u30e9\u30e0\u3060\u3051\u65b0\u3057\u3044collection tube\u306b\u79fb\u3057\u3001\u30ab\u30e9\u30e0\u306bAW2(AW)\u3092500\u03bcl\u5165\u308c\u308b\u300210000rpm\u9060\u5fc31\u5206\u3002<\/li>\n
    9. \u843d\u6db2\u3092\u6368\u3066\u3001\u540c\u3058collection tube\u3092\u518d\u88c5\u7740\u3057,\u30ab\u30e9\u30e0\u306bAW\u3092500\u03bcl\u5165\u308c\u308b\u300214000rpm\u9060\u5fc32\u5206\u3002<\/li>\n
    10. \u9759\u304b\u306b\u30c1\u30e5\u30fc\u30d6\u3092\u673a\u306b\u6301\u3063\u3066\u304d\u3066,\u843d\u6db2\u30fbcollection tube\u3092\u6368\u3066\u3066,\u30ab\u30e9\u30e0\u3092\u65b0\u3057\u3044\u30a8\u30c3\u30da\u30f3\u306b\u79fb\u3057\u300165\u2103\u306b\u71b1\u3057\u305fAE\u309280\u03bcl\u819c\u306b\u304b\u3051\u308b\u3002<\/li>\n
    11. 5\u5206\u5ba4\u6e29\u3067\u5f85\u306110000rpm\u9060\u5fc31\u5206<\/li>\n<\/ol>\n

       <\/p>\n

      \u95a2\u6771\u5316\u5b66\u30b7\u30ab\u30b8\u30fc\u30cb\u30a2\u30b9DNA\u62bd\u51fa\u8a66\u85ac<\/h4>\n

      (\u77ed\u6642\u9593\u3067\u3001\u5bb9\u6613\u3067\u3001\u5b89\u4fa1\u3001\u9577\u671f\u4fdd\u5b58\u306b\u306f\u3080\u304b\u306a\u3044)<\/p>\n

        \n
      1. \u4e0a\u8ff0\u306e\u3088\u3046\u306b\u6db2\u4f53\u7a92\u7d20\u3067\u85fb\u4f53\u3092\u3064\u3076\u3059\u3002<\/li>\n
      2. 1\u30b5\u30f3\u30d7\u30eb\u8a66\u85aca\u6db210\u03bcl, \u8a66\u85acb\u6db2100\u03bcl\u306e\u6bd4\u7387\u3067\u6df7\u5408\u6db2\u3092\u4f5c\u308a\u3001\u3059\u308a\u3064\u3076\u3057\u305f\u85fb\u4f53\u306b110\u03bcl\u305a\u3064\u5165\u308c\u308b\u3002\u8efd\u304f\u6307\u3067\u5f3e\u3044\u3066\u6df7\u5408\u3002<\/li>\n
      3. 72\u2103 20min, 94\u2103 3m\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30c8<\/li>\n
      4. 14000rpm 3min\u9060\u5fc3\u3057\u3001\u4e0a\u6f84\u307f80\u03bcl\u3092\u5225\u30a8\u30c3\u30da\u30f3\u306b\u3002<\/li>\n<\/ol>\n

         <\/p>\n

        \u00a0<\/a>PCR\u53cd\u5fdc<\/h2>\n

        Takara Ex Taq<\/h4>\n
          \n
        1. \u6c37\u3092\u6e96\u5099\u3057\u3001PCR\u30c8\u30ec\u30a4\u3092\u306e\u305b\u3066\u304a\u304f\u3002<\/li>\n
        2. 0.2mlPCR\u7528\u30c1\u30e5\u30fc\u30d6\u306b\u540d\u524d\u3092\u66f8\u304f\u3002<\/li>\n
        3. \u4ee5\u4e0b\u306e\u92f3\u578bDNA\u4ee5\u5916\u3092\u3059\u3079\u3066\u6df7\u5408\u3057PCR\u7528\u30c1\u30e5-\u30d6\u306b29 \u03bcl\u5206\u6ce8\u3059\u308b(\u30cd\u30ac\u30c6\u30a3\u30d6\u30b3\u30f3\u30c8\u30ed\u30fc\u30eb\u3082)\u3002<\/li>\n<\/ol>\n

          \u3000\u3000\u300010xPCR buffer \u00a0 \u00a0 \u00a0 \u00a030.0\u03bcl
          \n\u3000\u3000\u3000dNTPmix\u00a0\u00a0 <\/sup>\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 24.0\u03bcl
          \n\u3000\u3000\u3000DMSO\u00a0*1<\/sup>\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 15.0\u03bcl
          \n\u3000\u3000\u3000DW \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 220.0\u03bcl
          \n\u3000\u3000\u3000Primer No.1 *2<\/sup>\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 5.0\u03bcl
          \n\u3000\u3000\u3000Primer No.2 *2<\/sup>\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 5.0\u03bcl
          \n\u3000\u3000\u3000Taq polymerase \u00a0 \u00a0 \u00a0 \u00a0 \u00a01.5\u03bcl<\/u><\/p>\n

          \u3000\u3000\u3000*1 18SrDNA, nrITS\u306e\u307f\u5165\u308c\u3001\u4ed6\u306f\u3053\u306e\u5206DW
          \n\u3000\u3000\u3000*2 \u30d7\u30e9\u30a4\u30de\u30fc20pmol\/\u03bcl\u3000(20 \u03bcM)\u00a0<\/p>\n

          \u00a04. \u00a0\u62bd\u51faDNA\u30921~2 \u03bcl\u52a0\u3048\u30dc\u30eb\u3002
          \n\u00a0 5. \u00a0\u30b5\u30fc\u30de\u30eb\u30b5\u30a4\u30af\u30e9-\u5404\u7a2e\u30d7\u30ed\u30b0\u30e9\u30e0\u3067\u5897\u5e45\u3002<\/p>\n

          \u3000\u300094\u2103\u30001\u5206\u3000(\u3053\u306e\u9593\u306b\u30b5\u30f3\u30d7\u30eb\u3092\u30bb\u30c3\u30c8)
          \n\u3000\u300094\u2103\u300015\u79d2\u3001\u300050\u2103\u300015\u79d2\u3001\u300068\u2103\u300030\u79d2\u00a0*3<\/sup>\u3001\u3000\u00d745\u30b5\u30a4\u30af\u30eb
          \n\u3000\u3000 \u00a0 4\u2103\u3000\u4fdd\u6301<\/p>\n

          \u3000\u3000*3 extention\u306e\u76ee\u5b89\u306f1kb=1imin
          \n\u3000\u2192\u57fa\u672c\u30d7\u30ed\u30b0\u30e9\u30e0\u3067\u5897\u5e45\u3057\u306a\u3044\u5834\u5408\u306f\u8981\u76f8\u8ac7\u00a0<\/p>\n

          \n
          \n<\/div>\n

          <\/a>PCR\u7523\u7269\u306e\u96fb\u6c17\u6cf3\u52d5<\/h2>\n

          1%\u30a2\u30ac\u30ed\u30fc\u30b9\u30b2\u30eb\u306e\u4f5c\u88fd(\u77ed\u3044\u65ad\u7247\u540c\u58eb\u306e\u8ddd\u96e2\u3092\u51fa\u3057\u305f\u3044\u5834\u5408\u306f\u6fc3\u5ea6\u3092\u3042\u3052\u308b)<\/h4>\n
            \n
          1. \u30b2\u30eb\u30c8\u30ec\u30a4\u30b9\u30bf\u30f3\u30c9\u306b\u30b2\u30eb\u30c8\u30ec\u30a4\u3092\u306f\u3081\u3066\u3001\u30b3\u30fc\u30e0\u30922\u672c\u633f\u3059\u30022\u672c\u76ee\u306f\u4e0b\u304b\u30894\u672c\u76ee\u306e\u7d30\u7dda\u306e\u6240\u3002(16\u30b5\u30f3\u30d7\u30eb\u4ee5\u4e0b\u2192\u5c0f\u3055\u3044\u30b2\u30eb\u30c8\u30ec\u30a4)<\/li>\n
          2. \u30a2\u30ac\u30ed\u30fc\u30b9150 mg\u30011xTAE Buffer 15 ml(10xTAE\u3058\u3083\u306a\u3044)\u3092\u306f\u304b\u308a\u3068\u308a\u3001\u4e09\u89d2\u30d5\u30e9\u30b9\u30b3\u306b\u5165\u308c\u306690\u79d2\u30c1\u30f3\u3002 (\u5927\u30b2\u30eb\u30c8\u30ec\u30a4\u306e\u5834\u5408:\u30a2\u30ac\u30ed\u30fc\u30b9250 mg\u3001TAE Buffer 25 ml\u30021\u5206\u307b\u3069\u3067\u6cb8\u9a30\u3059\u308b\u306e\u3067\u53d6\u308a\u51fa\u3057\u3066\u8efd\u304f\u632f\u308a\u6cb8\u9a30\u3092\u93ae\u3081\u3001\u6b8b\u308a\u6642\u9593\u30c1\u30f3)<\/li>\n
          3. \u826f\u304f\u304b\u304d\u6df7\u305c\u3001\u3057\u305a\u304b\u306b\u30c8\u30ec\u30a4\u306b\u6d41\u3057\u5165\u308c\u30b3\u30fc\u30e0\u3092\u3055\u3059\u3002\u6ce1\u304c\u306a\u3044\u304b\u78ba\u8a8d\u3002<\/li>\n
          4. 30\u5206\u5f85\u3063\u3066\u304b\u3089TAE\u3000\u3092\u5c11\u3057\u5165\u308c\u3066\u30b3\u30fc\u30e0\u3092\u306f\u305a\u3059\u3002<\/li>\n<\/ol>\n

             <\/p>\n

            \u96fb\u6c17\u6cf3\u52d5<\/h4>\n
              \n
            1. \u30d1\u30e9\u30d5\u30a3\u30eb\u30e0\u306bLoadingBuffer 1\u03bc\u3065\u3064\u5206\u6ef4\u3002<\/li>\n
            2. \u5fc5\u8981\u306a\u3060\u3051\u30b2\u30eb\u3092\u30ab\u30c3\u30c8\u3002\u4f7f\u308f\u306a\u3044\u30b2\u30eb\u306f\u4e09\u89d2\u30d5\u30e9\u30b9\u30b3\u3078\u3002<\/li>\n
            3. PCR\u7523\u7269 4\u03bc\u3092\u3068\u308aLoadingBuffer\u3068\u6df7\u305c\u3066Loading\u3002<\/li>\n
            4. Mupid-2 plus \u3092\u4f7f\u7528\u3002135V 10\u5206\u3000\u96fb\u6c17\u6cf3\u52d5\u3002(\u611f\u96fb\u306b\u6ce8\u610f)<\/li>\n
            5. \u30b2\u30eb\u3092\u53d6\u308a\u51fa\u3057\u30a8\u30c1\u30d6\u30ed\u306b10\u5206\u3002(\u30c7\u30a3\u30b9\u30dd\u30b0\u30ed\u30fc\u30d6\u3092\u5fc5\u305a\u7740\u7528)<\/li>\n
            6. \u30b5\u30e9\u30f3\u30e9\u30c3\u30d7\u306b\u30b2\u30eb\u3092\u8f09\u305b\u3066\u30c8\u30e9\u30f3\u30b9\u30a4\u30eb\u30df\u30cd\u30fc\u30bf\u30fc\u3067\u89b3\u5bdf\u3002<\/li>\n
            7. \u30dd\u30e9\u3067\u5199\u771f\u3092\u64ae\u308b\u3002\u30b2\u30eb\u306f\u30b7\u30f3\u30af\u306e\u53ef\u71c3\u30b4\u30df\u3002<\/li>\n<\/ol>\n

