{"id":3566,"date":"2013-04-11T11:36:25","date_gmt":"2013-04-11T02:36:25","guid":{"rendered":"http:\/\/cell.brc.riken.jp:7321\/?page_id=3566"},"modified":"2014-02-13T10:24:55","modified_gmt":"2014-02-13T01:24:55","slug":"htlv_1","status":"publish","type":"page","link":"http:\/\/cell.brc.riken.jp\/ja\/quality\/htlv_1","title":{"rendered":"HTLV-I\u691c\u67fb"},"content":{"rendered":"

\u30ec\u30c8\u30ed\u30a6\u30a4\u30eb\u30b9\u306e\u4e00\u7a2e\u3067\u3042\u308bHTLV-I\uff08\u6210\u4ebaT\u7d30\u80de\u767d\u8840\u75c5\u30a6\u30a4\u30eb\u30b9\uff09\u306f\u3001\u6210\u4ebaT\u7d30\u80de\u767d\u8840\u75c5\uff08adult T-cell leukemia, ATL\uff09\u306e\u539f\u56e0\u30a6\u30a4\u30eb\u30b9\u3067\u3042\u308b\u3002 HTLV-I\u306eX\u907a\u4f1d\u5b50\u3092\u6a19\u7684\u3068\u3057\u305fprimer\u3092\u7528\u3044\u3066\u3001\u57f9\u990a\u7d30\u80de\u30b2\u30ce\u30e0\u306b\u30a4\u30f3\u30c6\u30b0\u30ec\u30fc\u30c8\u3055\u308c\u305fHTLV-I\u30d7\u30ed\u30a6\u30a4\u30eb\u30b9\u3092 PCR\u306b\u3088\u3063\u3066\u691c\u51fa\u3059\u308b\u3002\u8840\u6db2\u7531\u6765\u306e\u30d2\u30c8\u7d30\u80de\u682a\u306b\u3064\u3044\u3066\u5b9f\u65bd\u3057\u3066\u3044\u308b\u3002<\/p>\n

A. PCR Primer<\/p>\n

HTLV-I X\u3000forward 5\u2019-CGGATACCCAGTCTACGTGT-3\u2019<\/p>\n

HTLV-I X\u3000reverse 5\u2019-GAGCCGATAACGCGTCCATCG-3\u2019<\/p>\n

\u5185\u90e8\u30b3\u30f3\u30c8\u30ed\u30fc\u30eb<\/p>\n

\u03b2-globin forward 5\u2019-GGTTGGCCAATCTACTCCCAGG-3\u2019<\/p>\n

\u03b2-globin reverse 5\u2019-TGGTCTCCTTAAACCTGTCTTG-3\u2019<\/p>\n

\n

B. \u30b2\u30ce\u30e0DNA\u62bd\u51fa<\/p>\n

\u304a\u3088\u305d6cm dish 1\u679a\u5206\uff082\u00d7106<\/sup>\u500b\u7a0b\u5ea6\uff09\u306e\u7d30\u80de\u3092\u56de\u53ce\u3057\u3001PBS(-)\u3067\u6d17\u6d44\u3057\u305f\u7d30\u80de\u30da\u30ec\u30c3\u30c8\u304b\u3089DNeasy Blood & Tissue Kit(QIAGEN Cat#69506)\u3092\u7528\u3044\u3066\u30b2\u30ce\u30e0DNA\u3092\u62bd\u51fa\u3059\u308b\u3002<\/p>\n

 <\/p>\n

C. PCR<\/p>\n

\u62bd\u51fa\u5f8c\u306e\u6fc3\u5ea6\u6e2c\u5b9a\u306730\u03bcg\/\u03bcl\u4ee5\u4e0a\u3067\u3042\u3063\u305f\u30b2\u30ce\u30e0DNA\u306f10\u500d\u5e0c\u91c8\u3057\u3066\u7528\u3044\u308b\u3002<\/p>\n

\u30fbPCR mixture<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
<\/td>\n<\/td>\n1 sample\u3042\u305f\u308a\u306e\u6db2\u91cf<\/td>\n<\/tr>\n
<\/td>\nTemplate DNA<\/td>\n5.0\u03bcl<\/td>\n<\/tr>\n
<\/td>\nPrimer HTLV-I X FW (10pmol\/\u03bcl)<\/td>\n0.5\u03bcl<\/td>\n<\/tr>\n
<\/td>\nPrimer HTLV-I X RV (10pmol\/\u03bcl)<\/td>\n0.5\u03bcl<\/td>\n<\/tr>\n
<\/td>\nPrimer \u0392-globin FW (10pmol\/\u03bcl)<\/td>\n0.5\u03bcl<\/td>\n<\/tr>\n
<\/td>\nPrimer \u0392-globin RV (10pmol\/\u03bcl)<\/td>\n0.5\u03bcl<\/td>\n<\/tr>\n
<\/td>\n10\u00d7Ex Taq Buffer<\/td>\n2.5\u03bcl<\/td>\n<\/tr>\n
<\/td>\n2.5mM each dNTP mix<\/td>\n2.0\u03bcl<\/td>\n<\/tr>\n
<\/td>\nDDW<\/td>\n13.25\u03bcl<\/td>\n<\/tr>\n
<\/td>\nEx Taq Hot Start Version<\/td>\n0.25\u03bcl<\/td>\n<\/tr>\n
<\/td>\n\n
\n<\/td>\n<\/tr>\n
<\/td>\nTotal volume<\/td>\n25.0\u03bcl<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

