{"id":2409,"date":"2011-08-25T14:00:56","date_gmt":"2011-08-25T05:00:56","guid":{"rendered":"http:\/\/dna.brc.riken.jp:8002\/?page_id=2409"},"modified":"2020-12-09T10:27:30","modified_gmt":"2020-12-09T01:27:30","slug":"manual_05_01","status":"publish","type":"page","link":"http:\/\/dna.brc.riken.jp\/ja\/manual\/manual_05_01","title":{"rendered":"BAC \u30af\u30ed\u30fc\u30f3\u306e\u53d6\u308a\u6271\u3044"},"content":{"rendered":"<h2>BAC \u30af\u30ed\u30fc\u30f3\u306e\u53d6\u308a\u6271\u3044<\/h2>\n<h3>BAC \u30af\u30ed\u30fc\u30f3\u306e\u691c\u7d22<\/h3>\n<ul>\n<li><a href=\"\/DataSheet\/GRP0002j\">BAC\u30d6\u30e9\u30a6\u30b6\u30fc\u3092\u7528\u3044\u308b<\/a>\u3053\u3068\u3067\u3001\u907a\u4f1d\u5b50\u306egene symbol\u3092\u30ad\u30fc\u30ef\u30fc\u30c9\u3068\u3057\u3066BAC\u30af\u30ed\u30fc\u30f3\u3092\u691c\u7d22\u3057\u3001\u30b2\u30ce\u30e0\u9818\u57df\u4e0a\u306eBAC\u30af\u30ed\u30fc\u30f3\u306e\u4f4d\u7f6e\u3092\u78ba\u8a8d\u3059\u308b\u3053\u3068\u304c\u3067\u304d\u307e\u3059\u3002<\/li>\n<\/ul>\n<h3>BAC \u30af\u30ed\u30fc\u30f3\u306e\u53d6\u308a\u6271\u3044<\/h3>\n<ul>\n<li>\u53d7\u3051\u53d6\u3063\u305fBAC\u30af\u30ed\u30fc\u30f3\u306e\u30d0\u30a4\u30a2\u30eb\u3092\u4e00\u6642\u4fdd\u5b58\u3057\u305f\u3044\u3002<br \/>\nBAC\u30af\u30ed\u30fc\u30f3\u306f\u3001\u5927\u8178\u83cc\u7d44\u63db\u3048\u4f53\u3092\u5bd2\u5929\u57f9\u5730\u306b\u7a7f\u523a\u3057\u3066\u57f9\u990a\u3057\u3066\u3044\u307e\u3059\u3002\u5ba4\u6e29\u3067\u8f38\u9001\u3057\u3066\u3044\u307e\u3059\u3002\u53d7\u3051\u53d6\u3063\u305f\u30d0\u30a4\u30a2\u30eb\u306f\u30014\u2103\u3067\u4e00\u6642\u4fdd\u5b58\u53ef\u80fd\u3067\u3059\u3002\u304a\u53d7\u3051\u53d6\u308a\u5f8c1\u9031\u9593\u4ee5\u5185\u3092\u76ee\u5b89\u306b\u30b0\u30ea\u30bb\u30ed\u30fc\u30eb\u30b9\u30c8\u30c3\u30af\u3092\u4f5c\u88fd\u3057\u3001\u4fdd\u5b58\u3057\u3066\u304f\u3060\u3055\u3044\u3002\u83cc\u306e\u6b7b\u6ec5\u306e\u6050\u308c\u304c\u3042\u308a\u307e\u3059\u306e\u3067\u3001\u51cd\u7d50\u4fdd\u5b58\u306f\u4e0d\u53ef\u3067\u3059\u3002<\/li>\n<li>Viability of recombinant <i>E.coli<\/i> in stab culture.<br \/>\n<img decoding=\"async\" src=\"https:\/\/dnaconda.riken.jp\/images\/manual_05_01.png\" alt=\"\" width=\"400\" \/><br \/>\nStab cultures of a recombinant <i>E.coli<\/i>, DH10B carrying a BAC clone of mouse MSM\/Ms strain (MSMg01-174K01), were stored at different temperatures. Total and only dead cells were stained thiazole orange (TO) and propidium iodide (PI) , respectively.