              \u00a0<\/p>\n

              Loading Buffer<\/span><\/div>\n
              BPB 62.5mg, \u00a0DW 35mL, \u00a0\u30b0\u30ea\u30bb\u30ed\u30fc\u30eb 15 mL<\/div>\n
              \u00a0<\/div>\n
              10xTAE<\/span><\/div>\n
              Tris 48.4g (\u6c37)\u9162\u9178 \u00a0 \u00a0 \u00a0 \u00a011.4 ml<\/div>\n
              0.5M EDTA (pH 8.0) \u00a0 \u00a020.0 ml<\/div>\n
              DW\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 800.0 ml<\/div>\n
              \u7d14\u6c34(\u30e1\u30b9\u30a2\u30c3\u30d7\u7528) \u3000 \u00a0 \u00a0 +\u03b1 <\/span><\/div>\n
              \u8a08 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a01L<\/div>\n
              1. 1L\u30d3\u30fc\u30ab\u30fc\u306bDW800 ml\u3092\u5165\u308c\u3001\u30b9\u30bf\u30e9-\u3067\u64b9\u62cc\u3057\u306a\u304c\u3089\u5404\u8a66\u85ac\u3092\u52a0\u3048\u308b\u3002<\/div>\n
              2. \u30e1\u30b9\u30a2\u30c3\u30d7<\/div>\n
              \u00a0<\/div>\n
              \u00a01xTAE\u306f\u3053\u308c\u3092100ml+DW900ml\u3067\u4f5c\u308a\u51b7\u8535\u5eab\u4fdd\u7ba1\u3002<\/div>\n
              \u00a0<\/div>\n

              Takara Mighty Amp<\/h4>\n
              \n

              \u5897\u5e45\u304c\u56f0\u96e3\u306a\u5834\u5408\u3001\u8a66\u3059\u3002<\/p>\n

              2xBuffer \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 25.0 \u03bcl
              \nPrimer 1 (20\u03bcM) \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a00.5 \u03bcl
              \nPrimer 2 (20\u03bcM) \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a00.5 \u03bcl
              \nMighty Amp DNA polymerase \u00a0 1.0 \u03bcl
              \ntemDNA \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 1.0 \u03bcl
              \nDW \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 22.0 \u03bcl<\/span>
              \nTotal \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 50.0 \u03bcl<\/p>\n

              PCR\u30d7\u30ed\u30b0\u30e9\u30e0<\/p>\n

              98\u2103\u30002\u5206\u3000(Hot Start)
              \n98\u2103\u300010\u79d2\u3001\u300060\u2103\u300015\u79d2\u3001\u300068\u2103\u30001 min\/1kb\u3001\u3000\u00d740\u3000\u30b5\u30a4\u30af\u30eb\u3000\u3000
              \n4\u2103\u3067\u4fdd\u6301<\/p>\n<\/div>\n

              \n

              <\/a>\u00a0PCR\u7523\u7269\u306e\u7cbe\u88fd<\/h2>\n

              Wizard\u00ae SV Gel and PCR Clean-Up System – Promega<\/h4>\n

              [\u30b2\u30eb\u306e\u5207\u308a\u51fa\u3057\u306e\u5834\u5408]<\/p>\n

                \n
              1. \u5207\u308a\u51fa\u3057\u305f\u30b2\u30eb\u30925ml\u30a8\u30c3\u30da\u30f3\u306b\u5165\u308c,\u30b2\u30eb10mg\u3042\u305f\u308a10\u03bcl\u306eMenbrane Binding Solution\u3092\u5165\u308c\u308b\u300255\u21038\u5206\u3002(\u6eb6\u3051\u308c\u3070OK)<\/li>\n<\/ol>\n

                \u305d\u308c\u4ee5\u964d\u306f\u4e0b\u306e2)\u4ee5\u964d\u3068\u540c\u3058\u3002<\/p>\n

                [PCR\u7523\u7269\u306e\u5834\u5408]<\/p>\n

                  \n
                1. PCR\u53cd\u5fdc\u6db2\u3068\u540c\u91cf(26\u03bcl)\u306eMenbraneBindingSolution\u3092PCR\u7523\u7269\u306e\u5165\u3063\u305fPCRtube\u306b\u5165\u308c,\u30dc\u30eb\u30fb\u30c1\u30d3\u3002<\/li>\n
                2. SV minicolumn\u3092Collection Tube\u306b\u5165\u308c,sample\u756a\u53f7\u3092\u3069\u3046\u306b\u304b\u66f8\u3044\u3066\u30011\u306e\u5168\u91cf\u3092\u30ab\u30e9\u30e0\u306b\u5165\u308c,\u5ba4\u6e291\u5206\u3002<\/li>\n
                3. \u9060\u5fc313,500rpm,1\u5206\u3002<\/li>\n
                4. 700\u03bcl\u306eMembrane Wash Solution(\u30a8\u30bf\u30ce\u30fc\u30eb\u304c\u52a0\u3048\u3089\u308c\u305f\u3082\u306e)\u3092\u5165\u308c\u9060\u5fc313,500rpm,1\u5206\u3002\u843d\u6db2\u3092\u6368\u3066,\u518d\u5ea6Collection Tube\u306b\u88c5\u7740\u3002<\/li>\n
                5. 500\u03bcl\u306eMembrane Wash Solution(\u30a8\u30bf\u30ce\u30fc\u30eb\u304c\u52a0\u3048\u3089\u308c\u305f\u3082\u306e)\u3092\u5165\u308c\u9060\u5fc314,000rpm,5\u5206\u3002<\/li>\n
                6. \u843d\u6db2\u3092\u6368\u3066,\u518d\u88c5\u7740\u3057\u3001\u304b\u3089\u306e\u307e\u307e\u9060\u5fc313,500rpm,1\u5206\u3002<\/li>\n
                7. \u3084\u3055\u3057\u304f,SV minicolumn\u3092\u65b0\u3057\u30441.5ml\u30a8\u30c3\u30da\u30f3\u306b\u88c5\u7740\u3057,Nuclease-free Water\u309250\u03bc\u5165\u308c,\u5ba4\u6e291\u5206\u300213,500rpm,1\u5206\u3002<\/li>\n<\/ol>\n

                   <\/p>\n

                  Cycle Sequence\u3001\u7cbe\u88fd<\/h4>\n
                  16sample\u5206<\/div>\n

                  \u3000BigDye v.3.1 \u00a034.0\u00b5l\u3000(\u5206\u6ce8\u6e08\u307f1\u672c\u5206)
                  \n\u3000DW \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 26.0\u00b5l\u3000
                  \n\u30005x\u00a0<\/span>Buffer \u00a0 \u00a0 \u00a0 17.0\u00b5l<\/u><\/p>\n

                    \n
                  1. \u4e0a\u8a18\u30df\u30c3\u30af\u30b9\u3092\u30dc\u30eb\u30c6\u30c3\u30af\u30b9\u3001\u30c1\u30d3\u30bf\u30f3\u30014.5\u00a0\u00b5l\u305a\u3064PCR\u30c1\u30e5\u30fc\u30d6\u306b\u5206\u6ce8(\u6b8b\u308a\u306f\u30d5\u30bf\u3092\u9ed2\u304f\u5857\u3063\u3066\u7bb1\u306b\u623b\u3059)<\/li>\n
                  2. primer(1-2picoM)\u00a0\u00a0 1.6 \u00b5l\u3000\u3000(1\u30b5\u30f3\u30d7\u30ebF or R\u3069\u3063\u3061\u304b\u306e\u307f!)<\/li>\n
                  3. tempDNA \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 3.9 \u00b5l\u3000\u3000<\/li>\n<\/ol>\n

                    \u3092\u52a0\u3048\u3001\u30dc\u30eb\u30c6\u30c3\u30af\u30b9\u3001\u30c1\u30d3\u30bf\u30f3\u3000PCR\u30de\u30b7\u30fc\u30f3\u3067\u300cCycle Seq\u300d\u7d042.5\u6642\u9593<\/p>\n

                    [\u7cbe\u88fd]<\/p>\n

                    12sample\u5206<\/p>\n

                    \u3000DW \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u3000\u3000 \u00a0130 \u00b5l
                    \n\u3000EDTA(125mM) \u00a0 \u00a0 26 \u00b5l
                    \n\u3000\u9162\u9178Na(3M) \u00a0 \u00a0 \u00a0 \u00a0 \u00a026 \u00b5l<\/u><\/p>\n

                      \n
                    1. \u30dc\u30eb\u300114\u00b5 \u3065\u3064PCR\u30c1\u30e5\u30fc\u30d6\u306b\u5206\u6ce8\u3057\u3066\u30dc\u30eb\u3001\u30a8\u30bf\u30ce\u30fc\u30eb50 \u00b5l \u52a0\u3048\u30dc\u30eb<\/li>\n
                    2. \u5ba4\u6e29\u3067\u906e\u5149\u300015min\u5f85\u3061\u300112500rpm\u300120min\u30015\u2103<\/li>\n
                    3. \u4e0a\u6f84\u307f\u3092\u6368\u3066\u3066\u300170%\u30a8\u30bf\u30ce\u30fc\u30eb\u3092100\u00b5\u300112500rpm\u30015min\u30015\u2103<\/li>\n
                    4. \u4e0a\u6f84\u307f\u3092\u6368\u3066\u3066\u3001\u30a2\u30eb\u30df\u7b94\u3067\u8986\u3044\u3001Dry up 10min<\/li>\n<\/ol>\n

                       <\/p>\n

                      0.5M EDTA (pH 8.0) (\u51b7\u8535)<\/span>
                      \nEDTA=ethlenediaminetetraacetic acid\u30fb2Na\u30fb2H2O; MW=372.24<\/p>\n

                      EDTA \u00a046.5 g
                      \nDW \u00a0 \u00a0 \u00a0\u00a0200 ml +\u03b1(\u30e1\u30b9\u30a2\u30c3\u30d7)<\/span>
                      \n\u8a08 \u3000\u3000 250 ml<\/p>\n

                        \n
                      1. \u30d3\u30fc\u30ab\u30fc\u306b\u6c34\u3092\u5165\u308c\u30b9\u30bf\u30e9\u30fc\u3067\u64b9\u62cc\u3057\u306a\u304c\u3089EDTA\u3092\u52a0\u3048\u308b\u3002<\/li>\n
                      2. pH\u30e1\u30fc\u30bf\u30fc\u3067\u6e2c\u308a\u306a\u304c\u3089\u7c92\u4e0a\u306eNaOH\u30921\u3064\u3076\u305a\u3064\u52a0\u3048\u3001EDTA\u3092\u6eb6\u304b\u3059\u3002(\u6700\u7d42\u7684\u306b4,5\u3064\u3076)<\/li>\n
                      3. pH8\u306b\u8fd1\u3065\u3044\u3066\u304d\u305f\u30895N NaOH\u3092\u52a0\u3048\u3001pH\u3092\u8abf\u6574\u3059\u308b\u3002<\/li>\n
                      4. \u30e1\u30b9\u30a2\u30c3\u30d7\u3002\u8a66\u85ac\u30d3\u30f3\u306b\u79fb\u3057\u3001\u30aa\u30fc\u30c8\u30af\u30ec\u30fc\u30d6\u3002<\/li>\n<\/ol>\n

                        125mM EDTA\u3000(\u3082\u3068\u306f\u51b7\u8535\u3001\u5c0f\u5206\u3051\u5e38\u6e29\u53ef) <\/span><\/p>\n

                          \n
                        1. 0.5M EDTA\u3092\u7d14\u6c34\u30674\u500d\u306b\u5e0c\u91c8<\/li>\n
                        2. \u30aa\u30fc\u30c8\u30af\u30ec\u30fc\u30d6\u3002<\/li>\n<\/ol>\n

                          3M \u9162\u9178\u30ca\u30c8\u30ea\u30a6\u30e0 (pH 5.2, pH 7.0)\u3000 (\u3082\u3068\u306f\u51b7\u8535\u3001\u5c0f\u5206\u3051\u5e38\u6e29\u53ef)
                          \n<\/span>(Cycle sequence\u7cbe\u88fd\u6642\u306fpH 5.2) sodium acetate (NaOac)\u30fb3H2O; MW=136.08<\/p>\n

                          NaOAc\u30fb3H2O 3M \u300020.4 g
                          \nDW\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 40 ml +\u03b1<\/span>
                          \n\u8a08 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 250 ml<\/p>\n

                          \u6c37\u9162\u9178(\u9162\u9178)(pH\u8abf\u6574\u7528) pH 7.0\u306e\u6642 \u7d0464\u03bcl
                          \n\u00a0\u3000\u3000\u3000\u3000\u3000\u3000\u3000\u3000\u3000\u3000\u3000\u3000pH 5.2\u306e\u6642 \u7d045.7ml<\/p>\n

