{"id":4501,"date":"2018-10-23T08:19:16","date_gmt":"2018-10-22T23:19:16","guid":{"rendered":"http:\/\/dna.brc.riken.jp\/ja\/?page_id=4501"},"modified":"2018-10-23T08:19:37","modified_gmt":"2018-10-22T23:19:37","slug":"technicalnotes06","status":"publish","type":"page","link":"http:\/\/dna.brc.riken.jp\/ja\/manual\/technicalnotes06","title":{"rendered":"\u5927\u8178\u83cc\u7d44\u63db\u3048\u86cb\u767d\u8cea\u306e\u8abf\u88fd"},"content":{"rendered":"

\u5927\u8178\u83cc\u7d44\u63db\u3048\u86cb\u767d\u8cea\u306e\u8abf\u88fd<\/h3>\n

\u25a0\u3000\u5927\u8178\u83cc\u3067\u7d44\u63db\u3048\u86cb\u767d\u8cea\u3092\u767a\u73fe\u3055\u305b\u308b\u306b\u306fBL21 (DE3)\u306a\u3044\u3057BL21 (DE3) pLysS \u3092\u7528\u3044\u307e\u3059\u3002\u89aa\u682a\u306eBL21 \u682a\u306fOmpT\u3068Lon protease\u907a\u4f1d\u5b50\u306e\u6b20\u640d\u682a\u3067\u3042\u308a\u3001\u7d44\u63db\u3048\u86cb\u767d\u8cea\u306e\u5206\u89e3\u3092\u6291\u3048\u3064\u3064\u767a\u73fe\u3055\u305b\u308b\u3053\u3068\u304c\u3067\u304d\u307e\u3059\u3002\u3053\u308c\u3089\u306fT7 RNA polymerase promoter \u3092\u6301\u3064\u767a\u73fe\u30d9\u30af\u30bf\u30fc\u3067\u306e\u7d44\u63db\u3048\u86cb\u767d\u8cea\u7523\u751f\u306b\u6709\u52b9\u3067\u3059\u3002T7 RNA polymerase promoter \u3067\u306f\u306a\u3044\u767a\u73fe\u30d9\u30af\u30bf\u30fc\u3067\u3082\u5229\u7528\u53ef\u80fd\u3067\u3059\u3002\u5229\u7528\u6761\u4ef6\u306f\u30d9\u30af\u30bf\u30fc\u4ed8\u968f\u306e\u60c5\u5831\u3092\u3054\u78ba\u8a8d\u304f\u3060\u3055\u3044\u3002
\n\u25a0\u3000\u7d44\u63db\u3048\u5927\u8178\u83cc\u306e\u8abf\u88fd
\n\u5927\u8178\u83cc\u56de\u53ce\u5f8c\u306f\u6c37\u4e0a\u3067\u64cd\u4f5c\u3057\u307e\u3059\u3002
\n1. \u5bd2\u5929\u30d7\u30ec\u30fc\u30c8\u4e0a\u306e\u5927\u8178\u83cc\u30b3\u30ed\u30cb\u30fc\u30922 mL\u306eLB (Mg)+50 ug\/mL\u30a2\u30f3\u30d4\u30b7\u30ea\u30f3\u57f9\u5730\u306b\u690d\u3048\u3001\u4e00\u6669\u57f9\u990a\u3059\u308b\u3002
\n2. \u305d\u306e\u57f9\u990a\u6db2200 uL \u309240 mL\u306eLB (Mg)+25 ug\/mL\u30a2\u30f3\u30d4\u30b7\u30ea\u30f3\u57f9\u5730\u306b\u690d\u3048\u308b\u3002\u3067\u304d\u308c\u3070\u679d\u4ed8\u304d\u30d5\u30e9\u30b9\u30b3\u3092\u4f7f\u7528\u3059\u308b\uff08\u30d5\u30bf\u3092\u958b\u3051\u308b\u3053\u3068\u306a\u304f\u57f9\u990a\u4e2d\u306e\u83cc\u6fc3\u5ea6\u3092\u6e2c\u5b9a\u3067\u304d\u308b\u306e\u3067\uff09\u3002
\n3. 30\u2103\u3001160 rpm\u3067\u9707\u76ea\u57f9\u990a\u3059\u308b\u3002
\n4. OD600\u304c\u304a\u304a\u3088\u305d0.2 \u306b\u306a\u3063\u305f\u3089400 uL\u306e0.1 M IPTG\u3092\u52a0\u3048\u308b\u3002