\u30fbCycle\u8a2d\u5b9a<\/p>\n\n\n\n\n\n\n\n\n\n\n
<\/td>\n94\u2103<\/td>\n5min<\/td>\n<\/td>\n<\/tr>\n
<\/td>\n<\/td>\n<\/tr>\n
<\/td>\n94\u2103<\/td>\n10sec<\/td>\n\"kakko\"<\/td>\nfor 40 cycles<\/td>\n<\/tr>\n
<\/td>\n60\u2103<\/td>\n1sec<\/td>\n<\/tr>\n
<\/td>\n72\u2103<\/td>\n15sec<\/td>\n<\/tr>\n
<\/td>\n<\/td>\n<\/tr>\n
<\/td>\n72\u2103<\/td>\n5min<\/td>\n<\/td>\n<\/tr>\n
<\/td>\n4\u2103<\/td>\nsoak<\/td>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

D. \u00a0\u30a2\u30ac\u30ed\u30fc\u30b9\u30b2\u30eb\u96fb\u6c17\u6cf3\u52d5<\/p>\n

PCR\u5897\u5e45\u30b5\u30f3\u30d7\u30eb10\u03bcl\u306b10\u00d7Loading Buffer\u30921\u03bcl\u52a0\u3048\u30013% NuSieve GTG\u30a2\u30ac\u30ed\u30fc\u30b9\u30b2\u30eb\u96fb\u6c17\u6cf3\u52d5\u3092\u884c\u306a\u3063\u3066\u3001EtBr\u67d3\u8272\u306b\u3088\u308aDNA\u306e\u30d0\u30f3\u30c9\u3092\u78ba\u8a8d\u3059\u308b\u3002<\/p>\n

\u6cf3\u52d5\u7d50\u679c \u4f8b
\n\"\u6cf3\u52d5\u7d50\u679c<\/p>\n\n\n\n\n\n\n\n
<\/td>\nLane No.<\/td>\n<\/td>\n<\/tr>\n
<\/td>\n1<\/td>\nSize Marker<\/span>
\n(1Kb Plus DNA Ladder\u3001Invitrogen)<\/td>\n<\/tr>\n
<\/td>\n2\uff5e10<\/td>\nSample<\/td>\n<\/tr>\n
<\/td>\n11<\/td>\nNegative Control<\/span><\/td>\n<\/tr>\n
<\/td>\n12<\/td>\nPositive Control<\/span><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n","protected":false},"excerpt":{"rendered":"

\u30ec\u30c8\u30ed\u30a6\u30a4\u30eb\u30b9\u306e\u4e00\u7a2e\u3067\u3042\u308bHTLV-I\uff08\u6210\u4ebaT\u7d30\u80de\u767d\u8840\u75c5\u30a6\u30a4\u30eb\u30b9\uff09\u306f\u3001\u6210\u4ebaT\u7d30\u80de\u767d\u8840\u75c5\uff08adult T-cell leukemia, ATL\uff09\u306e\u539f\u56e0\u30a6\u30a4\u30eb\u30b9\u3067\u3042\u308b\u3002 HTLV-I\u306eX\u907a\u4f1d\u5b50\u3092\u6a19\u7684\u3068\u3057\u305fprimer\u3092\u7528\u3044\u3066\u3001\u57f9\u990a\u7d30\u80de\u30b2\u30ce\u30e0\u306b\u30a4\u30f3\u30c6\u30b0\u30ec\u30fc\u30c8\u3055\u308c\u305fHTLV-I\u30d7\u30ed\u30a6\u30a4\u30eb\u30b9\u3092 PCR\u306b\u3088\u3063\u3066\u691c\u51fa\u3059\u308b\u3002\u8840\u6db2\u7531\u6765\u306e\u30d2\u30c8\u7d30\u80de\u682a\u306b\u3064\u3044\u3066\u5b9f\u65bd\u3057\u3066\u3044\u308b\u3002 A. PCR Primer HTLV-I X\u3000 […]<\/p>\n","protected":false},"author":8,"featured_media":0,"parent":3425,"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-3566","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/3566","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\/8"}],"replies":[{"embeddable":true,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/comments?post=3566"}],"version-history":[{"count":5,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/3566\/revisions"}],"predecessor-version":[{"id":6295,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/3566\/revisions\/6295"}],"up":[{"embeddable":true,"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/3425"}],"wp:attachment":[{"href":"http:\/\/cell.brc.riken.jp\/ja\/wp-json\/wp\/v2\/media?parent=3566"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}