                            \n
                          1. \u30d3\u30fc\u30ab\u30fc\u306b\u6c34\u3092\u5165\u308c\u3001\u30b9\u30bf\u30e9-\u3067\u64b9\u62cc\u3057\u306a\u304c\u3089NaOAc\u30fb3H2O\u3092\u6eb6\u304b\u3059\u3002<\/li>\n
                          2. \u76ee\u7684\u306epH\u306b\u5408\u308f\u305b\u308b\u3002<\/li>\n
                          3. \u30e1\u30b9\u30a2\u30c3\u30d7\u3002\u8a66\u85ac\u30d3\u30f3\u306b\u79fb\u3057\u3001\u30aa\u30fc\u30c8\u30af\u30ec\u30fc\u30d6\u3002<\/li>\n<\/ol>\n
                            \n

                            <\/a>\u30b5\u30d6\u30af\u30ed2007<\/h2>\n

                            LBA\u57f9\u5730\u306e\u4f5c\u6210\u3000(10 plates\u7528)<\/h4>\n
                              \n
                            1. LB agar 8g\u3092DW250ml\u306b\u3044\u308c\u3001\u305d\u306e\u307e\u307e\u30aa\u30fc\u30c8\u30af\u30ec\u30fc\u30d6(121\u210315\u5206)<\/li>\n
                            2. 55\u2103\u307e\u3067\u306b12.5mg\/ml\u306b\u8abf\u6574\u3055\u308c\u305f\u30a2\u30f3\u30d4\u30b7\u30ea\u30f3\u30921ml\u5165\u308c\u6df7\u305c\u308b.<\/li>\n
                            3. \u30d7\u30ec\u30fc\u30c8\u306b\u9069\u91cf\u6ce8\u304e\u30b2\u30eb\u5316\u3055\u305b\u308b\u3002<\/li>\n
                            4. \u30a2\u30f3\u30d4\u30b7\u30ea\u30f3\u306f\u5931\u6d3b\u3057\u3084\u3059\u3044\u306e\u30672\u9031\u9593\u304c\u9650\u5ea6<\/li>\n<\/ol>\n

                              Cloning samples\u306e\u7528\u610f<\/h4>\n

                              \u901a\u5e38\u306ePCR sample\u3092\u7528\u610f\u3002Final Extension (72\u210310\u5206\u3068\u304b)\u304c\u3042\u308b\u307b\u3046\u304c\u826f\u3044\u3002<\/p>\n

                              \u30e9\u30a4\u30b2\u30fc\u30b7\u30e7\u30f3\u53cd\u5fdc<\/h4>\n

                              2x Rapid ligation buffer 5.0 \u03bcl
                              \npGEM-T Easy vector \u00a0 \u00a0 \u00a00.5 \u03bcl
                              \nPCR\u7523\u7269 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 4.0 \u03bcl
                              \nT4 DNA Ligase \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a01.0 \u03bcl<\/p>\n

                              \u4e0a\u8a18\u53cd\u5fdc\u6db2\u3092\u5ba4\u6e29\u30671\u6642\u9593\u53cd\u5fdc\u3055\u305b\u308b\u3002<\/p>\n

                              \u5f62\u8cea\u8ee2\u63db<\/h4>\n
                                \n
                              1. \u30bb\u30eb\u3092-80\u2103\u304b\u3089\u53d6\u308a\u51fa\u3057\u3066\u6c37\u4e2d\u3067\u89e3\u51cd\u3002<\/li>\n
                              2. \u30e9\u30a4\u30b2\u30fc\u30b7\u30e7\u30f3\u53cd\u5fdc\u6db2\u3092\u30a8\u30c3\u30da\u30f3\u30c1\u30e5\u30fc\u30d6\u306b\u5206\u6ce8\u3002<\/li>\n
                              3. \u30bb\u30eb\u30922\u306e\u30a8\u30c3\u30da\u30f3\u30c1\u30e5\u30fc\u30d6\u306b50\u03bcl\u305a\u3064\u5206\u6ce8\u3057\u3001\u5f37\u304f\u6307\u30673\u56de\u306f\u3058\u304f\u3002<\/li>\n
                              4. \u6c37\u4e0a30\u5206\u3002<\/li>\n
                              5. 2\u2103\u306745\uff5e50\u79d2\u9593\u30d2\u30fc\u30c8\u30b7\u30e7\u30c3\u30af\u3002<\/li>\n
                              6. \u6c37\u4e0a2\u5206\u3002<\/li>\n
                              7. SOC(\u51b7\u51cd\u5eab\u306e\u5225\u888b\u3000\u9ec4\u8272\u306e\u6db2\u4f531.5\u30c1\u30e5\u30fc\u30d6)\u3092250\u03bcl\u305a\u3064\u3044\u308c\u300137\u2103\u30671.5\uff5e2\u6642\u9593\u3002<\/li>\n
                              8. \u3000(X-gal\u3092LB plate\u306b60\u03bcl\u3065\u3064\u5857\u308a\u4e7e\u71e5)<\/li>\n
                              9. 120\u03bcl\u305a\u3064\u3001\u30d7\u30ec\u30fc\u30c8\u306b\u6492\u304f\u3002<\/li>\n
                              10. 37\u2103\u300116-24\u6642\u9593\u57f9\u990a\u3002<\/li>\n<\/ol>\n

                                Colony Direct PCR<\/h4>\n

                                \u3000LBplate\u4e0a\u306e\u767d\u3044conoly\u3092\u767d\u30c1\u30c3\u30d7\u3067\u3061\u3087\u3093\u3063\u3068\u3064\u3051\u3066<\/p>\n

                                \u00a0\u00a0 \u901a\u5e38\u306ePCRsampl\u306b\u3064\u3063\u3053\u307f\u30d4\u30da\u30c3\u30c6\u30a3\u30f3\u30b0\u3002 [30cycle]<\/p>\n

                                Grow in a liquid LB medium<\/h4>\n

                                \u4e7e\u71b1\u3057\u305f\u8a66\u9a13\u7ba1\u306bLB\u57f9\u5730\u30923mL\u30c7\u30a3\u30b9\u30dd\u30d4\u30da\u30c3\u30c8\u3067\u5165\u308c\u308b\u3002<\/p>\n

                                LBplate\u4e0a\u306e\u767d\u3044conoly\u3092\u30d7\u30e9\u30b9\u30c1\u30c3\u30af\u767d\u91d1\u8033\u3067\u3061\u3087\u3093\u3063\u3068\u3064\u3051\u3066\u8a66\u9a13\u7ba1\u306b\u3064\u3051\u308b\u3002<\/p>\n

                                37\u2103\u3001\u4e00\u6669\u3000\u3000\u3000(\u5404\u5668\u5177\u306f\u30d6\u30ea\u30fc\u30c1)<\/p>\n

                                QIAprep Spin Miniprep Kit Protocol<\/h4>\n

                                (Kit\u304c\u5c4a\u3044\u305f\u3089)<\/p>\n

                                  \n
                                1. Rnasae A\u3092Buffer P1\u306b\u5165\u308c\u6df7\u305c\u30012-8\u2103\u3067\u4fdd\u5b58\u3002Buffer PE\u306b\u30a8\u30bf\u30ce\u30fc\u30eb\u309224mL\u3002(\u6ce8\u610f\u4e8b\u9805)P2\u306f\u5f37\u304f\u632f\u3089\u306a\u3044\u3001\u3059\u3050\u84cb\u3092\u9589\u3081\u308b\u3002P2, N3, PB\u306f\u624b\u888b\u7740\u7528\u3002<\/li>\n
                                2. \u9060\u5fc3\u306f13000rpm\u3001\u5ba4\u6e29<\/li>\n
                                3. \u8a66\u9a13\u7ba1\u3092\u56de\u30571.5mL\u3092tube\u306b\u3044\u308c\u9060\u5fc31\u5206\u3001\u4e0a\u6f84\u307f\u3092\u30d6\u30ea\u30fc\u30c1\u5c02\u7528\u30c7\u30a3\u30b9\u30dd\u306b\u3044\u308c\u308b\u3002<\/li>\n
                                4. \u6b8b\u308a\u3092\u5165\u308c\u3066(1)\u3092\u3082\u3046\u4e00\u5ea6\u3002\u30da\u30ec\u30c3\u30c8\u72b6cell\u306b250\u03bcl\u306eP1\u3067\u30d4\u30da\u30c3\u30c8\u3067\u518d\u6fc1<\/li>\n
                                5. 250\u03bcl\u306eP2\u3092\u5165\u308c5\u56de\u3084\u3055\u3057\u304ftube\u3092\u3072\u3063\u304f\u308a\u8fd4\u3059<\/li>\n
                                6. 350\u03bcl\u306eN3\u3092\u5165\u308c5\u56de\u3084\u3055\u3057\u304ftube\u3092\u3072\u3063\u304f\u308a\u8fd4\u3059<\/li>\n
                                7. 10\u5206\u9060\u5fc3\u3057\u3001\u4e0a\u6f84\u307f\u3092QIAprep Spin Colum\u3078<\/li>\n
                                8. 1\u5206\u9060\u5fc3\u3001flow-through\u306f\u6368\u3066\u308b<\/li>\n
                                9. 750\u03bcl\u306ePE\u3092\u5165\u308c\u30011\u5206\u9060\u5fc3<\/li>\n
                                10. flow-through\u306f\u6368\u3066\u3001\u7a7a\u306e\u307e\u307e1\u5206\u9060\u5fc3<\/li>\n
                                11. \u65b0\u3057\u3044tube\u306b\u30b5\u30f3\u30d7\u30eb\u540d\u3092\u66f8\u304d\u3001 Colum\u3092\u88c5\u7740<\/li>\n
                                12. 50\u03bcl\u306eEB\u3092\u5165\u308c\u30011\u5206\u5f85\u3063\u3066\u30011\u5206\u9060\u5fc3<\/li>\n<\/ol>\n
                                  \n

                                  <\/a>\u30b7\u30fc\u30af\u30a8\u30f3\u30b5\u30fc\u306e\u4f7f\u3044\u65b9<\/h2>\n

                                  \u4e88\u7d04<\/h4>\n

                                  6\u968e\u306e\u30b7\u30fc\u30af\u30a8\u30f3\u30b5\u30fc\u5ba4\u524d\u306e\u30ab\u30ec\u30f3\u30c0\u30fc\u3067\u4e88\u7d04\u3092\u5165\u308c\u308b\u3002 \u30e9\u30f3\u56de\u6570\u3068\u540d\u524d\u3092\u66f8\u304d\u4f7f\u7528\u6642\u9593\u5206\u306e\u7dda\u3092\u5f15\u304f\u3002<\/p>\n

                                  \u30b5\u30f3\u30d7\u30eb\u6e96\u5099<\/h4>\n
                                    \n
                                  1. \u5206\u6ce8\u6e08\u307f\u306eHi-Di Formamide\u3092-20\u2103\u304b\u3089\u5fc5\u8981\u91cf\u51fa\u3057\u3066\u6eb6\u304b\u3059\u3002<\/li>\n
                                  2. \u4e7e\u71e5\u6e08\u307f\u306e\u30b5\u30a4\u30af\u30eb\u30b7\u30fc\u30af\u30a8\u30f3\u30b9\u30b5\u30f3\u30d7\u30eb\u306b\u3068\u3051\u305fHi-Di Formamide\u309220\u03bcL\u3044\u308c\u3001\u30dc\u30eb\u30fb\u30c1\u30d3\u3002<\/li>\n
                                  3. 96well \u30d7\u30ec\u30fc\u30c8\u306b\u5168\u91cf\u79fb\u3059\u30021A, 1B, 1C…..2A, 2B\u306e\u9806\u3002 16\u30b5\u30f3\u30d7\u30eb\u305a\u3064\u30ad\u30e3\u30d4\u30e9\u30ea\u30fc\u3067\u5438\u308f\u308c\u308b\u306e\u3067\u3001\u6b8b\u7a74\u306b\u3082Hi-Di Formamide\u309220\u03bcL\u3044\u308c\u308b\u3002\u30d7\u30ec\u30fc\u30c8\u30bb\u30d7\u30bf\u3092\u3057\u3063\u304b\u308a\u306f\u3081\u308b\u3002<\/li>\n<\/ol>\n

