Team:KIT-Kyoto/Protocol
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{{Template:KIT-Kyoto/menu}} | {{Template:KIT-Kyoto/menu}} | ||
<table border=0 width="965px" align="center"><tr><td> | <table border=0 width="965px" align="center"><tr><td> | ||
- | <div aling="left">[[Team:KIT-Kyoto|Home]] > [[Team:KIT-Kyoto/Note|Notebook]] > [[Team:KIT-Kyoto/Protocol|Protocol]]</div></td><td><div align="right">Language : [[Team:KIT-Kyoto/Protocol|English]] / [[Team:KIT-Kyoto/ProtocolJ|Japanese]]</div></td></tr></table> | + | <div aling="left">[[Team:KIT-Kyoto/Home|Home]] > [[Team:KIT-Kyoto/Note|Notebook]] > [[Team:KIT-Kyoto/Protocol|Protocol]]</div></td><td><div align="right">Language : [[Team:KIT-Kyoto/Protocol|English]] / [[Team:KIT-Kyoto/ProtocolJ|Japanese]]</div></td></tr></table> |
<table border="0" width="965px" align="center"><tr><td width="165px" valign="top" | <table border="0" width="965px" align="center"><tr><td width="165px" valign="top" | ||
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<table width="700px" align=center> | <table width="700px" align=center> | ||
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- | [[Image:PROT.PNG|left]]As it is of common knowledge, iGEM is carried out within the framework of a protocol common to all teams. Whereas most of the teams are registered on this page, we have uploaded not only the English protocol but also the Japanese one, e.g., our mother tongue version. This is the common protocol with some improvements after we translated it into Japanese.Publishing the Japanese protocol in will be of help to other new Japanese teams in the future. Furthermore, this will be linked to an improvement of iGEM’s name identification in Japan and will enhance the public recognition of synthetic biology. It will also promote Science and Communication among people within not only the scientific but with the non-scientific communities as well. We believe that these improvements will constitute a contribution to the illustration of the public in general. | + | [[Image:PROT.PNG|left]]As it is of common knowledge, iGEM is carried out within the framework of a protocol common to all teams. Whereas most of the teams are registered on this page, we have uploaded not only the English protocol but also the Japanese one, e.g., our mother tongue version. This is the common protocol with some improvements after we translated it into Japanese. Publishing the Japanese protocol in will be of help to other new Japanese teams in the future. Furthermore, this will be linked to an improvement of iGEM’s name identification in Japan and will enhance the public recognition of synthetic biology. It will also promote Science and Communication among people within not only the scientific but with the non-scientific communities as well. We believe that these improvements will constitute a contribution to the illustration of the public in general. |
</td></tr></table> | </td></tr></table> | ||
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<tr><td>↓ 42 °Cで45秒間熱ショックを与える</td><td> </td><td>↓ Heat shock the cells at 42 °C for 45 seconds. </td></tr> | <tr><td>↓ 42 °Cで45秒間熱ショックを与える</td><td> </td><td>↓ Heat shock the cells at 42 °C for 45 seconds. </td></tr> | ||
<tr><td>↓ 0.9 mlのSOC培地を加える</td><td> </td><td>↓ Add 0.9 ml SOC medium.</td></tr> | <tr><td>↓ 0.9 mlのSOC培地を加える</td><td> </td><td>↓ Add 0.9 ml SOC medium.</td></tr> | ||
