{"id":117,"date":"2015-08-03T13:04:15","date_gmt":"2015-08-03T04:04:15","guid":{"rendered":"http:\/\/www-p.cc.ocha.ac.jp\/bio-shimada-lab\/?page_id=117"},"modified":"2023-07-03T13:45:06","modified_gmt":"2023-07-03T04:45:06","slug":"pr_01baiyo","status":"publish","type":"page","link":"https:\/\/www-p.sci.ocha.ac.jp\/bio-shimada-lab\/ex_protocol\/pr_01baiyo.html","title":{"rendered":"\u57f9\u990a\u95a2\u4fc2"},"content":{"rendered":"<h2>\u4eba\u5de5\u6d77\u6c34(SF-1)\u306e\u4f5c\u308a\u65b9<\/h2>\n<p>\u30fb25L\u5bb9\u5668\u306b\u30af\u30e9\u30c3\u30b7\u30e5\u30a2\u30a4\u30b9\u3092\u5165\u308c10\u56de\u632f\u308a\u6c34\u9053\u6c34\u30673\u56de\u6d17\u6d44\u3067DW\u3092\u304b\u3051\u308b(\u6c5a\u308c\u304c\u3072\u3069\u3044\u5834\u5408\u2192\u5148\u751f)<br \/>\n\u30fbDW\u309222L\u7dda\u307e\u3067\u5165\u308c\u3001\u4eba\u5de5\u6d77\u6c34(SF-1)\u306e\u7c89\u3092\u3053\u307c\u3055\u306a\u3044\u3088\u3046\u306b1\u888b\u5165\u308c\u3001\u30a8\u30a2\u30ec\u30fc\u30b7\u30e7\u30f3\u3067\u6570\u65e5\u64b9\u62cc\u3057,\u6ffe\u904e\u5668\u3067\u6ffe\u904e\u3059\u308b<br \/>\n\u30fb1L\u30dc\u30c8\u30eb\u306b\u30af\u30e9\u30c3\u30b7\u30e5\u30a2\u30a4\u30b9\u3092\u5e95\u304b\u30893cm\u307b\u3069\u5165\u308c\u3001100\u56de\u307b\u3069\u4e0a\u4e0b\u5de6\u53f3 \u6c34\u9053\u6c34\u30673\u56de\u6d17\u6d44\u3057\u3001DW\u3092\u304b\u3051\u308b<br \/>\n\u30fb\u6d77\u6c34\u3092\u30dc\u30c8\u30eb\u306b\u5165\u308c\u3001\u30aa\u30fc\u30c8\u30af\u30ec\u30fc\u30d6(121\u210320\u5206)<\/p>\n<p>&nbsp;<\/p>\n<h2>PESI, PES\u306e\u4f5c\u308a\u65b9 1L\u306e\u5834\u5408<\/h2>\n<p style=\"font-weight: 400\"><strong>PESI medium 1L<\/strong><\/p>\n<p style=\"font-weight: 400\">\u30fb1L\u30d3\u30fc\u30ab\u30fc\u306bDW\u3092300mL\u5165\u308c\u3001\u30b9\u30bf\u30fc\u30e9\u30fc\u3092\u5165\u308c\u3001\u652a\u62cc\u6a5f\u3067\u56de\u3059<\/p>\n<p style=\"font-weight: 400\">\u30fb\u4ee5\u4e0b\u306e\u9806\u3067\u52a0\u3048\u308b<\/p>\n<p style=\"font-weight: 400\">Tris hydroxymethyl aminomethane \u2460&#8212;- 5.0 g<br \/>\nNaNO3\u00a0\u2461&#8212;&#8212;&#8211; 3.5 g<br \/>\nNa2-glycerophosphate \u2462&#8212;&#8212;&#8211; 500 mg<br \/>\nFe stock solution\u00a0\u00a0&#8212;&#8212;&#8212; 250 ml<br \/>\nP-2 metal mix\u00a0\u00a0&#8212;&#8212;&#8212;- 250 ml<br \/>\nKI stock solution\u00a0(0.1 mg \/ ml)\u00a0&#8212;&#8212; 10 ml<\/p>\n<p