                                    \u30d1\u30bd\u30b3\u30f3\u3067\u306e\u6e96\u5099<\/h4>\n
                                      \n
                                    1. \u30d1\u30bd\u30b3\u30f3\u306e\u96fb\u6e90\u3001\u30b7\u30fc\u30af\u30a8\u30f3\u30b5\u30fc\u306e\u96fb\u6e90\u306e\u9806\u3067\u5165\u308c\u308b\u3002Run 3130 Data Collection\u3000\u3092\u30c0\u30d6\u30eb\u30af\u30ea\u30c3\u30af\u3002<\/li>\n
                                    2. \u5de6\u30a2\u30a4\u30b3\u30f3\u5de6\u306e+\u30a2\u30a4\u30b3\u30f3\u3092\u62bc\u3057\u3066\u3059\u3079\u3066\u8868\u793a\u3055\u305b\u308b\u3002 Plate Manager\u3092\u62bc\u3057\u3066\u3001\u81ea\u5206\u306ePlate ID\u3092\u30c0\u30d6\u30eb\u30af\u30ea\u30c3\u30af\u3002 (\u8ffd\u52a0\u3059\u308b\u5834\u5408\u306f\u3001\u81ea\u5206\u306ePlate ID\u3092\u30af\u30ea\u30c3\u30af(\u9752\u5909)\u3057\u3001\u4e0b\u306e\u300cDuplication\u300d\u3092\u62bc\u3057\u3066\u3001ID\u540d\u3092\u5909\u66f4\u3057\u3066\u3001\u8868\u3092\u5168\u3066\u9078\u3093\u3067\u300cEdit\u300d\u304b\u3089\u300cClear row\u300d\u3067\u767d\u7d19\u306b\u3082\u3069\u3057\u3001\u300cOK\u300d)<\/li>\n
                                    3. \u8868\u306e\u300csample name\u300d\u306b\u30b5\u30f3\u30d7\u30eb\u540d\u3001\u300cresults Group1\u300d\u306b\u30aa\u30fc\u30ca\u30fc\u540d\u3001\u300cInstrument Protocol 1\u300d\u306b\u306f\u3001600bp\u4ee5\u4e0b\u306e\u5834\u5408\u306f\u300cUltraSeq36_POP7_v3\u300d\u3001\u305d\u308c\u4ee5\u4e0a\u306e\u5834\u5408\u306f\u300cRapidSeq36_POP7_v3\u300d\u3001 [Analysis Protocol 1]\u306b\u306f\u300c3130KB_POP7_v3\u300d\u3092\u9078\u629e\u3057\u3001\u300cOK\u300d\u3002(\u30b3\u30d4\u30da\u53ef\u80fd)<\/li>\n<\/ol>\n

                                      \u30b7\u30fc\u30af\u30a8\u30f3\u30b5\u30fc\u3067\u306e\u6e96\u5099<\/h4>\n
                                        \n
                                      1. \u30d7\u30ec\u30fc\u30c8\u3092\u9ed2\u3044\u30d7\u30ec\u30fc\u30c8\u30d9\u30fc\u30b9\u306b\u306f\u3081\u3066\u3001\u767d\u3044\u30d7\u30ec\u30fc\u30c8\u30ea\u30c6\u30a4\u30ca\u30fc\u3092\u4e0a\u304b\u3089\u300c\u30ab\u30c1\u30c3\u300d\u3068\u306f\u3081\u308b\u3002<\/li>\n
                                      2. \u30b7\u30fc\u30af\u30a8\u30f3\u30b5\u30fc\u6b63\u9762\u5de6\u4e0b\u300cTRAY\u300d\u30dc\u30bf\u30f3\u3092\u62bc\u3059\u3068\u30aa\u30fc\u30c8\u30b5\u30f3\u30d7\u30e9\u30fc\u304c\u624b\u524d\u306b\u51fa\u308b\u306e\u3067\u3001\u30c9\u30a2\u3092\u958b\u3051\u3066\u30d7\u30ec\u30fc\u30c8\u30d9\u30fc\u30b9\u306e\u51f9\u3092\u5965\u306b\u3057\u3066\u53d6\u308a\u4ed8\u3051\u308b\u3002<\/li>\n
                                      3. \u30c9\u30a2\u3092\u9589\u3081\u308c\u3070\u3001\u30aa\u30fc\u30c8\u30b5\u30f3\u30d7\u30e9\u30fc\u306f\u81ea\u52d5\u7684\u306b\u5143\u306e\u4f4d\u7f6e\u306b\u623b\u308b\u3002<\/li>\n<\/ol>\n

                                        \u30dd\u30ea\u30de\u30fc\u304c\u8db3\u308a\u306a\u305d\u3046\u3060\u3063\u305f\u3089\u3001\u51b7\u8535\u88dc\u304b\u3089\u300cPOP7\u300d\u3092\u3060\u3057\u3066\u3001\u6c17\u6ce1\u3092\u3060\u3055\u306a\u3044\u3088\u3046\u306b\u9759\u304b\u306b\u5145\u586b\u3059\u308b\u3002 2\u9031\u9593\u306b1\u56de\u306f\u3001\u30aa\u30fc\u30c8\u30b5\u30f3\u30d7\u30e9\u30fc\u306e\u6c34\u30fbBuffer\u304a\u3088\u3073\u5de6\u4e0b\u306e\u967d\u6975\u30d0\u30c3\u30d5\u30a1\u30fc\u30ea\u30b6\u30fc\u30d0\u306eBuffer\u3092\u4ea4\u63db\u3059\u308b\u3002\u30aa\u30fc\u30c8\u30b5\u30f3\u30d7\u30e9\u30fc\u306e\u5de6\u624b\u524d\u304c Buffer\u3067\u6b8b\u308a\u306e3\u3064\u304cDW\u7528\u3002Buffer\u306fx10\u3092\u51b7\u8535\u5eab\u304b\u3089\u53d6\u308a\u51fa\u3057\u300150mL\u30b3\u30cb\u30ab\u30eb(\u5c02\u7528\u304c\u30b7\u30fc\u30af\u30a8\u30f3\u30b5\u30fc\u5ba4\u306b\u3042\u308a)\u306bx10 Buffer\u30923.25ml\u3092\u30d4\u30da\u30c3\u30c8\u3067\u3068\u308a\u300132.5ml\u307e\u3067DW\u3067\u30e1\u30b9\u30a2\u30c3\u30d7\u3002\u967d\u6975\u30d0\u30c3\u30d5\u30a1\u30fc\u30ea\u30b6\u30fc\u30d0\u306b\u5148\u306b\u5165\u308c\u3001\u6b8b\u308a\u3092\u9670\u6975\u3078\u3002\u30bb\u30d7\u30bf\u306f\u3057\u3063\u304b\u308a\u3002<\/p>\n

                                        \u6ce8\u610f!\u30b7\u30fc\u30af\u30a8\u30f3\u30b5\u30fc\u5185\u306e\u6c34\u6f0f\u308c\u306f\u6f0f\u96fb\u306e\u539f\u56e0\u3068\u306a\u308a\u5371\u967a\u3067\u3059\u3002\u3057\u3063\u304b\u308a\u3053\u307c\u308c\u305f\u6c34\u6c17\u306f\u53d6\u308a\u9664\u304d\u307e\u3057\u3087\u3046\u3002<\/span><\/p>\n

                                        \u6708\u306f\u3058\u3081\u306e\u4eba\u306f\u3001\u30a6\u30a9\u30fc\u30bf\u30fc\u30b7\u30fc\u30eb\u90e8\u5206\u306e\u6c34\u3092\u5145\u586b\u3002\u5c02\u7528\u6ce8\u5c04\u5668\u306bDW\u3092\u5165\u308c\u3001\u30b7\u30ea\u30f3\u30b8\u30d5\u30a3\u30c3\u30c6\u30a3\u30f3\u30b0\u306b\u6ce8\u5c04\u5668\u3092\u306d\u3058\u3064\u3051\u3066\u5ec3\u6db2\u30d5\u30a3\u30c3\u30c6\u30a3\u30f3\u30b0\u3092\u3086\u308b\u3081\u3066\u30ad\u30e0\u30ef\u30a4\u30d7\u3067\u304a\u3055\u3048\u306a\u304c\u3089DW\u3092\u5145\u586b\u3057\u3001\u5ec3\u6db2\u30d5\u30a3\u30c3\u30c6\u30a3\u30f3\u30b0\u3092\u9589\u3081\u3001\u6ce8\u8336\u5668\u3092\u53d6\u308a\u5916\u3059\u3002<\/p>\n

                                        \u30e9\u30f3<\/h4>\n
                                          \n
                                        1. [Run Scheduler]\u3067\u6253\u3061\u8fbc\u3093\u3060Plate Name\u3092\u9078\u3073\u30af\u30ea\u30c3\u30af(\u9752\u5909)\u3002<\/li>\n
                                        2. \u53f3\u306e\u56f3\u3092\u62bc\u3057\u3001\u30de\u30c3\u30d7\u304c\u9ec4\u304b\u3089\u7dd1\u3078\u5909\u308f\u3063\u305f\u3089\u3001\u5de6\u4e0a([File]\u306e\u4e0b)\u306e\u25b3\u304c\u7dd1\u306b\u5909\u308f\u308b\u306e\u3067\u3001\u62bc\u3059\u3068\u30e9\u30f3\u304c\u958b\u59cb\u3002<\/li>\n
                                        3. \u30c7\u30fc\u30bf\u306f\u81ea\u52d5\u7684\u306b\u89e3\u6790\u3055\u308c\u3001\u4fdd\u5b58\u3055\u308c\u308b\u3002\u5fc5\u8981\u306b\u5fdc\u3058\u3066\u30d7\u30ea\u30f3\u30c8\u30a2\u30a6\u30c8\u3001\u30e1\u30e2\u30ea\u306b\u843d\u3068\u3057\u3001\u30de\u30c3\u30af\u3078 \u305d\u306e\u65e5\u306e\u3046\u3061\u306b\u4f7f\u7528\u8005\u304c\u3044\u306a\u3044\u5834\u5408\u306f\u3001\u30b7\u30fc\u30af\u30a8\u30f3\u30b5\u30fc\u3001\u30d1\u30bd\u30b3\u30f3\u306e\u9806\u306b\u96fb\u6e90\u3092\u843d\u3068\u3059\u3002 \u5229\u7528\u8005\u30ce\u30fc\u30c8\u306b\u5229\u7528\u8a18\u9332\u3092\u8a18\u5165\u3059\u308b\u3002<\/li>\n<\/ol>\n

                                          \u521d\u3081\u3066\u306e\u4eba\u306f\u300c\u30e6\u30fc\u30b6\u30fc\u30ac\u30a4\u30c9\u300d\u3092\u719f\u8aad\u3057\u7d4c\u9a13\u8005\u306b\u898b\u3066\u3082\u3089\u3044\u306a\u304c\u3089\u884c\u3046\u3053\u3068\u3002<\/span> \u30ad\u30e3\u30d4\u30e9\u30ea\u30fc\u4ea4\u63db\u3084\u4e0d\u660e\u306a\u70b9\u306f\u3001\u81ea\u5206\u306e\u5224\u65ad\u3067\u884c\u308f\u305a\u3001\u5fc5\u305a\u6559\u54e1\u306e\u6307\u793a\u3092\u4ef0\u3050\u3053\u3068\u3002<\/span><\/p>\n