\n5. 30\u2103\u300190 rpm\u3067\u9707\u76ea\u57f9\u990a\u3059\u308b\u3002
\n6. 6\u6642\u9593\u5f8c\u3001\u57f9\u990a\u6db2\u309250 mL\u30c1\u30e5\u30fc\u30d6\u306b\u79fb\u3059\u3002
\n7. 3,000 rpm\u30014\u2103\u300110\u5206\u306e\u9060\u5fc3\u306b\u3088\u308a\u4e0a\u6e05\u3092\u5206\u96e2\u3059\u308b\u3002
\n8. \u6c88\u6fb1\u309210 mL\u306ePBS\u306b\u3051\u3093\u6fc1\u3059\u308b\u3002
\n9. 3,000 rpm\u30014\u2103\u300110\u5206\u306e\u9060\u5fc3\u306b\u3088\u308a\u4e0a\u6e05\u3092\u5206\u96e2\u3059\u308b\u3002
\n10. \u6c88\u6fb1\u30920.6 mL\u306eTPBS\u306b\u3051\u3093\u6fc1\u3059\u308b\u3002
\n11. \u3051\u3093\u6fc1\u6db2\u30921.5 mL\u30c1\u30e5\u30fc\u30d6\u306b\u79fb\u3059\u3002
\n12. -80\u2103\u3067\u4fdd\u5b58\u3059\u308b\u3002
\n\u25a0\u3000\u7d44\u63db\u3048\u5927\u8178\u83cc\u306e\u7834\u7815\u7269\u306e\u8abf\u88fd
\n\u4fdd\u5b58\u307e\u3067\u306f\u6c37\u4e0a\u3067\u64cd\u4f5c\u3057\u307e\u3059\u3002
\n1. \u4fdd\u5b58\u3057\u305f\u5927\u8178\u83cc\u3051\u3093\u6fc1\u6db2\u306b\u6b21\u306e\u3082\u306e\u3092\u52a0\u3048\u308b\u3002
\n0.5 uL 1 mg\/mL leupeptin
\n0.5 uL 1 mg\/mL pepstatin A
\n10 uL 100 mM PMSF
\n2. \uff3b30\u79d2\uff0f30\u79d2\uff3d\u30a4\u30f3\u30bf\u30fc\u30d0\u30eb\uff13\u30b5\u30a4\u30af\u30eb\u306e\u8d85\u97f3\u6ce2\u306b\u3088\u308a\u5927\u8178\u83cc\u3092\u7834\u7815\u3059\u308b\u3002
\n3. 15,000 rpm\u30014\u2103\u300110\u5206\u306e\u9060\u5fc3\u306b\u3088\u308a\u4e0a\u6e05\u3092\u5206\u96e2\u3059\u308b\u3002
\n4. \u4e0a\u6e05\u3092130 uL\u305a\u30641.5 mL\u30c1\u30e5\u30fc\u30d6\u306b\u53d6\u308a\u5206\u3051\uff085\u672c\uff09\u3001-80\u2103\u4fdd\u5b58\u3059\u308b\u3002
\n5. \u6b8b\u3063\u3066\u3044\u308b\u4e0a\u6e05\u306e20 uL\u30921.5 mL\u30c1\u30e5\u30fc\u30d6\u306b\u53d6\u308a\u5206\u3051\u300120 uL\u306e2x Laemmli sample buffer\u3092\u52a0\u3048\u308b\u3002
\n6. 95\u2103\u30015\u5206\u9593\u3001\u4fdd\u6e29\u3057\u3001\u6c37\u4e0a\u3067\u51b7\u5374\u3059\u308b\u3002
\n7. \u305d\u306e2 uL\u309210%\u30a2\u30af\u30ea\u30eb\u30a2\u30df\u30c9SDS-PAGE\u3067\u96fb\u6c17\u6cf3\u52d5\u3059\u308b\uff08\u30b2\u30eb2\u679a\u5206\uff09\u3002
\n8. \u4e00\u65b9\u306e\u30b2\u30eb\u306fCBB\u67d3\u8272\u306b\u3088\u308a\u89e3\u6790\u3059\u308b\u3002
\n9. \u3082\u3046\u4e00\u65b9\u306e\u30b2\u30eb\u304b\u3089\u86cb\u767d\u8cea\u3092PVDF\u819c\u306b\u8ee2\u5199\u3059\u308b\u3002\u30bb\u30df\u30c9\u30e9\u30a4\u6cd5\u30921.5 mA\/cm2\u306e\u96fb\u6d41\u30671\u6642\u9593\u884c\u3046\u3002