- | <tr><td>↓ 37 °Cで1時間、振りながら回復培養する</td><td> </td><td>↓ | + | <tr><td>↓ 37 °Cで1時間、振りながら回復培養する</td><td> </td><td>↓ Recover at 37 °C for 1 hour, shaking.</td></tr> |
<tr><td>↓ 1 mlをLBプレート(+amp,+kan,+camのいずれか)にまく</td><td> </td><td>↓ Spread 1 ml on LB plates(Either +amp,+kan or +cam)</td></tr> | <tr><td>↓ 1 mlをLBプレート(+amp,+kan,+camのいずれか)にまく</td><td> </td><td>↓ Spread 1 ml on LB plates(Either +amp,+kan or +cam)</td></tr> | ||
<tr><td>↓ 37 °Cで一晩培養する</td><td> </td><td>↓ Cultivate overnight in 37 °C.</td></tr></table> | <tr><td>↓ 37 °Cで一晩培養する</td><td> </td><td>↓ Cultivate overnight in 37 °C.</td></tr></table> | ||
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<span id="al">'''アルカリミニプレップ'''</span></td><td width="20px"> </td><td width="340px"> | <span id="al">'''アルカリミニプレップ'''</span></td><td width="20px"> </td><td width="340px"> | ||
<span id="aleng">'''DNA miniprep'''</span></td></tr> | <span id="aleng">'''DNA miniprep'''</span></td></tr> | ||
- | <tr><td><table border=1 width="300px"><tr><td width="100px" align=center>Solution I</td><td width="200px" align=right>50 mM グルコース (MW 180)</td></tr><tr | + | <tr><td><table border=1 width="300px"><tr><td width="100px" align=center rowspan=3>Solution I</td><td width="200px" align=right>50 mM グルコース (MW 180)</td></tr><tr><td align=right>10 mM EDTA(pH 8.0)</td></tr><tr><td align=right>25 mM Tris-HCl (pH 8.0)</td></tr> |
- | <tr><td align=center>Solution II</td><td align=right>0.2 N NaOH</td></tr><tr | + | <tr><td align=center rowspan=2>Solution II</td><td align=right>0.2 N NaOH</td></tr><tr><td align=right>1% SDS</td></tr> |
- | border=1 width="300px"><tr><td width="100px" align=center>Solution I</td><td width="200px" align=right>50 mM glucose (MW 180)</td></tr> | + | <tr><td align=center rowspan=2>Solution III</td><td align=right>3 M 酢酸カリウム</td></tr><tr><td align=right>1.8 M 酢酸</td></tr></table></td><td> </td><td><table |
- | <tr | + | border=1 width="300px"><tr><td width="100px" align=center rowspan=3>Solution I</td><td width="200px" align=right>50 mM glucose (MW 180)</td></tr> |
- | <tr><td align=center>Solution II</td><td align=right>0.2 N NaOH</td></tr><tr | + | <tr><td align=right>10 mM EDTA(pH 8.0)</td></tr><tr><td align=right>25 mM Tris-HCl (pH 8.0)</td></tr> |
+ | <tr><td align=center rowspan=2>Solution II</td><td align=right>0.2 N NaOH</td></tr><tr><td align=right>1% SDS</td></tr><tr><td align=center rowspan=2>Solution III</td><td align=right>3 M potassium acetate</td></tr><tr><td align=right>1.8 M acetic acid</td></tr></table></td></tr> | ||
<tr><td>↓ LBプレート(+amp,+kan,+camのいずれか)に大腸菌をまき、37 °Cで一晩培養する</td><td> </td><td>↓ Streak <I>E.coli</I> to LB plates(Either +amp,+kan or +cam) and cultivate overnight in 37 °C.</td></tr> | <tr><td>↓ LBプレート(+amp,+kan,+camのいずれか)に大腸菌をまき、37 °Cで一晩培養する</td><td> </td><td>↓ Streak <I>E.coli</I> to LB plates(Either +amp,+kan or +cam) and cultivate overnight in 37 °C.</td></tr> | ||
<tr><td>↓ プレートからシングルコロニーを分離する</td><td> </td><td>↓ Pick up a single colony from plate.</td></tr> | <tr><td>↓ プレートからシングルコロニーを分離する</td><td> </td><td>↓ Pick up a single colony from plate.</td></tr> | ||
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<tr><td><table border=1 width="300px"><tr><td width="200px" align=center>Primer Mix</td><td width="100px" align=right>1.5 μl</td></tr> | <tr><td><table border=1 width="300px"><tr><td width="200px" align=center>Primer Mix</td><td width="100px" align=right>1.5 μl</td></tr> | ||