                                          \n
                                          \n

                                          <\/a>\u7cfb\u7d71\u6a39\u306e\u69cb\u7bc9\u65b9\u6cd5<\/h2>\n<\/div>\n

                                          \u30a2\u30e9\u30a4\u30e1\u30f3\u30c8<\/h4>\n
                                            \n
                                          1. \u30d1\u30bd\u30b3\u30f3\u3092512\u304b\u3089\u6301\u3063\u3066\u304f\u308b(\u30c6\u30fc\u30d6\u30eb\u30671\u53f0)<\/li>\n
                                          2. \u30d1\u30bd\u30b3\u30f3\u3092\u7acb\u3061\u4e0a\u3052\u3066\u3001\u30d5\u30a9\u30eb\u30c0\u3092\u30b3\u30d4\u30fc(2012-07-11_Shimada-lab142<\/li>\n
                                          3. BioEdit\u3067fas(\u30a2\u30e9\u30a4\u30e1\u30f3\u30c8\u30d5\u30a1\u30a4\u30eb)\u3092\u958b\u304f<\/li>\n
                                          4. Sequences in color\u3092\u62bc\u3059<\/li>\n
                                          5. File\u304b\u3089Open B03_\u540d\u524d<\/strong>_2012-07-11.ab1\u3000\u3000<\/li>\n
                                          6. \u6ce2\u5f62\u3092\u78ba\u8a8d\u3000(\u6c5a\u3044\u90e8\u5206\u306f\u5f8c\u3067\u4fee\u6b63\u3059\u308b\u5fc5\u8981\u3042\u308a)<\/li>\n
                                          7. \u4e0b\u306b\u958b\u3044\u305f\u30d5\u30a1\u30a4\u30eb\u3092\u30af\u30ea\u30c3\u30af\u3000\u756a\u53f7\u3092\u30af\u30ea\u30c3\u30af\u3000Edit\u304b\u3089Copy sequence<\/li>\n
                                          8. \u30a2\u30e9\u30a4\u30e1\u30f3\u30c8\u30d5\u30a1\u30a4\u30eb\u3092\u30af\u30ea\u30c3\u30af\u3000Edit\u304b\u3089Paste sequence<\/li>\n
                                          9. Mode\u3092Edit\u306b\u3000\u53f3\u96a3\u306fInsert<\/li>\n
                                          10. \u81ea\u5206\u306e\u914d\u5217\u306e\u6700\u521d\u306e\u90e8\u5206\u3092delete\u3042\u308f\u305b\u308b\u3000\u9806\u6b21\u30cf\u30a4\u30d5\u30f3\u3092\u5165\u308c\u3066\u3042\u308f\u305b\u308b<\/li>\n
                                          11. \u3042\u308f\u305b\u306a\u304c\u3089\u8fd1\u3044\u914d\u5217\u306b\u79fb\u52d5\u3055\u305b\u308b(\u6570\u5b57\u3092\u30af\u30ea\u30c3\u30af\u5de6\u62bc\u3057\u306a\u304c\u3089\u30b9\u30e9\u30a4\u30c9)<\/li>\n
                                          12. \u5f8c\u308d\u306e\u3044\u3089\u306a\u3044\u90e8\u5206\u3082delete\u3067\u3042\u308f\u305b\u3001\u4fdd\u5b58<\/li>\n<\/ol>\n

                                             <\/p>\n

                                            \u7cfb\u7d71\u6a39\u306e\u69cb\u7bc9<\/h4>\n
                                              \n
                                            1. MEGA5\u3092\u30c0\u30d6\u30eb\u30af\u30ea\u30c3\u30af<\/li>\n
                                            2. File\u304b\u3089Open A File\/Sesson\u00a0\u00a0 ITS2Ulva2012\u3092\u30c0\u30d6\u30eb\u30af\u30ea\u30c3\u30af<\/li>\n
                                            3. Analyze\u3092\u30af\u30ea\u30c3\u30af\u3000OK\u3092\u30af\u30ea\u30c3\u30af\u3000No\u3092\u30af\u30ea\u30c3\u30af<\/li>\n
                                            4. Phylogeny\u304b\u3089Construct\/Test Neighbor-Joining Tree\u3092\u30af\u30ea\u30c3\u30af<\/li>\n
                                            5. Yes\u3092\u30af\u30ea\u30c3\u30af<\/li>\n
                                            6. Gaps\/Missing Data Treatment \u306fPairwise deletion\u3092\u9078\u629e(\u9ec4\u8272\u90e8\u5206\u3092\u30af\u30ea\u30c3\u30af\u3001\u9078\u629e)<\/li>\n
                                            7. Compute\u3092\u30af\u30ea\u30c3\u30af<\/li>\n
                                            8. DNA\u9451\u5b9a\u306e\u7d50\u679c\u3092\u78ba\u8a8d\u3000Image\u304b\u3089Copy to Clipboard<\/li>\n
                                            9. \u30b9\u30bf\u30fc\u30c8\u304b\u3089\u30d1\u30ef\u30dd\u3092\u958b\u304d\u3001\u30c7\u30b6\u30a4\u30f3\u304b\u3089\u30b9\u30e9\u30a4\u30c9\u306e\u5411\u304d\u3092\u7e26\u306b<\/li>\n
                                            10. \u3044\u3089\u306a\u3044\u306ewindow\u306f\u6d88\u3057\u3066\u3001\u30da\u30fc\u30b9\u30c8\u3000<\/li>\n
                                            11. \u4e0a\u4e0b\u3092\u30d5\u30a1\u30a4\u30eb\u30b5\u30a4\u30ba\u306b\u3042\u308f\u305b\u3066\u3000\u53f3\u30af\u30ea\u30c3\u30af\u3000\u30b0\u30eb\u30fc\u30d7\u89e3\u9664\u30002\u56de<\/li>\n
                                            12. \u52a0\u5de5\u3057\u3066\u7d42\u4e86\u3000\u30d7\u30ea\u30f3\u30c8\u30a2\u30a6\u30c8\u3057\u3066\u63d0\u51fa<\/li>\n<\/ol>\n
                                              \n

                                              <\/a>RNA\u306e\u62bd\u51fa<\/h3>\n
                                              2sample\u306a\u3089\u30011ml<\/span> Buffer RLT\u306810\u03bcl<\/span>\u3000\u03b2-Mercaptoethanol\u3092\u30c1\u30e5\u30fc\u30d6\u3067\u6df7\u305c\u3066\u304a\u304f(A\u6db2)<\/span><\/div>\n
                                              \n
                                              \u00a0<\/div>\n
                                                \n
                                              1. \u751f100mg\u85fb\u4f53\u5165\u308a\u306e\u30c1\u30e5\u30fc\u30d6\u306b\u6db2\u4f53\u7a92\u7d20\u3092\u3086\u3063\u304f\u308a\u9069\u91cf\u5165\u308c\u3001\u30c9\u30ea\u30eb\u3067\u7834\u7815 (\u6db2\u4f53\u7a92\u7d20\u4f7f\u7528\u6642\u306f\u63db\u6c17\u306b\u6ce8\u610f!)<\/li>\n
                                              2. \u6025\u3044\u3067\u7834\u7815\u30b5\u30f3\u30d7\u30eb\u306b450\u03bc\u3065\u3064A\u6db2\u3092\u5206\u6ce8\u3001\u30dc\u30eb\u30c6\u30c3\u30af\u30b9<\/li>\n
                                              3. \u00a0\u5168\u91cf\u3092QIAshredder\u30ab\u30e9\u30e0(\u7d2b\u8272)\u306b\u79fb\u3057\u3001\u6700\u901f\u9060\u5fc32\u5206<\/li>\n
                                              4. \u4e0b\u306e\u30b4\u30df\u3092\u63a1\u3089\u306a\u3044\u3088\u3046\u306b\u6ce8\u610f\u3057\u3066\u4e0a\u6f84\u307f450\u03bcl\u3092\u65b0\u3057\u3044\u30a8\u30c3\u30da\u30f3\u306b\u79fb\u3057\u3001 0.5\u500d(225\u03bcl)\u306eEthanol\u3092\u52a0\u3048\u3001\u6025\u3044\u3067\u30d4\u30da\u30c3\u30c6\u30a3\u30f3\u30b0<\/li>\n
                                              5. \u6025\u3044\u3067\u5168\u91cf\u3092Rneasy\u30ab\u30e9\u30e0(\u6843\u8272)\u306b\u79fb\u3057\u3001\u9759\u304b\u306b\u3075\u305f\u3092\u9589\u3081\u3001 10000rpm\u9060\u5fc315\u79d2\u3001\u843d\u5c04\u6db2\u3092\u6368\u3066\u3001\u540c\u3058collection tube\u3092\u518d\u88c5\u7740<\/li>\n
                                              6. \u30ab\u30e9\u30e0\u306bBuffer RW1\u3092700\u03bcl\u5165\u308c\u3001\u9759\u304b\u306b\u3075\u305f\u3092\u9589\u3081\u300110000rpm\u9060\u5fc315\u79d2 (\u843d\u5c04\u6db2\u3092\u6368\u3066\u3001collection tube\u3082\u6368\u3066\u308b)<\/li>\n
                                              7. \u65b0\u3057\u3044collection tube\u3092\u4ed8\u3051\u3001\u30ab\u30e9\u30e0\u306bBuffer RPE\u3092500\u03bcl\u5165\u308c\u3001\u9759\u304b\u306b\u3075\u305f\u3092\u9589\u3081\u3001 10000rpm\u9060\u5fc315\u79d2 (\u843d\u5c04\u6db2\u3092\u6368\u3066\u3001\u540c\u3058collection tube\u3092\u518d\u88c5\u7740)<\/li>\n
                                              8. \u30ab\u30e9\u30e0\u306bBuffer RPE\u3092500\u03bcl\u5165\u308c\u3001\u9759\u304b\u306b\u3075\u305f\u3092\u9589\u3081\u300110000rpm\u9060\u5fc32\u5206 (\u6eb6\u51fa\u6db2\u30fbcollection tube\u3092\u6368\u3066\u308b)<\/li>\n
                                              9. \u30ab\u30e9\u30e0\u306b\u65b0\u3057\u3044\u30a8\u30c3\u30da\u30f3(\u756a\u53f7\u8a18\u5165)\u3092\u4ed8\u3051\u3001DW30\u03bcl\u3092\u819c\u306b\u304b\u3051\u308b\u3001 \u9759\u304b\u306b\u3075\u305f\u3092\u9589\u3081\u300110000rpm\u9060\u5fc31\u5206 (\u5fc5\u8981\u306a\u30899\u3092\u7e70\u308a\u8fd4\u3059)<\/li>\n
                                              10. Nano Drop\u3067\u6fc3\u5ea6\u3092\u6e2c\u5b9a<\/li>\n
                                              11. 3M Na\u30a2\u30bb\u30c6\u30a4\u30c8(1\/10\u500d\u91cf)+\u30a8\u30bf\u30ce\u30fc\u30eb(2.5\u500d\u91cf)\u3067-80\u5ea6\u306b\u30b9\u30c8\u30c3\u30af<\/li>\n<\/ol>\n<\/div>\n
                                                \n

                                                <\/a>mRNA FingerPrinting Kit<\/h3>\n
                                                  \n
                                                1. 3\u2018-Anchor primer\u6bce\u306b\u4e0b\u8a18\u306e\u6eb6\u6db2\u3092\u6e96\u5099\u3059\u308b\u3002 DEPC Water\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u00a0\u00a0\u00a0\u00a0\u00a08.0\u03bcl 3\u2019-Anchor Primer\u3000\u3000\u3000\u00a01.0\u03bcl Total RNA\u3000\u3000\u3000\u3000\u00a0\u00a0 \u3000\u00a0\u00a01.0\u03bcl (2.5\u03bcg)<\/li>\n
                                                2. 70\u2103, 10min\u71b1\u5909\u6027\u3002<\/li>\n
                                                3. \u6c37\u51b73min\u3002\u30b9\u30d4\u30f3\u30c0\u30a6\u30f3\u3002<\/li>\n
                                                4. \u4e0b\u8a18\u306e\u6eb6\u6db2\u3092\u52a0\u3048\u308b\u3002 DEPC Water\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 2.0\u03bcl 10\u00d7RT Buffer\u3000\u3000\u3000\u3000\u3000\u3000\u3000 2.0\u03bcl 25mM MgCl2\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u00a02.0\u03bcl 100mM DTT\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u3000\u3000 \u30002.0\u03bcl 10mM dNTP\u3000\u3000\u3000\u3000\u3000\u3000\u3000 \u3000\u30001.0\u03bcl Reverse Transcriptase\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 1.0\u03bcl<\/li>\n
                                                5. \u30b5\u30fc\u30de\u30eb\u30b5\u30a4\u30af\u30e9\u30fc\u3067\u300ecDNA\u300f\u3092\u5b9f\u884c\u3002<\/li>\n
                                                6. PCR Purification Kit\u3067\u7cbe\u88fd\u3002\u6700\u7d42\u306e\u6db2\u91cf\u306f45\u03bcl\u3068\u3057\u3001\u3053\u3053\u306b3\u2019-Anchor Primer\u30925\u03bcl\u52a0\u3048\u308b\u3002<\/li>\n
                                                7. \u300eD.D. METHOD\u300f\u306ePCR\u3092\u5b9f\u884c\u3059\u308b\u3002 10% \u30dd\u30ea\u30a2\u30af\u30ea\u30eb\u30a2\u30df\u30c9\u30b2\u30eb (\u5927\u30b2\u30eb\u4e00\u679a\u5206\u306e\u5206\u91cf) 30% \u30dd\u30ea\u30a2\u30af\u30ea\u30eb\u30a2\u30df\u30c9\u3000\u3000\u30008.340ml 1.5M Tris-HCl (pH 8.9)\u30006.260ml DW\u3000\u3000\u3000\u3000\u3000\u3000\u3000\u3000\u3000\u3000\u3000\u3000 10.185ml 10% APS\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 200\u03bcl TEMED\u3000\u3000\u3000\u3000\u3000\u3000\u3000\u3000\u3000\u3000 \u300015\u03bcl<\/span> Total\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0\u00a0 \u00a0 25ml<\/li>\n
                                                8. \u5dee\u304c\u51fa\u305f\u30d0\u30f3\u30c9\u3092\u5207\u308a\u51fa\u3057\u3066\u3001\u30a2\u30af\u30ea\u30eb\u30a2\u30df\u30c9\u304b\u3089DNA\u3092\u62bd\u51fa\u3002<\/li>\n
                                                9. *\u300e\u30dd\u30ea\u30a2\u30af\u30ea\u30eb\u30a2\u30df\u30c9\u30b2\u30eb\u304b\u3089\u306eDNA\u65ad\u7247\u306e\u56de\u53ce\u300f\u3092\u53c2\u7167<\/li>\n
                                                10. Program\u300e2nd D.D.\u300f\u3092\u7528\u3044\u3066\u518d\u5ea6PCR\u3002<\/li>\n
                                                11. Anchor Primer\u306f0.2\u03bcl\u3001Arbitrary Primer\u306f0.5\u03bcl\u3001DNA\u306f1\u03bcl\u4f7f\u7528\u3059\u308b\u3002<\/li>\n
                                                12. \u30b2\u30eb\u3092\u5207\u308a\u51fa\u3057\u3001\u30af\u30ed\u30fc\u30cb\u30f3\u30b0\u3002<\/li>\n<\/ol>\n
                                                  \n