<br \/>\nThe viability of the <i>E.coli<\/i> were counted by flow cytometer at indicated days. (Okubo et al., unpublished)<\/li>\n<\/ul>\n<ul>\n<li>\u30b9\u30bf\u30d6\u30d0\u30a4\u30a2\u30eb\u304b\u3089\u3069\u306e\u3088\u3046\u306bBAC\u30af\u30ed\u30fc\u30f3\u3092\u57f9\u990a\u3059\u308b\u306e\u304b\u3002<br \/>\n\u767d\u91d1\u8033\u3092\u7528\u3044\u30012x LB\u5bd2\u5929\u57f9\u5730\u306b\u5857\u308a\u5e83\u3052\u3066\u57f9\u990a\u3057\u3001\u305d\u306e\u30b7\u30f3\u30b0\u30eb\u30b3\u30ed\u30cb\u30fc\u304b\u30892x LB\u6db2\u4f53\u57f9\u5730\u3067\u57f9\u990a\u3092\u3057\u3066\u304f\u3060\u3055\u3044\u3002\u83cc\u4f53\u306f\u57f9\u5730\u306e\u4e2d\u307b\u3069\u306b\u3042\u308a\u307e\u3059\u306e\u3067\u3001\u767d\u91d1\u8033\u306f\u57f9\u5730\u306e\u4e2d\u307b\u3069\u307e\u3067\u7a81\u304d\u523a\u3057\u3066\u304f\u3060\u3055\u3044\u3002<\/li>\n<li>BAC\u30af\u30ed\u30fc\u30f3\u3092\u9577\u671f\u4fdd\u5b58\u3057\u305f\u3044\u3002<br \/>\n\u5f53\u5ba4\u3067\u306f\u3001\u30b7\u30f3\u30b0\u30eb\u30b3\u30ed\u30cb\u30fc\u304b\u30892x LB\u6db2\u4f53\u57f9\u5730\u306737\u2103 16\u6642\u9593\u57f9\u990a\u3057\u3001\u57f9\u990a\u6db2\u3068\u7b49\u91cf\u306e\u6ec5\u83cc\u6e08\u307f40%\u30b0\u30ea\u30bb\u30ed\u30fc\u30eb\u6eb6\u6db2\u3092\u52a0\u3048\u3066\u6df7\u548c\u3057\u3001-80\u2103\u3067\u4fdd\u5b58\u3057\u3066\u3044\u307e\u3059\u3002<\/li>\n<li>BAC\u30af\u30ed\u30fc\u30f3\u3067\u4f7f\u7528\u3057\u3066\u3044\u308b2x LB\u306e\u7d44\u6210\u3092\u77e5\u308a\u305f\u3044\u3002<br \/>\n\u5f53\u5ba4\u3067\u4f7f\u7528\u3057\u3066\u3044\u308b2x LB\u306e\u7d44\u6210\u306f\u30011 L\u7528\u3067\u306f<br \/>\nbacto tryptone 20 g<br \/>\nyeast extract 10 g<br \/>\nNaCl 10 g<br \/>\n\u3053\u308c\u3089\u3092\u6df7\u548c\u5f8c\u3001NaOH\u6eb6\u6db2\u3067pH 7.0\u306b\u8abf\u6574\u3057\u3001\u30aa\u30fc\u30c8\u30af\u30ec\u30fc\u30d6\u6ec5\u83cc\u3057\u3066\u3044\u307e\u3059\u3002<\/li>\n<\/ul>\n<h3>BAC DNA Preparation<\/h3>\n<ul>\n<li>BAC\u30af\u30ed\u30fc\u30f3\u304b\u3089\u3069\u306e\u304f\u3089\u3044\u306eDNA\u304c\u56de\u53ce\u3067\u304d\u308b\u306e\u304b<br \/>\n\u30b7\u30f3\u30b0\u30eb\u30b3\u30ed\u30cb\u30fc\u304b\u30892 mL\u306e2x LB\u6db2\u4f53\u57f9\u5730\u306737\u2103 16\u6642\u9593\u57f9\u990a\u3057\u305f\u5834\u5408\u3001\u6982\u306d200 ng\u306eDNA\u304c\u56de\u53ce\u3067\u304d\u3066\u3044\u307e\u3059\u3002<\/li>\n<\/ul>\n<ol>\n<li>Ideally spread BAC on a LB plate to isolate single clolonies. Pick a single colony of BAC into 5mL of 2x LB culture containing a selective antibiotic &#8211; incubate at 37&deg;C with shaking for 18-20 hrs.<\/li>\n<li>Collect the culture into a microtube, pellet the cells by centrifuge at 5,000 rpm for 5 mins in a bench top centrifuge, decant the supernatant.<\/li>\n<li>Resuspend the pellet in 300 uL of Qiagen solution P1 (cat no:19051) containing RNAaseA by pipetting &#8211; make sure that the cells are completely resuspended and no cell clumps!\n<ul>\n<li>Buffer P1<br \/>\n50 mM Tris-Cl, pH 8.0; 10 mM EDTA; 100 ug\/mL RNaseA<\/li>\n<\/ul>\n<\/li>\n<li>Add 300 uL of Qiagen solution P2 (cat:19052), mix by hand-inversion 4 or 5 times (DO NOT VORTEX), incubate a room temp for 5 mins.