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eyfp\u57fa\u56e0\u5728brnF\u542f\u52a8\u5b50\u7684\u63a7\u5236\u4e0b\u8868\u8fbe\uff0c\u8be5\u542f\u52a8\u5b50\u542b\u6709 Lrp \u86cb\u767d\u7684\u7ed3\u5408\u4f4d\u70b9\u3002(25)\u56e0\u6b64\uff0c\u5f53\u542f\u52a8\u5b50\u5e8f\u5217\u7f29\u77ed\u6216\u5ef6\u957f\u65f6\uff0c\u53ef\u4ee5\u6dfb\u52a0\u6216\u79fb\u9664\u7ed3\u5408\u4f4d\u70b9\u3002\u7531\u4e8ebrnF\u8f6c\u5f55\u4e3a\u65e0\u524d\u5bfc\u8f6c\u5f55\u672c\uff0c\u5e76\u4e14brnF\u7684\u7b2c\u4e00\u90e8\u5206\u53ef\u80fd\u5177\u6709\u8c03\u63a7\u529f\u80fd\uff0c(26)\u6807\u51c6Lrp\u4f20\u611f\u5668\u7684eyfp\u542f\u52a8\u5b50\u5305\u542bbrnF\u7684\u524d30bp \u3002\u672c\u7814\u7a76\u8bbe\u8ba1\u4e86\u56db\u79cd\u4e0d\u540c\u7684\u53d8\u4f53\uff0c\u4ece+30\uff08\u6807\u51c6Lrp\u751f\u7269\u4f20\u611f\u5668\u542f\u52a8\u5b50\uff09\u5f00\u59cb\uff0c\u4ee515\u4e3a\u6b65\u957f\u8bbe\u8ba1\u7f29\u77ed\u7248\u672c\uff0c\u5373+15\u30010\u548c\u221215\u3002\u5bf9\u4e8eeyfp\u7684RBS\uff0c\u9009\u62e9\u4e86\u4e09\u79cd\u53d8\u4f53\uff1aAAAAGGAGAT\uff08\u6700\u5f3a\uff09\u3001AGAAGGAGAT\uff08\u5929\u7136\uff0c\u5f3a\u5ea6\u8f83\u4f4e\uff09\u548cATCCGACCAT\uff08\u6700\u5f31\uff09\u3002\u56db\u79cd\u542f\u52a8\u5b50\u957f\u5ea6\u548ceyfp\u4fa7\u4e09\u79cdRBS\u7684\u7ec4\u5408\u4ea7\u751f\u4e864\u00d73=12\u79cd\u4e0d\u540c\u7684\u5f15\u7269\u3002\u8fd9\u79cd\u8bbe\u8ba1\u7b56\u7565\u603b\u5171\u5305\u62ec9\u00d712=108\u79cd\u4e0d\u540c\u7684Lrp\u751f\u7269\u4f20\u611f\u5668\u53d8\u4f53\u3002
\u6bcf\u79cd\u53d8\u4f53\u90fd\u53ef\u4ee5\u901a\u8fc7\u5355\u4e2a PCR \u53cd\u5e94\u6784\u5efa\uff0c\u5e76\u4e14\u6240\u6709 PCR \u53cd\u5e94\u90fd\u53ef\u4ee5\u4f7f\u7528\u76f8\u540c\u7684 PCR \u6a21\u677f\uff0c\u5373\u8d28\u7c92 pJC1- lrp - brnF\u2032 - eyfp\u3002\u4e3a\u4e86\u7ec4\u88c5\u751f\u7269\u4f20\u611f\u5668\u8d28\u7c92\uff0c\u6bcf\u4e2a PCR \u53cd\u5e94\u4e4b\u540e\u90fd\u4f1a\u8fdb\u884c\u53cc\u7247\u6bb5 Gibson \u7ec4\u88c5\uff0c\u5176\u4e2d PCR \u4ea7\u7269\u88ab\u7ec4\u88c5\u5230 pEC-Tmob18- lrp - eyfp\u4e3b\u94fe\u4e2d\uff1b\u6240\u6709 PCR \u4ea7\u7269\u90fd\u7ecf\u8fc7\u8bbe\u8ba1\uff0c\u56e0\u6b64\u5b83\u4eec\u53ef\u4ee5\u7ec4\u88c5\u5230\u76f8\u540c\u7684\u4e3b\u94fe\u4e2d\u3002<\/p>\n\n\n\n