<tr><td align=center>鋳型 DNA</td><td align=right>0.5 μl</td></tr><tr><td align=center>10 x PCR Buffer for KOD-Plus-</td><td align=right>5 μl</td></tr><tr><td align=center>2 mM dNTPs</td><td align=right>5 μl</td></tr> | <tr><td align=center>鋳型 DNA</td><td align=right>0.5 μl</td></tr><tr><td align=center>10 x PCR Buffer for KOD-Plus-</td><td align=right>5 μl</td></tr><tr><td align=center>2 mM dNTPs</td><td align=right>5 μl</td></tr> | ||
- | <tr><td align=center>2 mM | + | <tr><td align=center>2 mM MgSO<sub>4</sub></td><td align=right>4 μl</td></tr><tr><td align=center>ddH<sub>2</sub>O</td><td align=right>33 μl</td></tr><tr><td align=center>KOD-Plus-</td><td align=right>1 μl</td></tr> |
- | <tr><td> </td><td align=right>全量 50 µl</td></tr></table></td><td> </td><td><table border=1 width="300px"><tr><td width="200px" align=center>Primer Mix</td><td width="100px" align=right>1.5 μl</td></tr><tr><td align=center>Template DNA</td><td align=right>0.5 μl</td></tr><tr><td align=center>10 x PCR Buffer for KOD-Plus-</td><td align=right>5 μl</td></tr><tr><td align=center>2 mM dNTPs</td><td align=right>5 μl</td></tr><tr><td align=center>2 mM | + | <tr><td> </td><td align=right>全量 50 µl</td></tr></table></td><td> </td><td><table border=1 width="300px"><tr><td width="200px" align=center>Primer Mix</td><td width="100px" align=right>1.5 μl</td></tr><tr><td align=center>Template DNA</td><td align=right>0.5 μl</td></tr><tr><td align=center>10 x PCR Buffer for KOD-Plus-</td><td align=right>5 μl</td></tr><tr><td align=center>2 mM dNTPs</td><td align=right>5 μl</td></tr><tr><td align=center>2 mM MgSO<sub>4</sub></td><td align=right>4 μl</td></tr><tr><td align=center>ddH<sub>2</sub>O</td><td align=right>33 μl</td></tr><tr><td align=center>KOD-Plus-</td><td align=right>1 μl</td></tr><tr><td> </td><td align=right>50 μl system</td></tr></table></td></tr> |
<tr><td>↓ PCRのプログラム設定</td><td> </td><td>↓ PCR program</td></tr> | <tr><td>↓ PCRのプログラム設定</td><td> </td><td>↓ PCR program</td></tr> | ||
<tr><td><table border=1 width="300px"><tr><td width="60px" align=center>Start</td><td width="240px" align=right>94 °C、2分</td></tr><tr><td align=center rowspan=3>Cycle x 35</td><td align=right>94 °C、15秒 (熱変性)</td></tr><tr><td align=right>(Tm-10) °C、0.5分 (アニーリング)</td></tr><tr><td align=right>68 °C、1 kb/min (伸長)</td></tr><tr><td align=center rowspan=2>End</td><td align=right>68 °C、2分</td></tr> | <tr><td><table border=1 width="300px"><tr><td width="60px" align=center>Start</td><td width="240px" align=right>94 °C、2分</td></tr><tr><td align=center rowspan=3>Cycle x 35</td><td align=right>94 °C、15秒 (熱変性)</td></tr><tr><td align=right>(Tm-10) °C、0.5分 (アニーリング)</td></tr><tr><td align=right>68 °C、1 kb/min (伸長)</td></tr><tr><td align=center rowspan=2>End</td><td align=right>68 °C、2分</td></tr> | ||
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<tr><td align=center> | <tr><td align=center> | ||
:[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=SPE-101&us_id=&us_pwd=?us_id=&bun_id=0200100100900&iv_trademark=SPE-1&kskh_kbn=01&ksk_jkn=SPE-1 <I>Spe</I> I]</td></tr> | :[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=SPE-101&us_id=&us_pwd=?us_id=&bun_id=0200100100900&iv_trademark=SPE-1&kskh_kbn=01&ksk_jkn=SPE-1 <I>Spe</I> I]</td></tr> | ||
- | <tr><td align=center> | + | <tr><td align=center>ddH<sub>2</sub>O</td><td>全量が20 μl になるように調整する</td></tr> |
<tr><td> </td><td align=right>全量 20 μl</td></tr></table></td><td> </td><td><table border=1 width="300px"> | <tr><td> </td><td align=right>全量 20 μl</td></tr></table></td><td> </td><td><table border=1 width="300px"> | ||
