{"id":9813,"date":"2016-09-01T10:12:43","date_gmt":"2016-09-01T01:12:43","guid":{"rendered":"http:\/\/cell.brc.riken.jp\/ja\/?page_id=9813"},"modified":"2024-10-29T09:21:45","modified_gmt":"2024-10-29T00:21:45","slug":"myco_nested_1","status":"publish","type":"page","link":"http:\/\/cell.brc.riken.jp\/ja\/quality\/myco_kensa\/myco_nested_1","title":{"rendered":"Nested-PCR\u306b\u3088\u308b\u30de\u30a4\u30b3\u30d7\u30e9\u30ba\u30de\u691c\u51fa\u65b9\u6cd5"},"content":{"rendered":"

1. \u6e96\u5099\u3059\u308b\u3082\u306e<\/strong><\/p>\n

(1) \u691c\u4f53
\n\u30fb\u30b5\u30f3\u30d7\u30eb\uff1a\u57f9\u990a\u4e0a\u6e05\u3092\u7cbe\u88fd\u3057\u305fDNA
\n\u30fb\u9670\u6027\u5bfe\u7167\uff1aPCR mixture\u306b\u7528\u3044\u305f\u7cbe\u88fd\u6c34
\n\u30fb\u967d\u6027\u5bfe\u71671\uff1a\u30de\u30a4\u30b3\u30d7\u30e9\u30ba\u30de\u6c5a\u67d3\u7d30\u80de\u306e\u57f9\u990a\u4e0a\u6e05\u3092\u7cbe\u88fd\u3057\u305fDNA
\n\u30fb\u967d\u6027\u5bfe\u71672\uff1a\u30de\u30a4\u30b3\u30d7\u30e9\u30ba\u30de\u30b2\u30ce\u30e0DNA<\/p>\n

(2)\u6a5f\u5668
\n\u30fbPCR Thermal Cycler
\n(Applied Biosystems®<\/sup> GeneAmp®<\/sup> PCR System 9700, Ramp speed: Max mode)
\n(Applied Biosystems®<\/sup> Veriti®<\/sup> Thermal Cycler)<\/p>\n

(3)PCR\u30d7\u30e9\u30a4\u30de\u30fc
\nPCR \u30d7\u30e9\u30a4\u30de\u30fc \uff084\u7a2e\u985e\uff09
\n1st step forward primer<\/p>\n

\n
\n
MCGpF11\n<\/div>\n
5\u2019-ACACCATGGGAG(C\/T)TGGTAAT-3\u2019<\/tt>\n<\/div>\n<\/div>\n<\/div>\n

1st step reverse primer<\/p>\n

\n
\n
MCGpR1\n<\/div>\n
5\u2019-CTTC(A\/T)TCGACTT(C\/T)CAGACCCAAGGCAT-3\u2019<\/tt>\n<\/div>\n<\/div>\n<\/div>\n

2nd step forward primer<\/p>\n

\n
\n
R16-2\n<\/div>\n
5\u2019-GTG(C\/G)GG(A\/C)TGGATCACCTCCT-3\u2019<\/tt>\n<\/div>\n<\/div>\n<\/div>\n

2nd step reverse primer<\/p>\n

\n
\n
MCGpR21\n<\/div>\n
5\u2019-GCATCCACCA(A\/T)A(A\/T)AC(C\/T)CTT-3\u2019<\/tt>\n<\/div>\n<\/div>\n<\/div>\n

\u203b()\u66f8\u304d\u306fMix\u5869\u57fa\u3068\u3057\u3066\u5408\u6210\u3059\u308b\u3002<\/p>\n

2.\u65b9\u6cd5<\/strong>
\n(1)1st-step PCR
\n\u30101st step PCR mixture\u3011<\/p>\n\n\n\n\n\n\n\n\n\n\n\n
DDW<\/td>\n12.9 \u03bcL<\/td>\n<\/tr>\n
10\u00d7AmpliTaq Gold\u00ae 360 Buffer<\/td>\n2.0 \u03bcL<\/td>\n<\/tr>\n
10\u00d7dNTP Mixture (2.5 mM each)<\/td>\n1.6 \u03bcL<\/td>\n<\/tr>\n
25 mM MgCl2<\/sub><\/td>\n1.6 \u03bcL<\/td>\n<\/tr>\n
10\u03bcM (10 pmol\/\u03bcL) MCGpF11<\/td>\n0.4 \u03bcL<\/td>\n<\/tr>\n
10\u03bcM (10 pmol\/\u03bcL) MCGpR1<\/td>\n0.4 \u03bcL<\/td>\n<\/tr>\n
AmpliTaq Gold®<\/sup> 360 DNA Polymerase<\/td>\n0.1 \u03bcL<\/td>\n<\/tr>\n
\n
\n<\/td>\n<\/tr>\n
Total<\/td>\n19.0 \u03bcL \/ sample<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