                                                  <\/a>\u30dd\u30ea\u30a2\u30af\u30ea\u30eb\u30a2\u30df\u30c9\u30b2\u30eb\u304b\u3089\u306eDNA\u65ad\u7247\u306e\u56de\u53ce<\/h3>\n
                                                    \n
                                                  1. \u76ee\u7684\u306e\u30b2\u30eb\u3092\u30ab\u30c3\u30bf\u30fc\u3067\u5207\u308a\u51fa\u3059\u3002<\/li>\n
                                                  2. \u5207\u308a\u51fa\u3057\u305f\u30b2\u30eb\u3092-20\u2103\u3067\u51cd\u3089\u305b\u308b\u3002<\/li>\n
                                                  3. \u51cd\u3063\u305f\u30b2\u30eb\u306b\u6db2\u4f53\u7a92\u7d20\u3092\u52a0\u3048\u3001\u30da\u30c3\u30b9\u30eb\u3067\u7834\u7815\u3002<\/li>\n
                                                  4. \u00a0600\u03bcl\u306eBuffer\u3092\u52a0\u3048\u3066Vortex\u3057\u3001\u30a6\u30a9\u30fc\u30bf\u30fc\u30d0\u30b9\u306737\u2103\u30011\u6669\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30c8\u3002 (Buffer=0.5M\u9162\u9178\u30a2\u30f3\u30e2\u30cb\u30a6\u30e01mMEDTA \u907a\u4f1d\u5b50\u51b7\u8535\u5eab070801)<\/li>\n
                                                  5. \u00a014000rpm\u30015min. \u4e0a\u6f84\u307f\u3092\u65b0\u3057\u3044\u30c1\u30e5\u30fc\u30d6\u306b\u79fb\u3059\u3002<\/li>\n
                                                  6. \u00a0\u30da\u30ec\u30c3\u30c8\u306b\u3055\u3089\u306b150\u03bcl\u306eBuffer\u3092\u52a0\u3048Vortex\u3002<\/li>\n
                                                  7. \u00a014000rpm\u30015min. \u4e0a\u6f84\u307f\u30925\u306e\u4e0a\u6f84\u307f\u3068\u6df7\u305c\u308b\u3002<\/li>\n
                                                  8. \u00a0\u4e0a\u6f84\u307f\u3092300\u03bcl\u305a\u3064\u30c1\u30e5\u30fc\u30d6\u306b\u79fb\u3057\u3001\u3053\u3053\u306b30\u03bcl\u306e3M \u9162\u9178\u30ca\u30c8\u30ea\u30a6\u30e0\u3001750\u03bcl\u306e100%\u30a8\u30bf\u30ce\u30fc\u30eb\u3092\u52a0\u3048\u3066\u3001\u5ba4\u6e29\u306715min.<\/li>\n
                                                  9. 4\u2103, 14000rpm, 20min.<\/li>\n
                                                  10. \u4e0a\u6f84\u307f\u3092\u6368\u306670%\u30a8\u30bf\u30ce\u30fc\u30eb\u30921ml\u52a0\u3048\u308b\u3002<\/li>\n
                                                  11. 4\u2103, 14000rpm, 5min.<\/li>\n
                                                  12. \u4e7e\u71e5\u3055\u305b\u300120\u03bcl\u306eDEPC WATER or EB \u306a\u3069\u306b\u6eb6\u304b\u3059\u3002<\/li>\n<\/ol>\n
                                                    \n
                                                    \n

                                                    <\/a>3′ RACE\u6cd5<\/h3>\n

                                                    1st strand cDNA\u306e\u5408\u6210<\/h4>\n
                                                      \n
                                                    1. RNA\u30b5\u30f3\u30d7\u30eb\/\u30d7\u30e9\u30a4\u30de\u30fc\u306e\u6df7\u5408\u6db2\u3092\u4f5c\u308b\u3002<\/li>\n
                                                    2. Total RNA(1ng\uff5e5\u03bcg) dNTP mix(10mM) 1\u03bcl 3\u2019-RACE CDS Primer\u30001\u03bcl DEPC\u51e6\u7406\u6c34 Total 12\u03bcl<\/li>\n
                                                    3. RNA\u30b5\u30f3\u30d7\u30eb\/\u30d7\u30e9\u30a4\u30de\u30fc\u6df7\u5408\u6db2\u309265\u2103\u30675\u5206\u9593\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u3068\u3059\u308b\u3002<\/li>\n
                                                    4. \u6c37\u4e0a\u306b\u79fb\u3057\u30661\u5206\u4ee5\u4e0a\u7f6e\u304f\u3002<\/li>\n
                                                    5. \u4ee5\u4e0b\u306e\u8a66\u85ac\u3092\u9806\u306b\u6df7\u5408\u3057\u3001\u30b5\u30f3\u30d7\u30eb\u5206\u306eMix\u3092\u4f5c\u308b\u3002<\/li>\n
                                                    6. RNA\u30b5\u30f3\u30d7\u30eb\/\u30d7\u30e9\u30a4\u30de\u30fc\u6df7\u5408\u6db2\u300012\u03bcl 5\u00d7cDNA buffer 4\u03bcl DTT(0.1M\u30b9\u30c8\u30c3\u30af)\u30002\u03bcl Total \u3000\u300018\u03bcl<\/li>\n
                                                    7. 42\u2103\u30672\uff5e5\u5206\u9593\u30d7\u30ec\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30c8\u3059\u308b\u3002<\/li>\n
                                                    8. \u9006\u8ee2\u5199\u9175\u7d20\u30921\u03bcl\u52a0\u3048\u3001\u30c1\u30e5\u30fc\u30d6\u306e\u5185\u5bb9\u7269\u3092\u30d4\u30da\u30c3\u30c6\u30a3\u30f3\u30b0\u3067\u3088\u304f\u6df7\u5408\u3059\u308b\u3002<\/li>\n
                                                    9. 42\u2103\u306750\u5206\u9593\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30c8\u3002<\/li>\n
                                                    10. 70\u2103\u306715\u5206\u9593\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30c8\u3057\u3001\u53cd\u5fdc\u3092\u6b62\u3081\u308b\u3002<\/li>\n
                                                    11. \u6c37\u4e0a\u3067\u51b7\u3084\u3057\u305f\u5f8c\u3001\u8efd\u304f\u9060\u5fc3\u3057\u3066\u53cd\u5fdc\u6db2\u3092\u30c1\u30e5\u30fc\u30d6\u306e\u305d\u3053\u306b\u96c6\u3081\u308b\u3002<\/li>\n
                                                    12. \u305d\u306e\u307e\u307ePCR\u306b\u7528\u3044\u308b\u304b\u3001-20\u2103\u3067\u30b9\u30c8\u30c3\u30af\u3059\u308b\u3002<\/li>\n<\/ol>\n

                                                      \u00a0<\/p>\n

                                                      3\u2019RACE\u6cd5<\/h4>\n
                                                        \n
                                                      1. \u901a\u5e38\u306ePCRmix(primer\u629c\u304d)\u3092\u4f5c\u6210\u3059\u308b(DMSO\u306e\u6709\u7121\u306f\u30b1\u30fc\u30b9\u30d0\u30a4\u30b1\u30fc\u30b9)\u3002<\/li>\n
                                                      2. 1.\u3067\u4f5c\u3063\u305fMix\u3092degenerate primer\u30fb3\u2019adapter primer\u304c1\u03bcl\u305a\u3064\u5165\u3063\u305fPCR\u30c1\u30e5\u30fc\u30d6\u306b\u5206\u6ce8\u3059\u308b\u3002<\/li>\n
                                                      3. \u6700\u521d\u306e\u71b1\u5909\u6027\u3060\u30512\u5206\u9577\u304f\u53d6\u308a\u3001\u5f8c\u306f\u901a\u5e38\u306ePCR\u3092\u884c\u3046([1PCR]\u306eprogram\u3092\u7528\u3044\u308c\u3070\u3088\u3044)\u3002 \u30a2\u30cb\u30fc\u30ea\u30f3\u30b0\u6e29\u5ea6\u306f\u500b\u3005\u306e\u30d7\u30e9\u30a4\u30de\u30fc\u306b\u3088\u3063\u3066\u6c7a\u5b9a\u3059\u308b\u3002\u30b5\u30a4\u30af\u30eb\u306f25\uff5e30\u56de\u7a0b\u5ea6\u3002<\/li>\n
                                                      4. \u30a2\u30ac\u30ed\u30fc\u30b9\u30b2\u30eb\u306b\u3088\u308b\u96fb\u6c17\u6cf3\u52d5\u3067\u30d0\u30f3\u30c9\u3092\u78ba\u8a8d\u3059\u308b\u3002<\/li>\n
                                                      5. \u30d0\u30f3\u30c9\u304c\u78ba\u8a8d\u3067\u304d\u305f\u3089\u30b5\u30d6\u30af\u30ed\u3057\u3066\u3001PCR\u3092\u884c\u3046\u3002<\/li>\n
                                                      6. PCR\u7523\u7269\u3092\u7cbe\u88fd\u3057Cycle Sequence\u3092\u884c\u3044(\u3053\u306e\u6642\u306eprimer\u306f\u30d7\u30e9\u30b9\u30df\u30c9\u306e\u3082\u306e\u3092\u4f7f\u3063\u305f\u307b\u3046\u304c\u5897\u5e45\u306f\u5b89\u5b9a\u3059\u308b)\u3001\u30b7\u30fc\u30b1\u30f3\u30b5\u30fc\u3078\u3002<\/li>\n
                                                      7. \u30b7\u30fc\u30b1\u30f3\u30b9\u306bpoly(A)\u304c\u78ba\u8a8d\u3067\u304d\u305f\u3089mRNA\u304b\u3089\u5897\u5e45\u3067\u304d\u305f\u3082\u306e\u3060\u3068\u8003\u3048\u3066\u3088\u3044\u3002\u914d\u5217\u304b\u30895\u2019RACE\u7528\u306eprimer\u3092\u4f5c\u6210\u3059\u308b\u3002<\/li>\n<\/ol>\n
                                                        \n
                                                        \u00a0<\/div>\n<\/div>\n

                                                        5′ RACE\u6cd5<\/h3>\n

                                                        cDNA\u306e\u5408\u6210<\/h4>\n

                                                        GSP1 (3’\u3067\u6c7a\u3081\u305f\u914d\u5217\u304b\u3089\u4f5c\u88fd\u3059\u308bprimer) 2.5pmoles Sample RNA 1-5\u03bcg DEPC-treated water 15.5\u03bcl<\/p>\n

