Team:KIT-Kyoto/Protocol
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+ | <tr><td> | ||
+ | <table width="700px" align=center> | ||
+ | <tr><td width="340px"> | ||
+ | <span id="gel">'''ゲルからのDNA抽出'''</span></td><td width="20px"> </td><td width="340px"> | ||
+ | <span id="eng">'''Isolation of DNA fragments from agarose gel'''</span></td></tr> | ||
+ | <tr><td>[http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx QIAquick Gel Extraction Kit]を使用する</td><td> </td><td>We use [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx QIAquick Gel Extraction Kit].</td></tr> | ||
+ | <tr><td>↓ 1 x TAEに溶かした低融点のアガロースゲルをつくる</td><td> </td><td>↓ Make a low-melt agarose gel in 1 x TAE.</td></tr> | ||
+ | <tr><td>↓ DNAとマーカーをゲルにのせる</td><td> </td><td>↓ Load DNA and standard onto gel.</td></tr> | ||
+ | <tr><td>↓ 75 Vで45分間電気泳動する</td><td> </td><td>↓ Electrophorese at 75 V for 45 minutes.</td></tr> | ||
+ | <tr><td>↓ EtBrでゲルを40分間染色する</td><td> </td><td>↓Dye gel with EtBr for 40 minutes.</td></tr> | ||
+ | <tr><td>↓ UVを照射してDNAのバンドを可視化する</td><td> </td><td>↓ Visualize DNA bands using UV lightbox.</td></tr> | ||
+ | <tr><td>↓ 清潔で鋭利な手術用メスでゲルからDNA断片を切りだす</td><td> </td><td>↓ Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.</td></tr> | ||
+ | <tr><td>↓ 1.5 mlチューブ中のゲルスライスの重さを量る</td><td> </td><td>↓ Weigh the gel slice in 1.5 ml tube.</td></tr> | ||
+ | <tr><td>↓ ゲル100 mgに対して、3倍量(300 μl)のBuffer QGを加える</td><td> </td><td>↓ Add 3 volumes of Buffer QG to 1 volume of gel (100 mg - 100 μl). </td></tr> | ||
+ | <tr><td>↓ 42-50 °Cで10分間放置し、ゲルを完全に溶かす</td><td> </td><td>↓ Incubate at 42-50 °C for 10 minutes or until gel is fully dissolved.</td></tr> | ||
+ | <tr><td>↓ ゲルが完全に溶けたら、溶液が黄色であることを確認する</td><td> </td><td>↓ After the gel slice has dissolved completely, check that the color of the mixture is yellow.</td></tr> | ||
+ | <tr><td>↓ ゲル100 mgに対して、当量(100 μl)の2-プロパノールを加え、混ぜる</td><td> </td><td>↓ Add 1 gel volume of 2-propanol to the sample and mix.</td></tr> | ||
+ | <tr><td>↓ カラムにサンプルをのせる</td><td> </td><td>↓ Apply sample to column in a collection tube.</td></tr> | ||
+ | <tr><td>↓ 15,000 rpmで1分間遠心し、ろ液を捨てる</td><td> </td><td>↓ Centrifuge for 1 minute at 15,000 rpm and discard flow-through.</td></tr> | ||
+ | <tr><td>↓ 500 μlのBuffer QGをカラムに加えね残りのゲルを溶かす</td><td> </td><td>↓ Add 500 μl of Buffer QG to column to dissolve residual agarose.</td></tr> | ||
+ | <tr><td>↓ 15,000 rpmで1分間遠心し、ろ液を捨てる</td><td> </td><td>↓ Centrifuge for 1 minute at 15,000 rpm and discard flow-through.</td></tr> | ||
+ | <tr><td>↓ 750 μlのwash Buffer PEをカラムに加える</td><td> </td><td>↓ Add 750 μl of wash Buffer PE to column.</td></tr> | ||
+ | <tr><td>↓ 15,000 rpmで1分間遠心し、ろ液を捨てる</td><td> </td><td>↓Centrifuge for 1 minute at 15,000 rpm and discard flow-through.</td></tr> | ||
+ | <tr><td>↓ 15,000 rpmで1分間遠心し、ろ液を捨てる(残りのエタノールを除くため)</td><td> </td><td>↓ Centrifuge for 1 minute at 15,000 rpm and discard flow-through (to eliminate residual ethanol).</td></tr> | ||
+ | <tr><td>↓ カラムを新しい1.5 mlチューブに移しかえる</td><td> </td><td>↓ Place column into a clean 1.5 ml tube.</td></tr> | ||
+ | <tr><td>↓ 30 μlのelution Buffer EBをカラムの中央に加える</td><td> </td><td>↓ Add 30 μl of elution Buffer EB to center of column.</td></tr> | ||
+ | <tr><td>↓ 1分間立たせておく</td><td> </td><td>↓Let stand for 1 minute.</td></tr> | ||
+ | <tr><td>↓ 15,000 rpmで1分間遠心する</td><td> </td><td>↓ Centrifuge for 1 minute at 15,000 rpm. </td></tr> | ||
+ | <tr><td>↓ 集めたDNAは1.5 mlチューブに入れて-20 °Cで保存する</td><td> </td><td>↓ Store DNA collected in 1.5 ml tube at -20 °C.</td></tr> | ||
+ | |||
+ | </table> | ||
+ | </td></tr> | ||
+ | <tr><td> | ||
+ | |||
+ | <div align="right"><span style="font- | ||
+ | |||
+ | size:10pt;">[[#Contents|>>back to Contents]] | ||
+ | |||
+ | </span></div> | ||
+ | |||
+ | </td></tr> | ||
Revision as of 04:43, 24 October 2010
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