1) 1\u306e(1)\u3067\u6e96\u5099\u3057\u305f\u691c\u4f53\uff08DNA\u7cbe\u88fd\u30b5\u30f3\u30d7\u30eb\u3001\u9670\u6027\u30fb\u967d\u6027\u5bfe\u7167\uff09\u30921\u03bcL\u305a\u3064\u52a0\u3048\u308b\u3002
\n2) PCR program
\n\u3000\u3000Stage1 : 95\u2103\u300010\u5206\uff081\u30b5\u30a4\u30af\u30eb\uff09
\n\u3000\u3000Stage2 : 95\u2103\u300030\u79d2\u3000\u2192\u300055\u2103\u30002\u5206\u3000\u2192\u300072\u2103\u30002\u5206\uff0830\u30b5\u30a4\u30af\u30eb\uff09
\n\u3000\u3000Stage3 : 72\u2103\u30005\u5206\uff081\u30b5\u30a4\u30af\u30eb\uff09
\n\u3000\u3000Stage4 : 4\u2103\u3000\uff08\u4fdd\u5b58\uff09<\/p>\n

(2)2nd-step PCR
\n\u30102st step PCR mixture\u3011<\/p>\n\n\n\n\n\n\n\n\n\n\n\n
DDW<\/td>\n12.9 \u03bcL<\/td>\n<\/tr>\n
10\u00d7AmpliTaq Gold®<\/sup> 360 Buffer<\/td>\n2.0 \u03bcL<\/td>\n<\/tr>\n
10\u00d7dNTP Mixture (2.5 mM each)<\/td>\n1.6 \u03bcL<\/td>\n<\/tr>\n
25 mM MgCl2<\/sub><\/td>\n1.6 \u03bcL<\/td>\n<\/tr>\n
10\u03bcM (10 pmol\/\u03bcL) R16-2<\/td>\n0.4 \u03bcL<\/td>\n<\/tr>\n
10\u03bcM (10 pmol\/\u03bcL) MCGpR21<\/td>\n0.4 \u03bcL<\/td>\n<\/tr>\n
AmpliTaq Gold®<\/sup> 360 DNA Polymerase<\/td>\n0.1 \u03bcL<\/td>\n<\/tr>\n
\n
\n<\/td>\n<\/tr>\n
Total<\/td>\n19.0 \u03bcL \/ sample<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u30001) 1st-step PCR\u7523\u7269\u30921\u03bcL\u305a\u3064\u52a0\u3048\u308b\u3002
\n\u30002)(1)2)\u3068\u540c\u3058\u30d7\u30ed\u30b0\u30e9\u30e0\u3092\u5b9f\u884c\u3059\u308b\u3002<\/p>\n

(3)\u96fb\u6c17\u6cf3\u52d5\uff082%\u30a2\u30ac\u30ed\u30fc\u30b9\u30b2\u30eb\u3092\u4f7f\u7528\uff09
\n<\/a><\/p>\n

3.\u5224\u5b9a<\/strong>
\n\u3000\u9670\u6027\u5bfe\u7167\u306b\u30d0\u30f3\u30c9\u304c\u306a\u3044\u3053\u3068\u3001\u967d\u6027\u5bfe\u7167\uff08\u57f9\u990a\u4e0a\u6e05\uff09\u3001\u967d\u6027\u5bfe\u7167\uff08\u30b2\u30ce\u30e0DNA\uff09\u306b\u304a\u3044\u3066\u3001200\uff5e750 bp\u306ePCR\u7523\u7269\u306e\u30d0\u30f3\u30c9\u304c\u3042\u308b\u3053\u3068\u3092\u78ba\u8a8d\u5f8c\u3001\u691c\u4f53\u306e\u5224\u5b9a\u3092\u3059\u308b\u3002
\n\u3000\u9670\u6027\uff1a\u30d0\u30f3\u30c9\u304c\u306a\u3044
\n\u3000\u967d\u6027\uff1a200\uff5e750 bp\u306ePCR\u7523\u7269\u306e\u30d0\u30f3\u30c9\u304c\u3042\u308b<\/p>\n","protected":false},"excerpt":{"rendered":"

1. \u6e96\u5099\u3059\u308b\u3082\u306e (1) \u691c\u4f53 \u30fb\u30b5\u30f3\u30d7\u30eb\uff1a\u57f9\u990a\u4e0a\u6e05\u3092\u7cbe\u88fd\u3057\u305fDNA \u30fb\u9670\u6027\u5bfe\u7167\uff1aPCR mixture\u306b\u7528\u3044\u305f\u7cbe\u88fd\u6c34 \u30fb\u967d\u6027\u5bfe\u71671\uff1a\u30de\u30a4\u30b3\u30d7\u30e9\u30ba\u30de\u6c5a\u67d3\u7d30\u80de\u306e\u57f9\u990a\u4e0a\u6e05\u3092\u7cbe\u88fd\u3057\u305fDNA \u30fb\u967d\u6027\u5bfe\u71672\uff1a\u30de\u30a4\u30b3\u30d7\u30e9\u30ba\u30de\u30b2\u30ce\u30e0DNA (2)\u6a5f\u5668 \u30fbPCR Thermal Cycler (Applied Biosystems® GeneAmp® PCR System 9700, Ramp […]<\/p>\n","protected":false},"author":14,"featured_media":0,"parent":3498,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_seopress_robots_primary_cat":"","_seopress_titles_title":"","_seopress_titles_desc":"","_seopress_robots_index":"","_mc_calendar":[],"footnotes":"","_wp_rev_ctl_limit":""},"class_list":["post-9813","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/9813","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/users\/14"}],"replies":[{"embeddable":true,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/comments?post=9813"}],"version-history":[{"count":2,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/9813\/revisions"}],"predecessor-version":[{"id":14583,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/9813\/revisions\/14583"}],"up":[{"embeddable":true,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/3498"}],"wp:attachment":[{"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/media?parent=9813"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}