style=\"font-weight: 400\">\u30fbpH\u30927.8\u306b\u8abf\u6574\u2192DW 1000 ml\u306b\u30e1\u30b9\u30a2\u30c3\u30d7\u2192\u30aa\u30fc\u30c8\u30af\u30ec\u30fc\u30d6(121\u2103,20\u5206)\u21924 \u2103\u3067\u51b7\u8535\u4fdd\u5b58<\/p>\n<p style=\"font-weight: 400\"><strong>Fe stock solution 1L<\/strong><\/p>\n<p style=\"font-weight: 400\">\u30fb1L\u30d3\u30fc\u30ab\u30fc\u306bDW\u3092500mL\u5165\u308c\u3001\u30b9\u30bf\u30fc\u30e9\u30fc\u3092\u5165\u308c\u3001\u652a\u62cc\u6a5f\u3067\u56de\u3059<\/p>\n<p style=\"font-weight: 400\">\u30fb\u4ee5\u4e0b\u306e\u9806\u3067\u52a0\u3048\u308b<\/p>\n<p style=\"font-weight: 400\">Fe(NH4)2(SO4)2\u30fb6H2O \u2464&#8212;&#8212;-702 mg<\/p>\n<p style=\"font-weight: 400\">Na2-EDTA\u30fb2H2O \u2463&#8212;&#8212;- 660 mg<\/p>\n<p style=\"font-weight: 400\">\u30fbDW\u30671L\u306b\u30e1\u30b9\u30a2\u30c3\u30d7\u21924 \u2103\u3067\u51b7\u8535\u4fdd\u5b58<\/p>\n<p style=\"font-weight: 400\"><strong>PII Metal Mix 1L<\/strong><\/p>\n<p style=\"font-weight: 400\">\u30fb1L\u30d3\u30fc\u30ab\u30fc\u306bDW\u3092900mL\u5165\u308c\u3001\u30b9\u30bf\u30fc\u30e9\u30fc\u3092\u5165\u308c\u3001\u652a\u62cc\u6a5f\u3067\u56de\u3059<\/p>\n<p style=\"font-weight: 400\">\u30fb\u4ee5\u4e0b\u306e\u9806\u3067\u89e3\u3051\u305f\u3089\u52a0\u3048\u89e3\u3051\u305f\u3089\u52a0\u3048\u308b<\/p>\n<p style=\"font-weight: 400\">Na2-EDTA\u30fb2H2O \u2463&#8212;&#8212;&#8212;&#8212;1g<\/p>\n<p style=\"font-weight: 400\">H<sub>3<\/sub>BO<sub>3<\/sub> \u2465&#8212;&#8212;1.14g<\/p>\n<p style=\"font-weight: 400\">FeCl<sub>3<\/sub>\u30fb6H<sub>2<\/sub>O \u2466&#8212;&#8212;49mg<\/p>\n<p style=\"font-weight: 400\">MnCl<sub>2<\/sub>\u30fb4H<sub>2<\/sub>O \u2467&#8212;&#8212;164mg<\/p>\n<p style=\"font-weight: 400\">CoSO<sub>4<\/sub>\u30fb7H<sub>2<\/sub>O\u6c34\u6eb6\u6db2(1mg\/ml) \u2468&#8212;&#8212;4.8ml<\/p>\n<p style=\"font-weight: 400\">ZnSO<sub>4<\/sub>\u30fb7H<sub>2<\/sub>O\u6c34\u6eb6\u6db2(10mg\/ml) \u2469&#8212;&#8212;2.2m<\/p>\n<p style=\"font-weight: 400\">\u30fbDW\u30671L\u306b\u30e1\u30b9\u30a2\u30c3\u30d7\u21924 \u2103\u3067\u51b7\u8535\u4fdd\u5b58\u3002<\/p>\n<p style=\"font-weight: 400\"><strong>KI stock solution<\/strong><\/p>\n<p style=\"font-weight: 400\">\u30fb100ml DW\u306b20mg\u306eKI\u3092\u6eb6\u304b\u3057,200ml\u306b\u30e1\u30b9\u30a2\u30c3\u30d7\u21924 \u2103\u3067\u51b7\u8535\u4fdd\u5b58<\/p>\n<p