<tr><td width="120px" align=center>10 x Buffer</td><td rowspan=3 width="180px">2 µl (1/10 of total)</td></tr> | <tr><td width="120px" align=center>10 x Buffer</td><td rowspan=3 width="180px">2 µl (1/10 of total)</td></tr> | ||
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<tr><td align=center> | <tr><td align=center> | ||
:[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=SPE-101&us_id=&us_pwd=?us_id=&bun_id=0200100100900&iv_trademark=SPE-1&kskh_kbn=01&ksk_jkn=SPE-1 <I>Spe</I> I]</td></tr> | :[http://www.bio.toyobo.co.jp/bio01/cgi-bin/catalog_shohin_index?item=SPE-101&us_id=&us_pwd=?us_id=&bun_id=0200100100900&iv_trademark=SPE-1&kskh_kbn=01&ksk_jkn=SPE-1 <I>Spe</I> I]</td></tr> | ||
- | <tr><td align=center> | + | <tr><td align=center>ddH<sub>2</sub>O</td><td>Adjust to become total 20 μl.</td></tr> |
<tr><td> </td><td align=right>20 μl system</td></tr></table></td></tr> | <tr><td> </td><td align=right>20 μl system</td></tr></table></td></tr> | ||
<tr><td>↓ 37 °Cで1時間放置する</td><td> </td><td>↓ Incubate for 1 hour in 37 °C.</td></tr> | <tr><td>↓ 37 °Cで1時間放置する</td><td> </td><td>↓ Incubate for 1 hour in 37 °C.</td></tr> | ||
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<span id="agareng">'''Agarose gel electrophoresis'''</span></td></tr> | <span id="agareng">'''Agarose gel electrophoresis'''</span></td></tr> | ||
<tr><td><table border=1 width="300px"><tr><td align=center rowspan=4 width="100px">50 x TAE</td><td width="200px">Tris base 242 g</td></tr><tr><td>無水酢酸 57.1 ml</td></tr> | <tr><td><table border=1 width="300px"><tr><td align=center rowspan=4 width="100px">50 x TAE</td><td width="200px">Tris base 242 g</td></tr><tr><td>無水酢酸 57.1 ml</td></tr> | ||
- | <tr><td>0.5 M EDTA (pH 8.0) 100ml</td></tr><tr><td> | + | <tr><td>0.5 M EDTA (pH 8.0) 100ml</td></tr><tr><td>ddH<sub>2</sub>Oを加えて1 Lにする</td></tr></table></td><td> </td><td><table border=1 width="300px"><tr><td align=center rowspan=4 width="100px">50 x TAE</td><td width="200px">242g Tris base</td></tr><tr><td>57.1 ml of glacial Acetic acid</td></tr> |
- | <tr><td>100ml of 0.5 M EDTA (pH 8.0)</td></tr><tr><td>Make up to 1 L with | + | <tr><td>100ml of 0.5 M EDTA (pH 8.0)</td></tr><tr><td>Make up to 1 L with ddH<sub>2</sub>O</td></tr></table></td></tr> |
- | <tr><td>1 x TAEを作るには、20 mlの50 x TAEに980 | + | <tr><td>1 x TAEを作るには、20 mlの50 x TAEに980 mlのH<sub>2</sub>Oを加える</td><td> </td><td>To make 1 x TAE from 50 x TAE stock, dilute 20 ml of stock into 980 ml of ddH<sub>2</sub>O.</td></tr> |
<tr><td>↓ ゲルに使用するアガロースの量は扱うDNAによって変わるので下記の表に従えばよい</td><td> </td><td>↓ The amount of agarose to use in your gel depends on the DNA in question. Use the following table as a rough guide.</td></tr> | <tr><td>↓ ゲルに使用するアガロースの量は扱うDNAによって変わるので下記の表に従えばよい</td><td> </td><td>↓ The amount of agarose to use in your gel depends on the DNA in question. Use the following table as a rough guide.</td></tr> | ||
<tr><td><table border=1 width="300px"><tr><td width="150px" align=center>アガロース濃度 | <tr><td><table border=1 width="300px"><tr><td width="150px" align=center>アガロース濃度 | ||
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<tr><td> | <tr><td> | ||
- | <div align="right"><span style="font- | + | <div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div> |
- | + | ||
- | size:10pt;">[[#Contents|>>back to Contents]] | + | |
- | + | ||