\n10. \u86cb\u767d\u8cea\u3092\u8ee2\u5199\u3057\u305fPVDF\u819c\u3092\u30a6\u30a8\u30b9\u30bf\u30f3\u30d6\u30ed\u30c3\u30c6\u30a3\u30f3\u30b0\u3067\u89e3\u6790\u3059\u308b\u3002
\n\u25a0\u3000\u4e0a\u8a18\u306e\u624b\u9806\u306f\u3001\u7528\u3044\u308b\u6a5f\u5668\u3001\u907a\u4f1d\u5b50\u767a\u73fe\u30d9\u30af\u30bf\u30fc\u3001\u30bf\u30b0\u306e\u7a2e\u985e\u3042\u308b\u3044\u306f\u7528\u3044\u308b\u8a66\u85ac\u306e\u30ed\u30c3\u30c8\u7b49\u306b\u3088\u3063\u3066\u6761\u4ef6\u304c\u7570\u306a\u308b\u5834\u5408\u304c\u3042\u308a\u307e\u3059\u3002\u57f9\u990a\u6642\u9593\u3001\u6e29\u5ea6\u3084IPTG\u6dfb\u52a0\u91cf\u306f\u3001\u76ee\u7684\u7d44\u63db\u3048\u86cb\u767d\u8cea\u306e\u53ce\u91cf\u3084\u5206\u89e3\u5ea6\u3092\u76ee\u5b89\u306b\u691c\u8a0e\u3057\u3066\u4e0b\u3055\u3044\u3002\u8d85\u97f3\u6ce2\u7834\u7815\u306f\u6eb6\u6db2\u306e\u900f\u660e\u5ea6\u3092\u76ee\u5b89\u306b\u3057\u3066\u4e0b\u3055\u3044\u3002<\/p>\n

\nReagents<\/span><\/p>\n\n\n\n
TPBS<\/span><\/td>\n1% triton X-100 in 1x PBS. <\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n\n\n\n\n
LB (Mg)<\/span><\/td>\n1 L<\/span><\/td>\n<\/tr>\n
MgSO4-7H2O<\/td>\n2 g<\/td>\n<\/tr>\n
NaCl<\/td>\n5 g<\/td>\n<\/tr>\n
Bacto yeast extract<\/td>\n5 g<\/td>\n<\/tr>\n
Bacto trypsin<\/td>\n10 g
\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n\n\n\n\n\n
2x Laemmli sample buffer<\/span><\/td>\nfinal<\/td>\n5.0 mL<\/span><\/td>\n<\/tr>\n
10% SDS<\/td>\n2%<\/td>\n1.0 mL<\/td>\n<\/tr>\n
0.5 M tris pH 6.8<\/td>\n60 mM<\/td>\n0.6 mL<\/td>\n<\/tr>\n
80% glycerol<\/td>\n10%<\/td>\n625 uL<\/td>\n<\/tr>\n
1% BPB<\/td>\n0.01%<\/td>\n50 uL<\/td>\n<\/tr>\n
2-ME<\/td>\n0.7 M<\/td>\n250 uL<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n\n\n\n
10x Laemmli EP buffer<\/td>\n1 L<\/span><\/td>\n<\/tr>\n
glycine<\/td>\n144.2 g<\/td>\n<\/tr>\n
tris-base<\/td>\n30.2 g<\/td>\n<\/tr>\n
SDS<\/td>\n10.0 g<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n\n\n\n\n
Transfer buffer<\/span><\/td>\nfinal<\/td>\n1L<\/span><\/td>\n<\/tr>\n
glycine<\/td>\n192 mM<\/td>\n14.4 g<\/td>\n<\/tr>\n
tris-base<\/td>\n25.2 mM<\/td>\n3.05 g<\/td>\n<\/tr>\n
methanol (optional)<\/td>\n20%<\/td>\n200 mL<\/td>\n<\/tr>\n
SDS (optional)<\/td>\n0.05%<\/td>\n0.5 g<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 
\n(2006.01.30 T.M.)<\/p>\n","protected":false},"excerpt":{"rendered":"

