{"id":1410,"date":"2021-05-24T11:13:59","date_gmt":"2021-05-24T02:13:59","guid":{"rendered":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/?p=1410"},"modified":"2021-05-24T11:13:59","modified_gmt":"2021-05-24T02:13:59","slug":"%e3%82%b8%e3%82%a7%e3%83%8e%e3%82%bf%e3%82%a4%e3%83%94%e3%83%b3%e3%82%b0%ef%bc%88%e4%b8%80%e3%83%8e%e7%80%ac%ef%bc%89","status":"publish","type":"post","link":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/?p=1410","title":{"rendered":"\u30b8\u30a7\u30ce\u30bf\u30a4\u30d4\u30f3\u30b0\uff08\u4e00\u30ce\u702c\uff09"},"content":{"rendered":"

\u8a66\u85ac
\n50mM NaOH
\nKOD FX Neo (TOYOBO) \u542b Buffer, dNTPs\u3000(\u2190KOD FX\u3067\u3082\u53ef)
\nAgarose LE (Wako)
\n50x TAE (Nacalai)
\nGene Ladder 100 (Nippon Gene)
\nGreenView Nucleic Acid Gel Stain (RELYON)<\/p>\n

\u30d7\u30ed\u30c8\u30b3\u30fc\u30eb
\n1.5mL\u30c1\u30e5\u30fc\u30d6\u30018\u9023PCR\u30c1\u30e5\u30fc\u30d6\u3069\u3061\u3089\u3067\u3082\u53ef
\nPCR\u53cd\u5fdc\u6642\u306fApplied BIosystems\u00ae MicroAmp\u00ae 8-Tube Strips\u7b49\u3092\u4f7f\u3046<\/p>\n

1. \u9069\u5207\u306a\u51e6\u7f6e\u306b\u3066\u7d44\u7e54\u7247\u3001\u30b9\u30ef\u30d6\u3092\u56de\u53ce\u3059\u308b
\n2. \u30b5\u30f3\u30d7\u30eb\u306b50mM NaOH 50\u03bcL\u3092\u52a0\u3048\u308b\uff08\u30b5\u30f3\u30d7\u30eb\u304c\u6d78\u3063\u3066\u3044\u308b\u3053\u3068\u3092\u78ba\u8a8d\uff09
\n3. 98\u2103 5min
\n4. \u5ba4\u6e29\u306b\u623b\u3059
\n5. \u30b9\u30d4\u30f3\u30c0\u30a6\u30f3
\n6. \u4e0a\u6e0510\u03bcL\u56de\u53ce\u3000(=Lysate)
\n\u2190\u30b5\u30f3\u30d7\u30eb\u306e\u5468\u56f2\u306fDNA\u304c\u58ca\u308c\u3066\u7c98\u6027\u304c\u3042\u308b\u306e\u3067\u5438\u308f\u306a\u3044\u3088\u3046\u306b\u6c17\u3092\u3064\u3051\u308b
\n7. Lysate\u4ee5\u5916\u3092\u6df7\u305c\u308b \uff08+10%\u76ee\u5b89\u3067\u4f5c\u6210\u3057\u3066\u5206\u6ce8\u3059\u308b\uff09
\n2x Buffer\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 10\u03bcL
\ndNTPs\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 4\u03bcL
\nPrimer(Fw)\u00a0 \u00a0 \u00a0 \u00a0 \u00a00.2\u03bcL
\nPrimer(Rv)\u00a0 \u00a0 \u00a0 \u00a0 \u00a00.2\u03bcL
\nKOD FX Neo\u00a0 \u00a0 \u00a0 \u00a00.4\u03bcL
\nDDW\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a03.2\u03bcL
\nTotal\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a018\u03bcL<\/p>\n

\u203bPrimer\u306e\u30da\u30a2
\n\u7cfb\u7d71\uff1a13, 65
\n\u2192 Fw: ex4-465F Rv: ex4-409R
\n\u7cfb\u7d71\uff1aep5
\n\u2192 Fw: ex29+564F Rv: ex29+871R<\/p>\n