\n<ul>\n<li>Buffer P2<br \/>\n200 mM NaOH; 1% SDS (w\/v)<\/li>\n<\/ul>\n<\/li>\n<li>Add 300 uL of Qiagen solution P3 (cat 19053), seal the tubes and mix by inversion &#8211; leave on ice for 10 mins.\n<ul>\n<li>Buffer P3<br \/>\n3 M potassium acetate, pH 5.5<\/li>\n<\/ul>\n<\/li>\n<li>Centrifuge the tubes in a centrifuge at 15 krpm for 15 mins at 4&deg;C.<\/li>\n<li>Transfer the entire supernatant to a fresh tube and precipitate the DNA by adding 700 uL of isopropanol, mix and leave at room temperature for 5 mins.<\/li>\n<li>Centrifuge the tubes in a centrifuge at 15 krpm for 15 mins at 4&deg;C, carefully decant off the supernatant &#8211; white pellet should be visible. Wash with 70% EtOH then aspirate off ethanol. You do not need to remove all of the ethanol at this step, but you should minimize it.<\/li>\n<li>Add 50 uL of sterile TE (10mM Tris, pH 8, 1mM EDTA).<\/li>\n<li>Digest 8 uL of the DNA with 10 units of an appropriate restriction enzyme in 10 uL reaction mixture.<\/li>\n<li>Analyze the DNA by a pulsed-field gel electrophoresis.<\/li>\n<\/ol>\n<h2>Related Information<\/h2>\n<h3>Related Website<\/h3>\n<ul>\n<li><a href=\"https:\/\/bacpacresources.org\">BACPAC Resources Center, Children&#8217;s Hospital Oakland Research Institute [link]<\/a><\/li>\n<\/ul>\n<h3>Related Articles<\/h3>\n<ul>\n<li>Sugimoto, M., Kondo, M., Hirose, M., Suzuki, M., Mekada, K., Abe, T., Kiyonari, H,. Ogura, A., Takagi, N., Artzt, K., Abe, K. Molecular identification of t(w5): Vps52 promotes pluripotential cell differentiation through cell-cell interactions. Cell Rep., 2 (5): 1363-1374 (2012). PMID: <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/23142660\/\">23142660<\/a><\/li>\n<li>Yamaguchi, S., Niwa, R., Kazuki, Y., Ohbayashi, T. Application of a bacterial artificial chromosome modification system for a human artificial chromosome vector. <a href=\"https:\/\/repository.lib.tottori-u.ac.jp\/ja\/list\/cjtitle\/yam\/54\/1\/item\/3040\"> Yonago Acta medica 54:021-031 (2011).<\/a><\/li>\n<li>Gong, S., Kus, L., Heintz, N. Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis. Nat. Protoc., 5 (10), 1678-1696 (2010). PMID: <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/20885380\/\"> 20885380<\/a><\/li>\n<li>Abe, K., Noguchi, H., Tagawa, K., Yuzuriha, M., Toyoda, A., Kojima, T., Ezawa, K., Saitou, N., Hattori, M., Sakaki, Y., Moriwaki, K., Shiroishi, T. Contribution of Asian mouse subspecies Mus musculus molossinus to genomic constitution of strain C57BL\/6J, as defined by BAC-end sequence-SNP analysis. Genome Res., 14, 2439-2447 (2004). PMID: <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/15574823\/\">15574823<\/a><\/li>\n<\/ul>\n<h4>BAC\u30af\u30ed\u30fc\u30f3\u306e\u6587\u732e\u30ea\u30b9\u30c8<\/h4>\n<p>\u6587\u732e\u30ea\u30b9\u30c8\u306f\u3001\u5404BAC\u30af\u30ed\u30fc\u30f3\u306e\u30ab\u30bf\u30ed\u30b0\u30da\u30fc\u30b8\u3092\u53c2\u7167\u3057\u3066\u304f\u3060\u3055\u3044\u3002<\/p>\n<ul>\n<li><a