                                                          \n
                                                        1. \u3053\u306emix\u309270\u2103\u300110min<\/li>\n
                                                        2. component (\u03bcl) 10\u00d7PCR buffer 2.5 25mM MgCl2 2.5 10mM dNTP mix 1 0.1 M DTT 2.5 Final volume 8.5 \u3053\u308c\u3092\u6e29\u3081\u305fsample\u306b\u52a0\u3048\u300142\u2103\u30011min<\/li>\n
                                                        3. SuperScript\u2161\u30921\u03bcl\u52a0\u3048\u3066\u300ecDNA\u300f\u306e\u30d7\u30ed\u30b0\u30e9\u30e0<\/li>\n<\/ol>\n

                                                          S.N.A.P Column Purification of cDNA<\/h4>\n
                                                            \n
                                                          1. binding solution\u3092\u5ba4\u6e29\u306b\u623b\u3059<\/li>\n
                                                          2. \u30b5\u30f3\u30d7\u30eb\u6570\u00d750\u03bcl\u306esterilized, distilled water\u309265\u2103\u306b\u6e29\u3081\u3066\u304a\u304f\u3002<\/li>\n
                                                          3. 120\u03bcl\u306ebinding solution\u3092first strand sample\u306b\u52a0\u3048\u308b\u3002<\/li>\n
                                                          4. sample\u3092S.N.A.P column\u306b\u79fb\u3057\u300113000rpm\u306720sec\u3002<\/li>\n
                                                          5. \u30ab\u30e9\u30e0\u3092\u306f\u305a\u3057\u3001\u843d\u6db2\u306fcDNA\u306e\u5b58\u5728\u304c\u78ba\u8a8d\u3055\u308c\u308b\u307e\u3067PCR\u30c1\u30e5\u30fc\u30d6\u306b\u4fdd\u5b58\u3002<\/li>\n
                                                          6. \u30ab\u30e9\u30e0\u306b4\u2103\u306ewash buffer\u3092400\u03bcl\u52a0\u3048\u300113000rpm\u300120sec\u3001\u843d\u6db2\u3092\u6368\u3066\u308b(\u00d73)\u3002<\/li>\n
                                                          7. 400\u03bcl\u30014\u2103\u306e70%\u30a8\u30bf\u30ce\u30fc\u30eb\u3092\u52a0\u3048\u3066\u300113000rpm\u300120sec\u3001\u843d\u6db2\u3092\u6368\u3066\u308b(\u00d72)\u3002<\/li>\n
                                                          8. \u7a7a\u306e\u307e\u307e13000rpm\u30011\u5206\u3002<\/li>\n
                                                          9. \u30ab\u30e9\u30e0\u306b\u65b0\u3057\u3044\u30c1\u30e5\u30fc\u30d6\u3092\u3064\u3051\u3066\u300165\u2103\u306b\u6e29\u3081\u305fsterilized, distilled water\u309250\u03bcl\u52a0\u3048\u300113000rpm\u300120sec\u3002<\/li>\n<\/ol>\n

                                                            TdT Tailing of cDNA<\/h4>\n
                                                              \n
                                                            1. \u00a0\u4ee5\u4e0b\u306e\u6eb6\u6db2\u3092PCR\u30c1\u30e5\u30fc\u30d6\u306b\u5165\u308c\u3066\u3084\u3055\u3057\u304f\u6df7\u5408\u3059\u308b\u3002 DEPC-trerted water 6.5\u03bcl 5\u00d7 tailing buffer 5\u03bcl 2mM dCTP 2.5\u03bcl cDNA sample 10\u03bcl Final volume 24\u03bcl<\/li>\n
                                                            2. 2\uff5e3\u520694\u2103\u3067\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30c8\u30021\u5206\u9593\u6c37\u4e0a\u3002\u30c1\u30d3\u30bf\u30f3\u3001\u6c37\u4e0a\u3002<\/li>\n
                                                            3. TdT\u30921\u03bcl\u52a0\u3048\u300137\u2103\u306710\u5206\u9593\u3002<\/li>\n
                                                            4. GSP2(GSP1\u3088\u308a5\u2019\u3067\u4f5c\u88fd\u3057\u305fprimer)\u3068\u30ad\u30c3\u30c8\u306e\u30a2\u30c0\u30d7\u30bf\u30fcprimer\u3067PCR\u3001\u30b7\u30fc\u30af\u30a8\u30f3\u30b9 \u30a2\u30e9\u30a4\u30e1\u30f3\u30c8\u30673\u2019\u3068\u304b\u3076\u308b\u304b\u78ba\u8a8d\u3002<\/li>\n<\/ol>\n
                                                              \n

                                                              <\/a>Northern Blot\u6cd5<\/h3>\n

                                                              ECL Direct Nucleic Acid Labeling and Detection System \u30d7\u30ed\u30fc\u30d6\u306e\u6a19\u8b58<\/p>\n

                                                                \n
                                                              1. \u6a19\u8b58\u3059\u308bDNA\u3092\u30ad\u30c3\u30c8\u9644\u5c5e\u306e\u6c34(\u307e\u305f\u306fDEPC WATER)\u306710ng\/\u03bcl\u306b\u5e0c\u91c8\u3059\u308b\u3002<\/li>\n
                                                              2. 100ng\u306eDNA(RNA)\u30b5\u30f3\u30d7\u30eb(10\u03bcl)\u3092\u3068\u308a\u3001\u6cb8\u9a30\u6c34\u4e2d\u30675min, \u71b1\u5909\u6027\u3002<\/li>\n
                                                              3. \u30b5\u30f3\u30d7\u30eb\u3092\u6c37\u4e2d\u30675min\u6025\u51b7\u5f8c\u3001\u30b9\u30d4\u30f3\u30c0\u30a6\u30f3\u3002<\/li>\n
                                                              4. \u71b1\u5909\u6027\u3057\u305f\u30b5\u30f3\u30d7\u30eb10\u03bcl\u306eDNA labeling reagent\u3092\u52a0\u3048\u3001\u7a4f\u3084\u304b\u306b\u5b8c\u5168\u306b\u6df7\u548c\u3002<\/li>\n
                                                              5. Labeling reagent\u3068\u7b49\u91cf(10\u03bcl)\u306eglutaraldehyde solution\u3092\u52a0\u3048\u3001\u5b8c\u5168\u306b\u6df7\u548c\u3001\u30b9\u30d4\u30f3\u30c0\u30a6\u30f3\u3002<\/li>\n
                                                              6. 37\u2103\u300110min\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30c8\u3002<\/li>\n
                                                              7. \u4f5c\u6210\u3057\u305f\u30d7\u30ed\u30fc\u30d6\u306f\u76f4\u3061\u306b\u4f7f\u7528\u3059\u308b\u304b\u3001\u6c37\u4e2d\u306b\u4fdd\u5b58\u3057\u300110\uff5e15min\u4ee5\u5185\u306b\u4f7f\u7528\u3059\u308b\u3002<\/li>\n<\/ol>\n

                                                                \u30cf\u30a4\u30d6\u30ea\u30c0\u30a4\u30bc\u30fc\u30b7\u30e7\u30f3 1\u6b21\u6d17\u6d44\u30d0\u30c3\u30d5\u30a1\u30fc Urea\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 36g SDS\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 0.4g<\/p>\n\n\n\n
                                                                \u00a0\u00a0 20\u00d7SSC\u00a0\u00a0 2.5ml<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

                                                                Fill up to\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 100ml (DW) 2\u6b21\u6d17\u6d44\u30d0\u30c3\u30d5\u30a1\u30fc<\/p>\n\n\n\n
                                                                \u00a0\u00a0 20\u00d7SSC\u00a0\u00a0 10ml<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

                                                                Fill up to\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 100ml (DW)<\/p>\n

                                                                  \n
                                                                1. \u30cf\u30a4\u30d6\u30ea\u30c0\u30a4\u30bc\u30fc\u30b7\u30e7\u30f3\u30d0\u30c3\u30d5\u30a1\u30fc\u3092\u5ba4\u6e29\u3067\u5fc5\u8981\u91cf\u306f\u304b\u308a\u3001\u6700\u7d42\u6fc3\u5ea65%\u3068\u306a\u308b\u3088\u3046\u306bBlocking reagent\u3092\u30010.5M\u306b\u306a\u308b\u3088\u3046\u306bNaCl\u3092\u6dfb\u52a0\u305742\u2103\u306b\u6e29\u3081\u306a\u304c\u3089\u652a\u62cc\u3057\u3001\u5b8c\u5168\u306b\u6eb6\u89e3\u3055\u305b\u308b\u3002(\u30d0\u30c3\u30d5\u30a1\u30fc\u306e\u91cf\u306f\u30e1\u30f3\u30d6\u30ec\u30f3\u306e\u9762\u7a4d\u00d70.125ml\u7a0b\u5ea6)<\/li>\n
                                                                2. 42\u2103\u306e\u30cf\u30a4\u30d6\u30ea\u30c0\u30a4\u30bc\u30fc\u30b7\u30e7\u30f3\u30d0\u30c3\u30d5\u30a1\u30fc\u306e\u4e2d\u306b\u30e1\u30f3\u30d6\u30ec\u30f3\u3092\u3044\u308c\u30011hr\u30d7\u30ec\u30cf\u30a4\u30d6\u30ea\u30c0\u30a4\u30bc\u30fc\u30b7\u30e7\u30f3\u3002<\/li>\n
                                                                3. \u6a19\u8b58\u30d7\u30ed\u30fc\u30d6\u3092\u6dfb\u52a0\u3002\u7a4f\u3084\u304b\u306b\u632f\u76ea\u3057\u306a\u304c\u308942\u2103\u3067\u4e00\u6669\u30cf\u30a4\u30d6\u30ea\u30c0\u30a4\u30bc\u30fc\u30b7\u30e7\u30f3\u3002(\u30d6\u30ed\u30c3\u30c8\u306b\u76f4\u63a5\u304b\u304b\u3089\u306a\u3044\u3088\u3046\u306b\u30d0\u30c3\u30d5\u30a1\u30fc\u306e\u4e00\u90e8\u3092\u53d6\u308a\u51fa\u3057\u3066\u30d7\u30ed\u30fc\u30d6\u3068\u6df7\u548c\u3057\u3066\u623b\u3059\u3068\u826f\u3044)<\/li>\n
                                                                4. \u4e00\u6b21\u6d17\u6d44\u30d0\u30c3\u30d5\u30a1\u30fc\u309242\u2103\u306b\u6e29\u3081\u3066\u304a\u304d\u3001\u30d6\u30ed\u30c3\u30c8\u3092\u6d78\u305742\u2103\u3092\u8d8a\u3048\u306a\u3044\u3088\u3046\u306b20min,\u632f\u76ea\u3002(\u30d0\u30c3\u30d5\u30a1\u30fc\u91cf\u306f\u30e1\u30f3\u30d6\u30ec\u30f3\u306e\u9762\u7a4d\u00d72\uff5e5ml)<\/li>\n
                                                                5. 4\u3092\u7e70\u308a\u8fd4\u3059\u3002<\/li>\n
                                                                6. \u304d\u308c\u3044\u306a\u5bb9\u5668\u306b\u30e1\u30f3\u30d6\u30ec\u30f3\u3092\u79fb\u30572\u6b21\u6d17\u6d44\u30d0\u30c3\u30d5\u30a1\u30fc\u3092\u5165\u308c\u5ba4\u6e29\u30675min, \u632f\u76ea\u3002<\/li>\n
                                                                7. 6\u3092\u7e70\u308a\u8fd4\u3059\u3002<\/li>\n
                                                                8. \u6d17\u6d44\u5f8c\u306f\u30b5\u30e9\u30f3\u30e9\u30c3\u30d7\u306b\u5305\u3093\u3067\u30e1\u30f3\u30d6\u30ec\u30f3\u304c\u4e7e\u304b\u306a\u3044\u3088\u3046\u306b2\uff5e8\u2103\u3067\u4fdd\u5b58\u3002<\/li>\n<\/ol>\n

