Team:KIT-Kyoto/yokoigawa

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<p><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; FONT-SIZE: 10.5pt; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA">↓反応液調整後、</span><span style="FONT-FAMILY: 'Century','serif'; FONT-SIZE: 10.5pt; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA" lang="EN-US">37</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; FONT-SIZE: 10.5pt; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA">℃で</span><span style="FONT-FAMILY: 'Century','serif'; FONT-SIZE: 10.5pt; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA" lang="EN-US">20</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; FONT-SIZE: 10.5pt; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA">時間静置</span></p>
<p><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; FONT-SIZE: 10.5pt; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA">↓反応液調整後、</span><span style="FONT-FAMILY: 'Century','serif'; FONT-SIZE: 10.5pt; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA" lang="EN-US">37</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; FONT-SIZE: 10.5pt; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA">℃で</span><span style="FONT-FAMILY: 'Century','serif'; FONT-SIZE: 10.5pt; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA" lang="EN-US">20</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; FONT-SIZE: 10.5pt; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA">時間静置</span></p>
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<body><span lang="EN-US"><font face="Century">
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<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt" class="MsoNormal" align="left"><span lang="EN-US">8/30(</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">火</span><span lang="EN-US">)</span></p>
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<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt" class="MsoNormal" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">武田、横井川</span></p>
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<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt" class="MsoNormal" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">【目的】</span></p>
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<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt" class="MsoNormal" align="left"><span lang="EN-US">PCR</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">産物と</span><span lang="EN-US">pUAST-flag vector</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">の制限酵素処理</span><span lang="EN-US">(</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">二回目：</span><span lang="EN-US">Xbal)</span></p>
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<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt" class="MsoNormal" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">【実験操作】</span></p>
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<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt" class="MsoNormal" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">制限酵素処理</span><span lang="EN-US">20h</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">後、フェノールクロロホルム処理を行う</span></p>
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<p style="TEXT-ALIGN: left; TEXT-INDENT: -10.5pt; MARGIN: 0mm 0mm 0pt 10.5pt; mso-char-indent-count: -1.0" class="MsoNormal" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">↓</span><span lang="EN-US">PCR</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">産物を</span><span lang="EN-US">400</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">μ</span><span lang="EN-US">l</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">まで</span><span lang="EN-US">milliQ</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">で希釈し、フェノール・クロロホルムを</span><span lang="EN-US">400</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">μ</span><span lang="EN-US">l</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">加えて</span><span lang="EN-US">voltex </span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">し</span><span lang="EN-US">10000rpm3min4</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">℃で遠心。</span></p>
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<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt" class="MsoNormal" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">↓水層のみを新しいチューブに移した。</span></p>
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<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt" class="MsoNormal" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">↓クロロホルムを等量加えて、</span><span lang="EN-US">voltex</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">し</span><span lang="EN-US">10000rpm3min4</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">℃で遠心。</span></p>
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<p style="TEXT-ALIGN: left; TEXT-INDENT: -10.5pt; MARGIN: 0mm 0mm 0pt 10.5pt; mso-char-indent-count: -1.0" class="MsoNormal" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">↓水層のみを新しいチューブに移し</span><span lang="EN-US">3M CH<sub>3</sub>COONa(PH 5.27)</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">を</span><span lang="EN-US">1/10</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">等量加え、</span><span lang="EN-US">isopropanol</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">を等量加えた。</span></p>
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<p style="TEXT-ALIGN: left; TEXT-INDENT: -10.5pt; MARGIN: 0mm 0mm 0pt 10.5pt; mso-char-indent-count: -1.0" class="MsoNormal" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">↓</span><span lang="EN-US">3</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">分静置後、</span><span lang="EN-US">14000rpm 10min 4</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">℃で遠心。</span></p>
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<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt" class="MsoNormal" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">↓遠心後、上清を捨て、</span><span lang="EN-US">70</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">％</span><span lang="EN-US">ethanol</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">を</span><span lang="EN-US">200</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">μ</span><span lang="EN-US">l</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">加え、</span><span lang="EN-US">15000rpm 5min 4</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">℃で遠心。</span></p>
