{"id":1594,"date":"2020-12-03T16:25:43","date_gmt":"2020-12-03T07:25:43","guid":{"rendered":"https:\/\/www.promega.co.jp\/resources\/faq\/?p=1594"},"modified":"2020-12-18T14:17:38","modified_gmt":"2020-12-18T05:17:38","slug":"q296","status":"publish","type":"post","link":"https:\/\/www.promega.co.jp\/resources\/faq\/proteomics\/invitro-transcription-translation\/kits\/tnt-system\/q296","title":{"rendered":"\u92f3\u578bDNA\u306f\u3069\u306e\u3088\u3046\u306b\u8abf\u88fd\u3059\u308c\u3070\u3088\u3044\u3067\u3059\u304b\uff1f"},"content":{"rendered":"\n<p>\u92f3\u578bDNA\u306f\u3001\u74b0\u72b6DNA (Plasmid DNA)\u307e\u305f\u306f\u76f4\u9396 DNA (DNA fragment\u3001PCR\u7523\u7269)\u3092\u8abf\u88fd\u3057\u307e\u3059\u3002<\/p>\n\n\n\n<p>1) \u74b0\u72b6 DNA \u3092\u4f7f\u7528\u3059\u308b\u5834\u5408<\/p>\n\n\n\n<ul><li>pTNT<sup>\u00ae<\/sup>&nbsp;Vector(L5610)\u307e\u305f\u306f\u3001pCMVTNT<sup>\u00ae<\/sup>&nbsp;Vector(L5620)\u306e\u30de\u30eb\u30c1\u30af\u30ed\u30fc\u30cb\u30f3\u30b0\u30b5\u30a4\u30c8\u306b\u907a\u4f1d\u5b50\u914d\u5217\u3092\u633f\u5165\u3059\u308b\u3053\u3068\u304c\u3067\u304d\u307e\u3059\u3002\u3053\u306e\u5834\u5408\u306f\u3001\u958b\u59cb\u30b3\u30c9\u30f3(ATG)\u3092\u907a\u4f1d\u5b50\u914d\u5217\u306b\u5165\u308c\u3066\u304f\u3060\u3055\u3044\u3002<\/li><li>pTNT<sup>\u00ae<\/sup>&nbsp;Vector, pCMVTNT<sup>\u00ae<\/sup>&nbsp;Vector\u4ee5\u5916\u306eVector\u3092\u4f7f\u7528\u3059\u308b\u5834\u5408\u306f\u3001TNT System\u3067\u4f7f\u7528\u3059\u308b\u30d7\u30ed\u30e2\u30fc\u30bf\u30fc\u914d\u5217\u3001\u958b\u59cb\u30b3\u30c9\u30f3\u3001\u7d42\u6b62\u30b3\u30c9\u30f3\u3001T7 terminator\u914d\u5217\uff08\u5fc5\u8981\u306b\u5fdc\u3058\u3066\uff09\u3092\u3054\u78ba\u8a8d\u304f\u3060\u3055\u3044\u3002<\/li><\/ul>\n\n\n\n<p>\u6ce8\u610f\uff1apTNT<sup>\u00ae<\/sup>&nbsp;Vector\u3001pCMVTNT<sup>\u00ae<\/sup>&nbsp;Vector\u306b\u306f\u7ffb\u8a33\u52b9\u7387\u3092\u4e0a\u3052\u308b\u305f\u3081\u306b<a href=\"https:\/\/www.promega.co.jp\/jp\/jp_tech\/FAQs\/Q&amp;A_TNT.html#invitro_06\">\u03b2-\u30b0\u30ed\u30d3\u30f35\u2019-\u30ea\u30fc\u30c0\u30fc\u30b7\u30fc\u30af\u30a8\u30f3\u30b9<\/a>\u914d\u5217\u3092\u542b\u3093\u3067\u3044\u307e\u3059\u3002\u92f3\u578bDNA\u306e\u914d\u5217\u306b\u3088\u3063\u3066\u306f\u3001\u8ee2\u5199\u30fb\u7ffb\u8a33\u53cd\u5fdc\u306e\u52b9\u7387\u304c\u4f4e\u3044\u3044\u5834\u5408\u3082\u3042\u308a\u307e\u3059\u3002\u4f7f\u7528\u3059\u308b\u30c6\u30b9\u30c8\u30b5\u30f3\u30d7\u30eb\u3068\u540c\u6642\u306b\u30b7\u30b9\u30c6\u30e0\u306b\u4ed8\u5c5e\u306eControl\u53cd\u5fdc\u3092\u5b9f\u65bd\u3057\u3066\u304f\u3060\u3055\u3044\u3002<\/p>\n\n\n\n<p>2) \u76f4\u9396DNA\u3092\u4f7f\u7528\u3059\u308b\u5834\u5408<\/p>\n\n\n\n<ul><li>\u74b0\u72b6DNA\u3092\u5236\u9650\u9175\u7d20\u3067\u5207\u65ad\u3057\u3001\u76f4\u9396DNA\u306b\u3059\u308b\u5834\u5408<br>\u2192\u5e73\u6ed1\u672b\u7aef\u3067\u5207\u65ad\u3067\u304d\u308b\u5236\u9650\u9175\u7d20\u3092\u4f7f\u7528\u3057\u3066\u304f\u3060\u3055\u3044\u3002\u7a81\u51fa\u672b\u7aef\u3067\u5207\u65ad\u3057\u305f\u5834\u5408\u306f\u3001Klenow