style=\"font-weight: 400\">\u00a0<\/p>\n<p style=\"font-weight: 400\"><strong>PES medium<\/strong><\/p>\n<p style=\"font-weight: 400\">\u30fb1L\u30d3\u30fc\u30ab\u30fc\u306bDW\u3092300mL\u5165\u308c\u3001\u30b9\u30bf\u30fc\u30e9\u30fc\u3092\u5165\u308c\u3001\u652a\u62cc\u6a5f\u3067\u56de\u3059<\/p>\n<p style=\"font-weight: 400\">\u30fb\u4ee5\u4e0b\u306e\u9806\u3067\u52a0\u3048\u308b<\/p>\n<p style=\"font-weight: 400\">Tris hydroxymethyl aminomethane &#8212;&#8212;&#8211; 5.0 g<br \/>\nNaNO<sub>3<\/sub>\u00a0&#8212;&#8212;&#8211; 3.5 g<br \/>\nNa<sub>2<\/sub>-glycerophosphate &#8212;&#8212;&#8211; 500 mg<br \/>\nFe stock solution * &#8212;&#8212;&#8212; 250 ml<br \/>\nP-2 metal mix ** &#8212;&#8212;&#8212;- 250 ml<br \/>\nVitamine B<sub>12<\/sub>\u00a0stock solution (0.1 mg \/ ml) &#8212;- 1.0 ml<br \/>\nThiamine-HCl stock solution (1 mg \/ ml) &#8212;- 5.0 ml<br \/>\nBiotine stock solution (0.1 mg \/ ml) &#8212;- 0.5 ml<\/p>\n<p style=\"font-weight: 400\">\u30fbpH\u30927.8\u306b\u8abf\u6574\u2192DW 1000 ml\u306b\u30e1\u30b9\u30a2\u30c3\u30d7\u2192\u30aa\u30fc\u30c8\u30af\u30ec\u30fc\u30d6(121\u2103,20\u5206)\u21924 \u2103\u3067\u51b7\u8535\u4fdd\u5b58<\/p>\n<p>&nbsp;<\/p>\n<h4 style=\"text-align: left\">1L\u30dc\u30c8\u30eb<\/h4>\n<p style=\"text-align: left\">\u30dc\u30c8\u30eb\u306f\u500b\u4eba\u5c02\u7528\u3000\u30b7\u30fc\u30eb\u3092\u8cbc\u308b<\/p>\n<p style=\"text-align: left\">\u00a0<\/p>\n<hr \/>\n<h2 style=\"text-align: left\">\u6297\u751f\u7269\u8cea<\/h2>\n<p style=\"text-align: left\">\u30da\u30cb\u30b7\u30ea\u30f3G(\u30b7\u30de\u30c0\u7814DNA\u5b9f\u9a13\u5ba4\u51b7\u8535\u5eab) (\u30b0\u30e9\u30e0\u967d\u6027\u83cc\u30b0\u30e9\u30e0\u9670\u6027\u7403\u83cc\u306b\u5bfe\u3057\u3066\u306f\u5f37\u3044\u6297\u83cc\u4f5c\u7528\u3092\u793a\u3059\u304c\u3001\u30b0\u30e9\u30e0\u9670\u6027\u687f\u83cc\u306b\u306f\u52b9\u304b\u306a\u3044)\u771f\u6b63\u7d30\u83cc\u306e\u7d30\u80de\u58c1\u306e\u4e3b\u8981\u6210\u5206\u3067\u3042\u308b\u30da\u30d7\u30c1\u30c9\u30b0\u30ea\u30ab\u30f3\u3092\u5408\u6210\u3059\u308b\u9175\u7d20\u3068\u7d50\u5408\u3057\u3001\u305d\u306e\u6d3b\u6027\u3092\u963b\u5bb3\u3059\u308b<\/p>\n<p style=\"text-align: left\">\u30b9\u30c8\u30ec\u30d7\u30c8\u30de\u30a4\u30b7\u30f3(\u30b7\u30de\u30c0\u7814DNA\u5b9f\u9a13\u5ba4\u51b7\u8535\u5eab) (\u30b0\u30e9\u30e0\u967d\u6027\u83cc\u3001\u30b0\u30e9\u30e0\u9670\u6027\u83cc\u306e\u767a\u80b2\u3092\u963b\u6b62\u3059\u308b)\u7d30\u83cc\u306eDNA\u304b\u3089\u8ee2\u5199\u3055\u308c\u305f\u30e1\u30c3\u30bb\u30f3\u30b8\u30e3\u30fcRNA\u306e\u907a\u4f1d\u60c5\u5831\u3092\u8aa4\u8aad\u3055\u305b\u3066\u3001\u30bf\u30f3\u30d1\u30af\u8cea\u306e\u751f\u5408\u6210\u3092\u963b\u5bb3\u3059\u308b<\/p>\n<p style=\"text-align: left\">\u901a\u5e38\u306f1L\u6d77\u6c34\u306b\u5bfe\u3057\u3066\u540430mg\u3065\u3064\u5165\u308c\u308b<\/p>\n<p>Wako\u306e\u30da\u30cb\u30b7\u30ea\u30f3-\u30b9\u30c8\u30ec\u30d7\u30c8\u30de\u30a4\u30b7\u30f3\u6eb6\u6db2\u306f,1ml\u3042\u305f\u308a\u30da\u30cb\u30b7\u30ea\u30f316mg(10,000units),\u30b9\u30c8\u30ec\u30d7\u30c8\u30de\u30a4\u30b7\u30f310mg\u304c\u5165\u3063\u3066\u3044\u308b<\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p style=\"text-align: left\">\u73ea\u85fb\u9664\u53bb\u306b\u306f\u4e8c\u9178\u5316\u30b2\u30eb\u30de\u30cb\u30a6\u30e0\u3092\u4f7f\u7528\u3059\u308b<\/p>\n<p style=\"text-align: left\">\u73ea\u85fb\u304cSi\u306e\u4ee3\u308f\u308a\u306bGe\u3092\u53d6\u308a\u8fbc\u307f\u3001\u88ab\u6bbb\u304c\u3067\u304d\u306a\u304f\u306a\u308b<\/p>\n<p style=\"text-align: left\">1N NaOH 200mL(NaOH 8g\u3092200mlDW\u3067\u6eb6\u304b\u3059)\u306b\u4e8c\u9178\u5316\u30b2\u30eb\u30de\u30cb\u30a6\u30e0\u3092500mg\u5165\u308c\u3001\u30aa\u30fc\u30c8\u30af\u30ec\u30fc\u30d6(121\u210320\u5206)<\/p>\n<p style=\"text-align: left\">10N HCl (35%\u5869\u9178\u30924\u500d\u5e0c\u91c8)\u3067pH10\u3050\u3089\u3044\u307e\u3067\u3055\u3052,1N HCl \u3067pH8\u307e\u3067\u3055\u3052\u308b\u3002<\/p>\n<p style=\"text-align: left\">\u6025\u6fc0\u306bpH\u3092\u4e0b\u3052\u308b\u3068\u767d\u6fc1\u3057\u3066\u3057\u307e\u3046\u306e\u3067\u3001\u6ce8\u610f!<\/p>\n<hr \/>\n<h2 style=\"text-align: left\">\u6d17\u3044\u7269\u30fb\u4e7e\u71b1\u6ec5\u83cc<\/h2>\n<p style=\"text-align: left\">\u5bb9\u5668\u30923\u3064\u30b7\u30f3\u30af\u306b\u306a\u3089\u3079\u3001\u5de6\u7aef\u306b\u6d17\u5264\u3001\u6b8b\u308a\u306f\u6c34\u9053\u6c34\u3067\u6e80\u305f\u3059<\/p>\n<p style=\"text-align: left\">\u57fa\u672c\u306f\u30b9\u30dd\u30f3\u30b8\u3068\u30d6\u30e9\u30b7\u3092\u4f7f\u7528\u3057\u3066\u6d17\u5264\u3067\u6d17\u3046\u3002 2\u5ea6\u3059\u3059\u304e\u3092\u3059\u308b\u3002\u3053\u306e\u6642\u3082\u30b9\u30dd\u30f3\u30b8\u3068\u30d6\u30e9\u30b7\u3092\u4f7f\u7528\u3059\u308b\u3002 \u6700\u5f8c\u306f\u8131\u30a4\u30aa\u30f3\u6c34\u3092\u304b\u3051\u308b\u3002 \u4e7e\u71e5\u3055\u305b\u308b\u3002 \u7fcc\u65e5\u3001\u304b\u3054\u306b\u5165\u308c\u3066\u4e7e\u71b1\u6ec5\u83cc(180\u5ea6\u30011\u6642\u9593)<\/p>\n<hr \/>\n<h2 style=\"text-align: left\">\u30a2\u30aa\u30b5\u985e\u306e\u6210\u719f\u8a98\u5c0e<\/h2>\n<p style=\"text-align: left\">\u5927\u304d\u3081\u306e\u5143\u6c17\u305d\u3046\u306a\u85fb\u4f53(\u91ce\u5916or20\u2103\u57f9\u990a)\u3092\u7b46\u3067\u30af\u30ea\u30fc\u30cb\u30f3\u30b0 (\u6d77\u6c34:5cm\u76f4\u5f84\u30c7\u30a3\u30b9\u30dd\u30b7\u30e3\u30fc\u30ec\u4e2d)<\/p>\n<p style=\"text-align: left\">\u5225\u30c7\u30a3\u30b9\u30dd\u30b7\u30e3\u30fc\u30ec\u306b\u79fb\u3057\u3001\u7d30\u304b\u304f\u523b\u3080(2mm\u89d2)<\/p>\n<p style=\"text-align: left\">\u305d\u3053\u306bDW(\u6d77\u6c34)\u3092\u9069\u91cf\u5165\u308c\u3066,\u30d4\u30f3\u30bb\u30c3\u30c8\u3067\u3057\u3083\u304b\u3057\u3083\u304b\u6df7\u305c\u308b<\/p>\n<p style=\"text-align: left\">\u9759\u7f6e\u3057\u3066,\u85fb\u4f53\u3092\u3057\u305a\u3081,\u304a\u304a\u3056\u3063\u3071\u306b\u3059\u3066,\u6b8b\u308a\u3092\u30d4\u30da\u30c3\u30c8\u3067\u5438\u3046<\/p>\n<p style=\"text-align: left\">\u3053\u308c\u30922\uff5e3\u5ea6\u7e70\u308a\u8fd4\u3059<\/p>\n<p style=\"text-align: left\">\u9069\u5ea6\u306b\u4e7e\u71e5(\u5ba4\u6e29 10\u5206\u7a0b\u5ea6)<\/p>\n<p style=\"text-align: left\">20\u2103\u306e1\uff5e2\u500dPES\u6d77\u6c34\u3092\u5165\u308c,25\u2103\u306b1\uff5e4\u65e5<\/p>\n<p style=\"text-align: left\">\u305d\u306e\u9593,\u78ba\u8a8d\u306e\u307f<\/p>\n<p style=\"text-align: left\">\u6210\u719f\u304c\u78ba\u8a8d\u3067\u304d\u305f\u3089,\u5149\u3092\u5f53\u3066\u3066\u96c6\u3081,\u30d4\u30da\u30c3\u30c8\u3067\u30a8\u30c3\u30da\u30f3\u306b\u79fb\u30572000rpm2min\u3067\u6fc3\u7e2e<\/p>\n<ul>\n<li style=\"text-align: left\">\u6210\u719f\u3057\u306a\u3044\u5834\u5408 PES\u30cc\u30ad\u306e\u6ec5\u83cc\u6d77\u6c34\u306b\u79fb\u3059 \u30e9\u30a4\u30c8\u3067\u6696\u3081\u308b(1\u6669\u3064\u3051\u3063\u3071\u306a\u3057\u3067,\u671d8\u6642\u306b\u6d88\u3057,10\u6642\u306b\u653e\u51fa\u4f8b\u30a2\u30ea)<\/li>\n<\/ul>\n","protected":false},"excerpt":{"rendered":"<p>\u4eba\u5de5\u6d77\u6c34(SF-1)\u306e\u4f5c\u308a\u65b9 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