href=\"\/DataSheet\/RDB07573\">B6N BAC [link]<\/a><\/li>\n<li><a href=\"\/DataSheet\/RDB04214\">MSM\/Ms BAC [link]<\/a><\/li>\n<li><a href=\"\/DataSheet\/RDB06273\">Rat F344\/Stm RNB1 BAC [link]<\/a><\/li>\n<li><a href=\"\/DataSheet\/RDB06273\">Rat LE\/Stm RNB2 BAC [link]<\/a><\/li>\n<li><a href=\"\/DataSheet\/RDB13444\">Chinese hamster BAC [link]<\/a><\/li>\n<li><a href=\"\/DataSheet\/RDB08484\">Drosophila melanogaster DME BAC [link]<\/a><\/li>\n<li><a href=\"\/DataSheet\/RDB08485\">Drosophila simulans DSM BAC [link]<\/a><\/li>\n<li><a href=\"\/DataSheet\/RDB08486\">Drosophila sechellia DSE BAC [link]<\/a><\/li>\n<li><a href=\"\/DataSheet\/RDB08487\">Drosophila ananassae DNB BAC [link]<\/a><\/li>\n<li><a href=\"\/DataSheet\/RDB08488\">Drosphila auraria DAB BAC [link]<\/a><\/li>\n<\/ul>\n<p style=\"text-align: right;\">(MAN0001j 2009.06.13 T.M.)<\/p>\n<p style=\"text-align: left;\">2020.11.15<\/p>\n","protected":false},"excerpt":{"rendered":"<p>BAC \u30af\u30ed\u30fc\u30f3\u306e\u53d6\u308a\u6271\u3044 BAC \u30af\u30ed\u30fc\u30f3\u306e\u691c\u7d22 BAC\u30d6\u30e9\u30a6\u30b6\u30fc\u3092\u7528\u3044\u308b\u3053\u3068\u3067\u3001\u907a\u4f1d\u5b50\u306egene symbol\u3092\u30ad\u30fc\u30ef\u30fc\u30c9\u3068\u3057\u3066BAC\u30af\u30ed\u30fc\u30f3\u3092\u691c\u7d22\u3057\u3001\u30b2\u30ce\u30e0\u9818\u57df\u4e0a\u306eBAC\u30af\u30ed\u30fc\u30f3\u306e\u4f4d\u7f6e\u3092\u78ba\u8a8d\u3059\u308b\u3053\u3068\u304c\u3067\u304d\u307e\u3059\u3002 BAC \u30af\u30ed\u30fc\u30f3\u306e\u53d6\u308a\u6271\u3044 \u53d7\u3051\u53d6\u3063\u305fBAC\u30af\u30ed\u30fc\u30f3\u306e\u30d0\u30a4\u30a2\u30eb\u3092\u4e00\u6642\u4fdd\u5b58\u3057\u305f\u3044\u3002 BAC\u30af\u30ed\u30fc\u30f3\u306f\u3001\u5927\u8178\u83cc\u7d44\u63db\u3048\u4f53\u3092\u5bd2\u5929\u57f9\u5730\u306b\u7a7f\u523a\u3057\u3066\u57f9\u990a\u3057\u3066\u3044\u307e\u3059\u3002\u5ba4\u6e29\u3067\u8f38\u9001\u3057\u3066\u3044\u307e\u3059\u3002\u53d7\u3051\u53d6\u3063\u305f [&hellip;]<\/p>\n","protected":false},"author":9,"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-2409","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/2409","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\/9"}],"replies":[{"embeddable":true,"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/comments?post=2409"}],"version-history":[{"count":8,"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/2409\/revisions"}],"predecessor-version":[{"id":5074,"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/2409\/revisions\/5074"}],"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=2409"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}