Fragment\u306a\u3069\u3067\u5e73\u6ed1\u672b\u7aef\u51e6\u7406\u3092\u3057\u3066\u304f\u3060\u3055\u3044\u3002<\/li><li>PCR\u306b\u3088\u308a\u92f3\u578bDNA\u3092\u8abf\u88fd\u3059\u308b\u5834\u5408\uff08TNT<sup>\u00ae<\/sup>&nbsp;Quick for PCR DNA\uff09<br>\u2192forward primer\u3068\u3057\u3066T7 promoter\u914d\u5217+\u30b9\u30da\u30fc\u30b5\u30fc\u914d\u5217+Kozak\u306e\u914d\u5217\u3092\u542b\u3080\u3088\u3046\u306b\u8a2d\u8a08\u3057\u307e\u3059\uff08\u88685\uff09\u3002<\/li><\/ul>\n\n\n\n<p>\u88685\u3000TNT<sup>\u00ae<\/sup>&nbsp;Quick for PCR DNA\u3067\u7528\u3044\u3089\u308c\u305fT7\u30d7\u30ed\u30e2\u30fc\u30bf\u30fc\u306e\u914d\u5217<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table><tbody><tr><th>\u5236\u9650\u9175\u7d20\u30b5\u30a4\u30c8<\/th><th>T7 Bacteriophage<br>Sequence<\/th><th>Spacer<\/th><th>Eukaryotic Translation<br>Initiation Sequence<\/th><th>\u53c2\u8003\u6587\u732e<\/th><\/tr><tr><td>GGATCC<\/td><td>TAA TAC GAC TCA CTA TAG GG<\/td><td>AG<\/td><td>CCC ACC ATG<\/td><td>5,6<\/td><\/tr><tr><td>GGATCC<\/td><td>TAA TAC GAC TCA CTA TAG GG<\/td><td>AG<\/td><td>CCC ACC ATG G<\/td><td>7,8<\/td><\/tr><tr><td>GGATCC<\/td><td>TAA TAC GAC TCA CTA TAG G<\/td><td>AA CAG<\/td><td>CCC ACC ATGG<\/td><td>9,10<\/td><\/tr><tr><td>nnn<\/td><td>TAA TAC GAC TCA CTA TAG G<\/td><td>AA CAG<\/td><td>CCC ACC ATG<\/td><td>11,12<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<p>3) TNT<sup>\u00ae<\/sup>&nbsp;System\u53cd\u5fdc\u306b\u7528\u3044\u308b\u92f3\u578bDNA\u306e\u7cbe\u88fd\u65b9\u6cd5<\/p>\n\n\n\n<p>\u74b0\u72b6DNA\u306f\u3001Wizard<sup>\u00ae<\/sup>&nbsp;Plus SV Minipreps (A1330\u306a\u3069)\u3092\u7528\u3044\u3066\u7cbe\u88fd\u3059\u308b\u3053\u3068\u3092\u63a8\u5968\u3057\u3066\u3044\u307e\u3059\u3002\u76f4\u9396DNA\u306f\u3001Wizard<sup>\u00ae<\/sup>&nbsp;SV Gel and PCR Clean-Up System (A9281)\u3067\u7cbe\u88fd\u3059\u308b\u3053\u3068\u3092\u63a8\u5968\u3057\u3066\u3044\u307e\u3059\u3002<\/p>\n\n\n\n<p>\u7cbe\u88fd\u5f8c\u306eDNA\u306f\u3001OD<sub>260nm<\/sub>\/OD<sub>280nm<\/sub>\u3092\u6e2c\u5b9a\u3057\u3001ratio=1.8\u4ee5\u4e0a\u3068\u306a\u308b\u3088\u3046\u306b\u3057\u3066\u304f\u3060\u3055\u3044\u3002ratio=1.8\u4ee5\u4e0b\u306e\u5834\u5408\u306f\u3001\u30a8\u30bf\u30ce\u30fc\u30eb\u6c88\u6bbf\u3092\u5b9f\u65bd\u3057\u3066\u304f\u3060\u3055\u3044\u3002ratio\u304c1.8\u3088\u308a\u3082\u5c0f\u3055\u3044\u92f3\u578bDNA\u3092\u4f7f\u7528\u3057\u305f\u5834\u5408\u3001\u8ee2\u5199\u30fb\u7ffb\u8a33\u306e\u52b9\u7387\u306f\u4f4e\u4e0b\u3057\u307e\u3059\u3002<\/p>\n\n\n\n<p>&lt;\u53c2\u8003&gt;<br>\u4e00\u822c\u7684\u306b\u306f\u3001\u74b0\u72b6DNA\u3092\u92f3\u578b\u3068\u3059\