8. 18\u03bcL\u5206\u6ce8
\n9. +2\u03bcL Lysate
\n10. \u3088\u304f\u6df7\u305c\u3066\u30b9\u30d4\u30f3\u30c0\u30a6\u30f3\u3057\u305f\u5f8c\u3001\u30b5\u30a4\u30af\u30e9\u30fc\u306b\u30bb\u30c3\u30c8
\n11. \u53cd\u5fdc\u6761\u4ef6
\nPre\u00a0 \u00a0 94\u2103\u00a0 \u00a02min
\nDen\u00a0 \u00a098\u2103\u00a0 \u00a010sec
\nAnn\u00a0 \u00a0xx\u2103\u00a0 \u00a030 sec
\nExt\u00a0 \u00a0 68\u2103\u00a0 \u00a0xx sec x30cycle
\nPost\u00a0 \u00a0 \u00a04\u2103\u00a0 \u221e
\n\u203b\u30d7\u30ed\u30b0\u30e9\u30e0\u4f5c\u6210\u6e08\u306a\u306e\u3067\u300113, 65\u306f\u300c1365\u300d, ep5\u306f\u300cep5\u300d\u3067PCR\u3059\u308c\u3070OK
\n12. \u30b2\u30eb\u4f5c\u6210
\n1.5\uff05\u30b2\u30eb
\n1x TAE\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a050 mL
\nAgarose LE\u00a0 \u00a0 \u00a0 \u00a00.75 g<\/p>\n

\u3092Bench Pint\u306b\u5165\u308c\u3066\u96fb\u5b50\u30ec\u30f3\u30b8\u3067\u52a0\u71b1
\n\uff08\u203b\u5439\u304d\u3053\u307c\u308c\u308b\u524d\u306b1\u56de\u6b62\u3081\u308b\uff09
\n13. + 5 \u03bcL GreenView Nucleic Acid Gel Stain
\n14. \u30c8\u30ec\u30a4\u306b\u6d41\u3057\u8fbc\u3080\u3001\u30b3\u30fc\u30e0(25 well)\u3092\u3055\u3059\u3001\u30b2\u30eb\u3092\u56fa\u3081\u308b
\n=====(\u56fa\u307e\u308b\u307e\u306720~30\u5206)=====
\n15. PCR\u53cd\u5fdc\u6db2 20 \u03bcL + 2\u03bcL Dye
\n16. \u30b2\u30eb\u306bapply \u3059\u308b 10\u03bcL\/well
\n17. \u7aef\u306bmarker\u3092apply\u3059\u308b5\u03bcL\/well
\n18. \u6cf3\u52d5 100V 25min\u3000(\u203b\u5fc5\u305a\u8a2d\u5b9a\u306e\u30c1\u30a7\u30c3\u30af\u3092\u884c\u3046\uff01\uff01\u7279\u306b\u96fb\u6d41\u306e\u5411\u304d)
\n19. \u7d50\u679c\u3092\u5224\u5b9a\u3059\u308b<\/p>\n

\"\"<\/a><\/p>\n

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

\u8a66\u85ac 50mM NaOH KOD FX Neo (TOYOBO) \u542b Buffer, dNTPs\u3000(\u2190KOD FX\u3067\u3082\u53ef) Agarose LE (Wako) 50x TAE (Nacalai) Gene Ladder <\/p>\n","protected":false},"author":6,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"inline_featured_image":false,"jetpack_post_was_ever_published":false,"_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":"","jetpack_publicize_message":"","jetpack_publicize_feature_enabled":true,"jetpack_social_post_already_shared":true,"jetpack_social_options":{"image_generator_settings":{"template":"highway","default_image_id":0,"font":"","enabled":false},"version":2}},"categories":[4],"tags":[],"class_list":["post-1410","post","type-post","status-publish","format-standard","hentry","category-molecular-biology"],"jetpack_publicize_connections":[],"jetpack_featured_media_url":"","jetpack_sharing_enabled":true,"jetpack_shortlink":"https:\/\/wp.me\/p4fFyn-mK","jetpack-related-posts":[],"_links":{"self":[{"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=\/wp\/v2\/posts\/1410","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=\/wp\/v2\/users\/6"}],"replies":[{"embeddable":true,"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=1410"}],"version-history":[{"count":5,"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=\/wp\/v2\/posts\/1410\/revisions"}],"predecessor-version":[{"id":1417,"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=\/wp\/v2\/posts\/1410\/revisions\/1417"}],"wp:attachment":[{"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=1410"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=1410"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/anatomy.med.gunma-u.ac.jp\/protocols\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=1410"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}