                                                                   <\/p>\n

                                                                  \u30b7\u30b0\u30ca\u30eb\u306e\u691c\u51fa<\/h4>\n
                                                                    \n
                                                                  1. Detection reagent1\u3068Detection reagent2\u3092\u7b49\u91cf\u6df7\u5408\u3057\u3001\u30e1\u30f3\u30d6\u30ec\u30f3\u4e0a\u306e\u4f59\u8a08\u306a2\u6b21\u6d17\u6d44\u30d0\u30c3\u30d5\u30a1\u30fc\u3092\u9664\u304d\u3001DNA\u304c\u30d6\u30ed\u30c3\u30c8\u3055\u308c\u3066\u3044\u308b\u9762\u306b\u691c\u51fa\u8a66\u85ac\u3092\u6dfb\u52a0\u3059\u308b\u3002(\u691c\u51fa\u8a66\u85ac\u306e\u91cf\u306f\u30e1\u30f3\u30d6\u30ec\u30f3\u306e\u9762\u7a4d\u00d70.125ml\u7a0b\u5ea6)<\/li>\n
                                                                  2. \u5ba4\u6e29\u30671min\u3002<\/li>\n
                                                                  3. \u30e1\u30f3\u30d6\u30ec\u30f3\u306e\u89d2\u3092\u6301\u3063\u3066\u4f59\u8a08\u306a\u691c\u51fa\u8a66\u85ac\u3092\u9664\u304d\u3001\u6c17\u6ce1\u304c\u5165\u3089\u306a\u3044\u3088\u3046\u306b\u30b5\u30e9\u30f3\u30e9\u30c3\u30d7\u3067\u5305\u3080\u3002<\/li>\n
                                                                  4. \u30d6\u30ed\u30c3\u30c8\u304c\u4e0a\u306b\u306a\u308b\u3088\u3046\u306b\u30b5\u30f3\u30d7\u30eb\u3092\u30d5\u30a3\u30eb\u30e0\u30ab\u30bb\u30c3\u30c8\u306e\u4e2d\u306b\u304a\u304dHyper film ECL\u3092\u30bb\u30c3\u30c8\u3059\u308b(\u6697\u4e2d)\u3002<\/li>\n
                                                                  5. 1min, \u9732\u5149\u3055\u305b\u308b\u3002<\/li>\n
                                                                  6. \u73fe\u50cf\u3001\u5b9a\u7740\u6db25min\u3001\u6d17\u6d4430min\u3002<\/li>\n<\/ol>\n
                                                                    \n

                                                                     <\/p>\n

                                                                    Northern Blot\u6cd5<\/h3>\n

                                                                    \u8a66\u85ac\u4f5c\u6210\u6642\u306b\u306f\u5c02\u7528\u306e\u5668\u5177\u3092\u7528\u3044\u308b\u3053\u3068\u3002 \u6e96\u5099\u3000\uff5e\u8a66\u85ac\u8abf\u6574\uff5e<\/p>\n

                                                                      \n
                                                                    1. 20\u00d7MOPS (pH 7.0) CH3COONa\u30fb3H2O\u00a0\u00a0\u00a0 1.36g MOPS\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 8.37g<\/li>\n<\/ol>\n\n\n\n
                                                                      \u00a0EDTA\u30fb2Na\u30fb2H2O\u00a0\u00a0\u00a0 0.75g<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n
                                                                        \n
                                                                      1. Fill up to 100ml(DW)<\/li>\n
                                                                      2. 2.20\u00d7SSC (pH 7.0)<\/li>\n
                                                                      3. NaCl\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u00a0\u00a0\u00a0\u00a087.6g<\/li>\n<\/ol>\n\n\n\n
                                                                        \u00a0\u00a0 Sodium Citrate\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 50g<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n
                                                                          \n
                                                                        1. Fill up to 500ml(DW)<\/li>\n
                                                                        2. \u4f5c\u6210\u5f8c\u30aa\u30fc\u30c8\u30af\u30ec\u30a4\u30d6\u51e6\u7406\u3057\u305f\u307b\u3046\u304c\u3088\u3044\u3002<\/li>\n<\/ol>\n

                                                                           <\/p>\n

                                                                          \u2606\u30b2\u30eb\u306e\u4f5c\u6210\u30001% 100ml<\/h4>\n
                                                                            \n
                                                                          1. \u30a2\u30ac\u30ed\u30fc\u30b9\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 1.0g 20\u00d7MOPS\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5ml DW\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 50ml \u3092\u6df7\u5408\u3057\u3001\u30ec\u30f3\u30b8\u3067\u6e29\u3081\u3066\u6eb6\u304b\u3057\u3001\u3055\u3089\u306b\u6b8b\u308a\u306eDW27ml\u3092\u52a0\u3048\u3066\u6e29\u3081\u308b\u3002<\/li>\n
                                                                          2. 55\u2103\u7a0b\u5ea6\u307e\u3067\u51b7\u307e\u3057\u3001\u30db\u30eb\u30e0\u30a2\u30eb\u30c7\u30d2\u30c918ml\u3092\u52a0\u3048\u308b\u3002<\/li>\n
                                                                          3. \u578b\u306b\u6d41\u3059\u3002<\/li>\n<\/ol>\n

                                                                            \u6ce8\u30fb\u30db\u30eb\u30e0\u30a2\u30eb\u30c7\u30d2\u30c9\u306f\u63ee\u767a\u3057\u3084\u3059\u304f\u6709\u5bb3!\u30b2\u30eb\u306f\u30c9\u30e9\u30d5\u30c8\u5185\u3067\u56fa\u3081\u308b\u3002 \u00a0<\/p>\n

                                                                            \u2606Sample\u8abf\u6574<\/h4>\n

                                                                            Sample Buffer\u300020sample\u5206<\/p>\n

                                                                            0.4mg\/ml EtBr\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 20\u03bcl<\/p>\n

                                                                            20\u00d7MOPS\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u300010\u03bcl<\/p>\n

                                                                            \u30db\u30eb\u30e0\u30a2\u30df\u30c9\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 200\u03bcl<\/p>\n

                                                                            \u30db\u30eb\u30e0\u30a2\u30eb\u30c7\u30d2\u30c9\u3000\u3000\u300070\u03bcl<\/p>\n

                                                                              \n
                                                                            1. S.B.15\u03bcl\u3068RNA 5\u03bcl\u3092\u6df7\u5408\u3057\u300165\u2103\u300115min\u3067\u5909\u6027\u3055\u305b\u308b\u3002<\/li>\n
                                                                            2. \u30ed\u30fc\u30c7\u30a3\u30f3\u30b0\u30d0\u30c3\u30d5\u30a1\u30fc\u3068\u6df7\u5408\u3057\u3001\u30a2\u30d7\u30e9\u30a4\u3002<\/li>\n<\/ol>\n

                                                                               <\/p>\n

                                                                              \u2606\u96fb\u6c17\u6cf3\u52d5<\/h4>\n
                                                                                \n
                                                                              1. 1\u00d7MOPS\u3067\u6cf3\u52d5\u69fd\u3092\u6e80\u305f\u3057\u3001\u96fb\u6c17\u6cf3\u52d5 (60V\u30672hr\u7a0b\u5ea6) \u3002<\/li>\n
                                                                              2. \u7d42\u4e86\u5f8c\u30b2\u30eb\u3092DW\u3067\u8efd\u304f\u6d17\u3044\u3001\u3055\u3089\u306b20\u00d7SSC\u306b5min\u6d78\u3059\u00d72\u56de(\u3053\u306e\u9805\u306f\u7701\u7565\u53ef)\u3002<\/li>\n<\/ol>\n

                                                                                 <\/p>\n

                                                                                \u2606\u30d6\u30ed\u30c3\u30c6\u30a3\u30f3\u30b0<\/h4>\n
                                                                                  \n
                                                                                1. Hybond H+\u3092\u30b2\u30eb\u306e\u5927\u304d\u3055\u306b\u5207\u308b\u3002<\/li>\n
                                                                                2. \u4e0b\u56f3\u306e\u3088\u3046\u306b\u30bb\u30c3\u30c8\u3059\u308b\u3002<\/li>\n<\/ol>\n

                                                                                  \"North<\/a> \u6ce8\u30fb\u30b2\u30eb\u3068\u30e1\u30f3\u30d6\u30ec\u30f3\u306e\u9593\u306b\u6c17\u6ce1\u3092\u5165\u308c\u306a\u3044\u3002 \u30a6\u30a7\u30eb\u3092\u4e0b\u5074\u306b\u3059\u308b\u3002 \u00a0<\/p>\n

                                                                                  \u2606\u30d7\u30ed\u30fc\u30d6\u306e\u4f5c\u6210<\/h4>\n

                                                                                  Amersham\u793eGene Images Random Prime Labeling Kit\u3092\u4f7f\u7528\u3057\u305f\u5834\u5408<\/p>\n

                                                                                    \n
                                                                                  1. \u6fc3\u5ea62-25ng\/\u03bcl\u306eDNA\u6eb6\u6db2\u3092\u7528\u610f\u3059\u308b\u3002<\/li>\n
                                                                                  2. DNA\u6eb6\u6db2\u30925min\u3001\u30b7\u30e3\u30f3\u30e1\u30eb\u3057\u6c37\u51b7\u3002<\/li>\n
                                                                                  3. Nucleotide mix\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 10\u03bcl Primer\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5\u03bcl Denatured DNA\u00a0\u00a0\u00a0\u00a0\u00a0 50ng Klenow\u3000\u3000\u3000\u3000\u3000\u3000\u30001\u03bcl Fill up to 50\u03bcl (DEPC Water)<\/li>\n
                                                                                  4. \u30d4\u30da\u30c3\u30c6\u30a3\u30f3\u30b0\u3067\u3084\u3055\u3057\u304f\u6df7\u5408\u3002<\/li>\n
                                                                                  5. 37\u2103\u30011hr\u3067\u53cd\u5fdc\u3055\u305b\u308b\u3002<\/li>\n<\/ol>\n

                                                                                     <\/p>\n

                                                                                    \u2606\u30d7\u30ed\u30fc\u30d6\u306e\u7cbe\u88fd<\/h4>\n

                                                                                     <\/p>\n","protected":false},"excerpt":{"rendered":"

                                                                                    DNA\u306e\u62bd\u51fa DNA\u62bd\u51fa\u30b5\u30f3\u30d7\u30eb\u306e\u6e96\u5099 \u85fb\u4f53\u306e\u4e00\u90e8(1cm\u89d2)\u3092\u3061\u304e\u308a\u3001\u6ec5\u83cc\u6d77\u6c34\u5165\u308a\u30c7\u30a3\u30b9\u30dd\u30b7\u30e3\u30fc\u30ec\u306b\u5165\u308c,\u5b9f\u4f53\u9855\u5fae\u93e1\u3067\u78ba\u8a8d\u3057\u306a\u304c\u3089\u7b46\u3067\u6c5a\u308c\u3092\u843d\u3068\u3059\u3002(\u6c5a\u308c\u304c\u3072\u3069\u3051\u308c\u3070\u7e70\u308a\u8fd4\u3059) \u30aa\u30fc\u30c8\u30af\u30ec\u30fc\u30d6\u6e08\u307f\u306e\u30a8\u30c3\u30da\u30f3\u30c1\u30e5\u30fc\u30d6(\u540d … \u7d9a\u304d\u3092\u8aad\u3080 →<\/span><\/a><\/p>\n","protected":false},"author":18,"featured_media":0,"parent":94,"menu_order":5,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-141","page","type-page","status-publish","hentry"],"aioseo_notices":[],"_links":{"self":[{"href":"https:\/\/www-p.sci.ocha.ac.jp\/bio-shimada-lab\/wp-json\/wp\/v2\/pages\/141","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www-p.sci.ocha.ac.jp\/bio-shimada-lab\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www-p.sci.ocha.ac.jp\/bio-shimada-lab\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www-p.sci.ocha.ac.jp\/bio-shimada-lab\/wp-json\/wp\/v2\/users\/18"}],"replies":[{"embeddable":true,"href":"https:\/\/www-p.sci.ocha.ac.jp\/bio-shimada-lab\/wp-json\/wp\/v2\/comments?post=141"}],"version-history":[{"count":38,"href":"https:\/\/www-p.sci.ocha.ac.jp\/bio-shimada-lab\/wp-json\/wp\/v2\/pages\/141\/revisions"}],"predecessor-version":[{"id":669,"href":"https:\/\/www-p.sci.ocha.ac.jp\/bio-shimada-lab\/wp-json\/wp\/v2\/pages\/141\/revisions\/669"}],"up":[{"embeddable":true,"href":"https:\/\/www-p.sci.ocha.ac.jp\/bio-shimada-lab\/wp-json\/wp\/v2\/pages\/94"}],"wp:attachment":[{"href":"https:\/\/www-p.sci.ocha.ac.jp\/bio-shimada-lab\/wp-json\/wp\/v2\/media?parent=141"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}