u308b\u5834\u5408\u306f\u3001RRL\u304c\u597d\u307e\u3057\u3044\u3068\u3044\u308f\u308c\u307e\u3059\u3002WGE\u306e\u53cd\u5fdc\u306f\u3001RRL\u3088\u308a\u3082\u9ad8\u3044\u30de\u30b0\u30cd\u30b7\u30a6\u30e0\u3001\u30ab\u30eb\u30b7\u30a6\u30e0\u6fc3\u5ea6\u3092\u5fc5\u8981\u3068\u3057\u307e\u3059\uff08\u793e\u5185\u30c7\u30fc\u30bf\uff09\u3002\u305d\u306e\u305f\u3081\u3001\u92f3\u578b\u3067\u3042\u308b\u74b0\u72b6DNA\u306f\u9ad8\u6b21\u69cb\u9020\uff08super coiled\uff09\u3092\u5f62\u6210\u3057\u3066\u3057\u307e\u3044\u3001\u305d\u306e\u7d50\u679c\u3068\u3057\u3066\u3001\u8ee2\u5199\u30fb\u7ffb\u8a33\u306e\u52b9\u7387\u304c\u4f4e\u4e0b\u3059\u308b\u53ef\u80fd\u6027\u304c\u3042\u308a\u307e\u3059\u3002<\/p>\n\n\n\n<p>\u53c2\u8003\u6587\u732e\uff1a<\/p>\n\n\n\n<ol start=\"5\"><li>Lancaster,J.M.et al.(1996)Nat.Genet.13, 238.<\/li><li>Sarkar,G.and Sommer,S.S.(1989)Science 244, 331.<\/li><li>Liu,B.et al.(1994)Cancer Res.54, 4590.<\/li><li>Papadopoulos,N.et al.(1994) Science 263,1625.<\/li><li>Roest,P.A.M.et al.(1993)Hum. Mol.Genet. 2, 1719.<\/li><li>van der Luijt,R.et al.(1994) Genomics 20, 1.<\/li><li>Hogervorst,F.B.L.et al.(1995) Nat.Genet.10, 208.<\/li><li>Lo Ten Foe,J.R.et al.(1996) Nat.Genet.14 ,320.<\/li><\/ol>\n","protected":false},"excerpt":{"rendered":"<p>\u92f3\u578bDNA\u306f\u3001\u74b0\u72b6DNA (Plasmid DNA)\u307e\u305f\u306f\u76f4\u9396 DNA (DNA fragment\u3001PCR\u7523\u7269)\u3092\u8abf\u88fd\u3057\u307e\u3059\u3002 1) \u74b0\u72b6 DNA \u3092\u4f7f\u7528\u3059\u308b\u5834\u5408 pTNT\u00ae&nbsp;Vector(L5610)\u307e\u305f\u306f\u3001 &#8230; <\/p>\n","protected":false},"author":16,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":[],"categories":[151],"tags":[],"_links":{"self":[{"href":"https:\/\/www.promega.co.jp\/resources\/faq\/wp-json\/wp\/v2\/posts\/1594"}],"collection":[{"href":"https:\/\/www.promega.co.jp\/resources\/faq\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.promega.co.jp\/resources\/faq\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.promega.co.jp\/resources\/faq\/wp-json\/wp\/v2\/users\/16"}],"replies":[{"embeddable":true,"href":"https:\/\/www.promega.co.jp\/resources\/faq\/wp-json\/wp\/v2\/comments?post=1594"}],"version-history":[{"count":1,"href":"https:\/\/www.promega.co.jp\/resources\/faq\/wp-json\/wp\/v2\/posts\/1594\/revisions"}],"predecessor-version":[{"id":1595,"href":"https:\/\/www.promega.co.jp\/resources\/faq\/wp-json\/wp\/v2\/posts\/1594\/revisions\/1595"}],"wp:attachment":[{"href":"https:\/\/www.promega.co.jp\/resources\/faq\/wp-json\/wp\/v2\/media?parent=1594"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.promega.co.jp\/resources\/faq\/wp-json\/wp\/v2\/categories?post=1594"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.promega.co.jp\/resources\/faq\/wp-json\/wp\/v2\/tags?post=1594"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}