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<p style="TEXT-ALIGN: left; TEXT-INDENT: -10.5pt; MARGIN: 0mm 0mm 0pt 10.5pt; mso-char-indent-count: -1.0" class="MsoNormal" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">↓遠心後、上清を捨て乾燥させる。乾燥後</span><span lang="EN-US">milliQ44</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">μ</span><span lang="EN-US">l</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">を加えて</span><span lang="EN-US">voltex</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">し、</span><span lang="EN-US">milliQ</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">中に</span><span lang="EN-US">DNA</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">を溶解させる。</span></p>
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<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt 21pt; mso-para-margin-left: 0gd" class="MsoListParagraph" align="left"><span lang="EN-US"><?xml:namespace prefix ="" o ns
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="" "urn:schemas-microsoft-com:office:office" /><o:p>&nbsp;</o:p></span></p>
 +
<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt" class="MsoNormal" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">下記の反応液を調整する</span></p>
 +
<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt 21pt; mso-para-margin-left: 0gd" class="MsoListParagraph" align="left"><span lang="EN-US"><o:p>&nbsp;</o:p></span></p>
 +
<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt 21pt; mso-para-margin-left: 0gd" class="MsoListParagraph" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">反応液調整（</span><span lang="EN-US">DIAP2</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">）</span></p>
 +
<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt 21pt; mso-para-margin-left: 0gd" class="MsoListParagraph" align="left"><span lang="EN-US"><span style="mso-spacerun: yes">&nbsp; </span>PCR</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">産物</span><span lang="EN-US"> in milliQ<span style="mso-spacerun: yes">&nbsp;
 +
</span>44</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">μ</span><span lang="EN-US">l</span></p>
 +
<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt 21pt; mso-para-margin-left: 0gd" class="MsoListParagraph" align="left"><span lang="EN-US"><span style="mso-spacerun: yes">&nbsp; </span>10x M Buffer<span style="mso-spacerun: yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span>5</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">μ</span><span lang="EN-US">l</span></p>
 +
<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt 21pt; mso-para-margin-left: 0gd" class="MsoListParagraph" align="left"><span lang="EN-US"><span style="mso-spacerun: yes">&nbsp; </span>Xba 1<span style="mso-spacerun: yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span>1</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">μ</span><span lang="EN-US">l</span></p>
 +
<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt 21pt; mso-para-margin-left: 0gd" class="MsoListParagraph" align="left"><span lang="EN-US"><o:p>&nbsp;</o:p></span></p>
 +
<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt 21pt; mso-para-margin-left: 0gd" class="MsoListParagraph" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">反応液調整（</span><span lang="EN-US">vector</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">）</span></p>
 +
<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt 21pt; mso-para-margin-left: 0gd" class="MsoListParagraph" align="left"><span lang="EN-US"><span style="mso-spacerun: yes">&nbsp; </span>pUAST-flag vector in milliQ<span style="mso-spacerun: yes">&nbsp; </span>44</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">μ</span><span lang="EN-US">l</span></p>
 +
<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt 21pt; mso-para-margin-left: 0gd" class="MsoListParagraph" align="left"><span lang="EN-US"><span style="mso-spacerun: yes">&nbsp; </span>10x H Buffer<span style="mso-spacerun: yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span>5</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">μ</span><span lang="EN-US">l</span></p>
 +
<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt 21pt; mso-para-margin-left: 0gd" class="MsoListParagraph" align="left"><span lang="EN-US"><span style="mso-spacerun: yes">&nbsp; </span>Xba 1<span style="mso-spacerun: yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span>1</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin">μ</span><span lang="EN-US">l</span></p>
 +
<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt 21pt; mso-para-margin-left: 0gd" class="MsoListParagraph" align="left"><span lang="EN-US"><o:p>&nbsp;</o:p></span></p>
 +
<p style="TEXT-ALIGN: left; MARGIN: 0mm 0mm 0pt" class="MsoNormal" align="left"><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; FONT-SIZE: 10.5pt; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA">↓反応液調整後、</span><span style="FONT-FAMILY: 'Century','serif'; FONT-SIZE: 10.5pt; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA" lang="EN-US">37</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; FONT-SIZE: 10.5pt; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA">℃で</span><span style="FONT-FAMILY: 'Century','serif'; FONT-SIZE: 10.5pt; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: 'ＭＳ 明朝'; mso-fareast-theme-font: minor-fareast; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA" lang="EN-US">20</span><span style="FONT-FAMILY: 'ＭＳ 明朝','serif'; FONT-SIZE: 10.5pt; mso-ascii-font-family: Century; mso-ascii-theme-font: minor-latin; mso-fareast-theme-font: minor-fareast; mso-hansi-font-family: Century; mso-hansi-theme-font: minor-latin; mso-bidi-font-size: 11.0pt; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: JA; mso-bidi-language: AR-SA">時間静置</span></font></span></p>
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Revision as of 09:38, 18 September 2011