\u5728\u6784\u5efa Lrp \u751f\u7269\u4f20\u611f\u5668\u53d8\u4f53\u4e4b\u524d\uff0c\u901a\u8fc7\u5c06lrp\u548ceyfp\u63d2\u5165\u53ef\u79fb\u52a8\u7684 pEC-Tmob18 \u9aa8\u67b6\u6765\u6784\u5efa\u9aa8\u67b6\u8d28\u7c92\u3002\u5728\u4e24\u4e2a\u57fa\u56e0\u4e4b\u95f4\u63d2\u5165\u4e00\u4e2a\u5305\u542b\u4e24\u4e2a NcoI \u9650\u5236\u4f4d\u70b9\u7684\u77ed\u95f4\u9694\u533a\u3002\u6b64\u5e8f\u5217\u5141\u8bb8\u8d28\u7c92\u7ebf\u6027\u5316\uff0c\u5e76\u5728\u4e0b\u4e00\u6b65\u4e2d\u63d2\u5165\u5305\u542b\u4e0d\u540clrp\u8d77\u59cb\u5bc6\u7801\u5b50\u3001lrp RBS\u3001brnF\u542f\u52a8\u5b50\u957f\u5ea6\u548ceyfp RBS \u7684\u4e0d\u540c\u7248\u672c\u7684\u5e8f\u5217\u53d8\u4f53\u3002
108 \u79cd\u53d8\u4f53\u4e2d\uff0c96 \u79cd\u88ab\u9009\u4e3a\u81ea\u52a8\u514b\u9686\u5de5\u4f5c\u6d41\u7a0b\u7684\u5e94\u7528\u793a\u4f8b\uff0c\u56e0\u4e3a\u5927\u591a\u6570\u81ea\u52a8\u5316\u8bbe\u5907\u90fd\u662f\u4e3a 96 \u5b54\u591a\u6ef4\u5b9a\u677f\u8bbe\u8ba1\u7684\u3002PCR \u53d8\u4f53\u6784\u5efa\u3001Gibson \u7ec4\u88c5\u548c\u5927\u80a0\u6746\u83cc\u70ed\u4f11\u514b\u8f6c\u5316\u8fde\u7eed\u8fdb\u884c\u3002Opentrons OT-2 \u80fd\u591f\u5728 85 \u5206\u949f\u5185\u81ea\u52a8\u79fb\u6db2 96 \u4e2a PCR \u53cd\u5e94\uff0c\u7136\u540e\u5c06\u5e73\u677f\u624b\u52a8\u8f6c\u79fb\u5230\u70ed\u5faa\u73af\u4eea\u4e2d\u3002\u70ed\u5faa\u73af\u540e\uff0c\u4f7f\u7528 OT-2 \u6df7\u5408 96 \u4e2a Gibson \u7ec4\u88c5\u53cd\u5e94\uff0c\u8fd9\u9700\u8981 50 \u5206\u949f\u3002\u901a\u8fc7\u624b\u52a8\u5c06\u5e73\u677f\u8f6c\u79fb\u5230\u70ed\u5faa\u73af\u4eea\u8fdb\u884c\u5b75\u80b2\u3002\u5728 50 \u00b0C \u5b75\u80b2\u6b65\u9aa4\u4e4b\u524d\u5bf9 Gibson \u4e3b\u6df7\u5408\u7269\u8fdb\u884c\u9002\u5f53\u51b7\u5374\u81f3\u5173\u91cd\u8981\uff1b\u5426\u5219\uff0c\u5c31\u65e0\u6cd5\u83b7\u5f97\u5927\u80a0\u6746\u83cc\u8f6c\u5316\u5b50\u3002\u5728\u6211\u4eec\u7684\u5de5\u4f5c\u6d41\u7a0b\u4e2d\uff0c\u51b7\u5374\u662f\u901a\u8fc7\u5c06\u6240\u6709\u5b9e\u9a8c\u5ba4\u5668\u76bf\u653e\u5165\u88c5\u6ee1\u51b0\u7684 3D \u6253\u5370\u5c0f\u76d2\u5b50\u4e2d\u5b9e\u73b0\u7684\u3002\u5927\u80a0\u6746\u83cc\u70ed\u4f11\u514b\u8f6c\u5316\u662f\u5728 TECAN EVO200 \u6db2\u4f53\u5904\u7406\u673a\u5668\u4eba\u4e0a\u8fdb\u884c\u7684\uff0c\u8be5\u673a\u5668\u4eba\u5b9e\u73b0\u4e86\u5b8c\u5168\u65e0\u4eba\u64cd\u4f5c\u7684\u8fc7\u7a0b\uff0c\u4ece Gibson \u7ec4\u88c5\u6df7\u5408\u7269\u548c\u611f\u53d7\u6001\u7ec6\u80de\u5f00\u59cb\uff0c\u5230\u5c06\u7ec6\u80de\u70b9\u5728\u743c\u8102\u677f\u4e0a\u51c6\u5907\u8fc7\u591c\u5b75\u80b2\u7ed3\u675f\u3002\u8fd9\u4e2a\u8fc7\u7a0b\u8017\u65f6 170 \u5206\u949f\u3002\u603b\u5171\u5728 8 \u5c0f\u65f6\u5185\u5b8c\u6210\u4e86 96 \u4e2a\u6784\u5efa\u4f53\u7684 PCR\u3001Gibson \u7ec4\u88c5\u548c\u8f6c\u5316\uff0c\u4ec5\u9700 40 \u5206\u949f\u7684\u624b\u52a8\u64cd\u4f5c\u65f6\u95f4\u3002
\u5b9e\u65bd\u70ed\u6fc0\u6b65\u9aa4\u7684\u5408\u9002\u65b9\u6cd5\u662f\u5c06 V \u578b\u5e95 96 \u5b54\u677f\u653e\u5728\u52a0\u70ed\u88c5\u7f6e\u4e0a\u9884\u70ed\uff0c\u5c06 8 \u79cd\u7ec6\u80de-\u8d28\u7c92\u6df7\u5408\u7269\u4ece\u51b7\u5374\u8f7d\u4f53\u4e00\u6b21\u4e00\u4e2a\u5730\u8f6c\u79fb\u5230\u52a0\u70ed\u677f\u4e0a\uff0c\u5b75\u80b2 30 \u79d2\uff0c\u7136\u540e\u5c06\u5176\u8f6c\u79fb\u56de\u51b7\u5374\u8f7d\u4f53\u3002\u867d\u7136\u5c06\u6574\u4e2a\u677f\u4ece\u51b7\u5374\u8f7d\u4f53\u8f6c\u79fb\u5230\u52a0\u70ed\u88c5\u7f6e\u66f4\u5feb\uff0c\u4f46\u8fd9\u4f1a\u4ea7\u751f\u4e0d\u592a\u53ef\u9760\u7684\u70ed\u4f20\u9012\u7ed3\u679c\u3002\u901a\u8fc7\u79bb\u5fc3\u6d53\u7f29\u7ec6\u80de\u60ac\u6d6e\u6db2\uff0c\u5c06\u5176\u91cd\u65b0\u60ac\u6d6e\u5728 LB \u57f9\u517b\u57fa\u4e2d\uff0c\u7136\u540e\u70b9\u5728\u743c\u8102\u677f\u4e0a\uff0c\u5373\u901a\u8fc7\u5728\u5355\u4e2a\u743c\u8102\u677f\u7684\u540c\u4e00\u884c\u4e0a\u79fb\u53d6 6 \u4e2a 5 \u03bcL \u7684\u70b9\u3002
\u6b64\u5916\uff0c\u4f7f\u7528\u5927\u80a0\u6746\u83cc\u611f\u53d7\u6001\u7ec6\u80de\uff08NEB 5-alpha \u611f\u53d7\u6001\u5927\u80a0\u6746\u83cc\uff09\u548c pUC19 \u6807\u51c6\u8f7d\u4f53\u6d4b\u8bd5\u4e86\u70ed\u4f11\u514b\u8f6c\u5316\u7684\u53c2\u6570\u3002\u6d4b\u8bd5\u4e86\u70ed\u4f11\u514b\u6e29\u5ea6\uff0837\u300142 \u548c 47 \u00b0C\uff09\u548c\u70ed\u4f11\u514b\u6301\u7eed\u65f6\u95f4\uff080 \u5230 30 \u79d2\uff0c\u6b65\u957f\u4e3a 5 \u79d2\uff09\u3002\u4ee4\u4eba\u60ca\u8bb6\u7684\u662f\uff0c\u5728\u6bcf\u79cd\u6761\u4ef6\u4e0b\u90fd\u6210\u529f\u8f6c\u5316\u7684\u60c5\u51b5\u4e0b\uff0c\u83cc\u843d\u5f62\u6210\u5355\u4f4d\u51e0\u4e4e\u6ca1\u6709\u5dee\u5f02\u3002\u6700\u7ec8\uff0c\u9009\u62e9\u4e86\u4e0e\u6807\u51c6\u624b\u52a8\u7a0b\u5e8f\u6700\u76f8\u4f3c\u7684\u6761\u4ef6\u3002<\/p>\n","protected":false},"excerpt":{"rendered":"