\u5927\u8178\u83cc\u7d44\u63db\u3048\u86cb\u767d\u8cea\u306e\u8abf\u88fd \u25a0\u3000\u5927\u8178\u83cc\u3067\u7d44\u63db\u3048\u86cb\u767d\u8cea\u3092\u767a\u73fe\u3055\u305b\u308b\u306b\u306fBL21 (DE3)\u306a\u3044\u3057BL21 (DE3) pLysS \u3092\u7528\u3044\u307e\u3059\u3002\u89aa\u682a\u306eBL21 \u682a\u306fOmpT\u3068Lon protease\u907a\u4f1d\u5b50\u306e\u6b20\u640d\u682a\u3067\u3042\u308a\u3001\u7d44\u63db\u3048\u86cb\u767d\u8cea\u306e\u5206\u89e3\u3092\u6291\u3048\u3064\u3064\u767a\u73fe\u3055\u305b\u308b\u3053\u3068\u304c\u3067\u304d\u307e\u3059\u3002\u3053\u308c\u3089\u306fT7 RNA polymerase promoter \u3092\u6301\u3064\u767a\u73fe\u30d9\u30af\u30bf\u30fc\u3067\u306e\u7d44\u63db\u3048\u86cb\u767d\u8cea\u7523\u751f\u306b\u6709\u52b9\u3067\u3059\u3002T7 RNA pol […]<\/p>\n","protected":false},"author":13,"featured_media":0,"parent":296,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_seopress_titles_title":"","_seopress_titles_desc":"","_seopress_robots_index":"","_seopress_robots_follow":"","_seopress_robots_imageindex":"","_seopress_robots_snippet":"","_seopress_robots_primary_cat":"","_seopress_robots_breadcrumbs":"","_seopress_robots_freeze_modified_date":"","_seopress_robots_custom_modified_date":"","_seopress_robots_canonical":"","_seopress_social_fb_title":"","_seopress_social_fb_desc":"","_seopress_social_fb_img":"","_seopress_social_fb_img_attachment_id":0,"_seopress_social_fb_img_width":0,"_seopress_social_fb_img_height":0,"_seopress_social_twitter_title":"","_seopress_social_twitter_desc":"","_seopress_social_twitter_img":"","_seopress_social_twitter_img_attachment_id":0,"_seopress_social_twitter_img_width":0,"_seopress_social_twitter_img_height":0,"_seopress_redirections_value":"","_seopress_redirections_enabled":"","_seopress_redirections_enabled_regex":"","_seopress_redirections_logged_status":"","_seopress_redirections_param":"","_seopress_redirections_type":0,"_seopress_analysis_target_kw":"","footnotes":"","_wp_rev_ctl_limit":""},"class_list":["post-4501","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/4501","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/users\/13"}],"replies":[{"embeddable":true,"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/comments?post=4501"}],"version-history":[{"count":2,"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/4501\/revisions"}],"predecessor-version":[{"id":4503,"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/4501\/revisions\/4503"}],"up":[{"embeddable":true,"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/296"}],"wp:attachment":[{"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/media?parent=4501"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}