8/29(月)

武田、横井川

【目的】

PCR産物とpUAST-flag vectorの制限酵素処理（一回目：Xho 1）

【実験操作】

～フェノールクロロホルム処理・エタノール沈殿～

↓DAIP2のPCR産物を400μlまでmilliQで希釈し、フェノール・クロロホルムを400μl加えてvoltex し10000rpm3min4℃で遠心。

↓水層のみを新しいチューブに移した。

↓クロロホルムを等量加えて、voltexし10000rpm3min4℃で遠心。

↓水層のみを新しいチューブに移し3M CH3COONa(PH 5.27)を1/10等量加え、isopropanolを等量加えた。

↓3分静置後、14000rpm 10min 4℃で遠心。

↓遠心後、上清を捨て、70％ethanolを200μl加え、15000rpm 5min 4℃で遠心。

↓遠心後、上清を捨て乾燥させる。乾燥後milliQ44μlを加えてvoltexし、milliQ中にDNAを溶解させる。

～制限酵素処理～

フェノールクロロホルム処理後下記の反応液を調整した

反応液調整（cDNA）

  PCR産物 in milliQ  44μl

  10x H Buffer　　　　5μl

  Xho 1               1μl

 

　　反応液調整（vector）

      pUAST-flag vector(500ng/μl)  10μl

      milliQ                       34μl

      10x H Buffer                 5μl

      Xho 1                        1μl

 

↓反応液調整後、37℃で20時間静置




8/30(火)

武田、横井川

【目的】

PCR産物とpUAST-flag vectorの制限酵素処理(二回目：Xbal)

【実験操作】

制限酵素処理20h後、フェノールクロロホルム処理を行う

↓PCR産物を400μlまでmilliQで希釈し、フェノール・クロロホルムを400μl加えてvoltex し10000rpm3min4℃で遠心。

↓水層のみを新しいチューブに移した。

↓クロロホルムを等量加えて、voltexし10000rpm3min4℃で遠心。

↓水層のみを新しいチューブに移し3M CH3COONa(PH 5.27)を1/10等量加え、isopropanolを等量加えた。

↓3分静置後、14000rpm 10min 4℃で遠心。

↓遠心後、上清を捨て、70％ethanolを200μl加え、15000rpm 5min 4℃で遠心。

↓遠心後、上清を捨て乾燥させる。乾燥後milliQ44μlを加えてvoltexし、milliQ中にDNAを溶解させる。

 

下記の反応液を調整する

 

反応液調整（DIAP2）

  PCR産物 in milliQ  44μl

  10x M Buffer         5μl

  Xba 1                1μl

 

反応液調整（vector）

  pUAST-flag vector in milliQ  44μl

  10x H Buffer                 5μl

  Xba 1                        1μl

 

↓反応液調整後、37℃で20時間静置

Retrieved from "http://2011.igem.org/Team:KIT-Kyoto/yokoigawa"