- | </span></div> | + | |
</td></tr> | </td></tr> | ||
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<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>↓ 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>↓ 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 | + | <tr><td>↓ 500 μlのBuffer QGをカラムに加え、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>↓ 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>↓ 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> | ||
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<tr><td> | <tr><td> | ||
- | <div align="right"><span style="font- | + | <div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div> |
- | + | </td></tr> | |
- | |||
+ | <tr><td> | ||
+ | <table width="700px" align=center> | ||
+ | <tr><td width="340px"> | ||
+ | <span id="lig">'''ライゲーション'''</span></td><td width="20px"> </td><td width="340px"> | ||
+ | <span id="ligeng">'''DNA ligation'''</span></td></tr> | ||
+ | <tr><td>[https://www.roche-applied-science.com/servlet/RCProductDisplay?storeId=10202&catalogId=10202&langId=-1&countryId=us&forCountryId=us&productId=3.6.14.2.2.3 Rapid DNA Ligation Kit]を使用する</td><td> </td><td>We use [https://www.roche-applied-science.com/servlet/RCProductDisplay?storeId=10202&catalogId=10202&langId=-1&countryId=us&forCountryId=us&productId=3.6.14.2.2.3 Rapid DNA Ligation Kit].</td></tr> | ||
+ | <tr><td>↓ 下記の組成に従って試薬を混ぜる</td><td> </td><td>↓ Mix the reagent according to the following components.</td></tr> | ||
+ | <tr><td><table border=1 width="300px"><tr><td align=center width="200px">5 x DNA dilution buffer</td><td align=right width="100px">2 μl</td></tr> | ||
+ | <tr><td align=center>ベクター(カット・CHIP処理後)</td><td align=right>0.25-1 μl</td></tr> | ||
+ | <tr><td align=center>インサート DNA</td><td align=right>0.5-6.5 μl</td></tr> | ||
+ | <tr><td align=center>ddH<sub>2</sub>O</td><td align=right>全量が10 μlになるように調整する</td></tr></table></td><td> </td><td><table border=1 wight="300px"><tr><td wight="200px" align=center>5x DNA dilution buffer</td><td wight="100px" align=right>2 μl</td></tr> | ||
+ | <tr><td align=center>Vector(cut and CHIP treated)</td><td align=right>0.25-1 μl</td></tr> | ||
+ | <tr><td align=center>Insert DNA</td><td align=right>0.5-6.5 μl</td></tr> | ||
+ | <tr><td align=center>ddH<sub>2</sub>O</td><td align=right>To final volume of 10 μl</td></tr></table></td></tr> | ||
+ | <tr><td>↓ よく混ぜる</td><td> </td><td>↓ Mix well.</td></tr> | ||
+ | <tr><td>↓ 10 μlの2 x rapid ligation bufferと1 μlのligaseを加える</td><td> </td><td>↓ Add 10 μl of | ||
+ | 2 x rapid ligation buffer and 1 μl ligase.</td></tr> | ||
+ | <tr><td>↓ よく混ぜる</td><td> </td><td>↓ Mix well.</td></tr> | ||
+ | <tr><td>↓ 室温で5分間放置する</td><td> </td><td>↓ Hold at room temperature for 5 minutes.</td></tr> | ||
+ | <tr><td>↓ トランスフォーメーションに使用するときは、100 μlのコンピテント細胞に対して10 μl使用する</td><td> </td><td>↓Then use 10 μl of the ligation reaction to transform 100 μl of chemically competent cells.</td></tr> | ||
+ | |||
+ | </table> | ||
</td></tr> | </td></tr> | ||
+ | <tr><td> | ||
+ | <div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div> | ||
+ | </td></tr> | ||
+ | <tr><td> | ||
+ | <table width="700px" align=center> | ||
+ | <tr><td width="340px"> | ||
+ | <span id="com">'''コンピテント細胞の作製'''</span></td><td width="20px"> </td><td width="340px"> | ||
+ | <span id="comeng">'''Preparing chemically competent cells'''</span></td></tr> | ||
+ | <tr><td>↓ <I>E.coli</I>(DH5 Alpha)をLBプレートにまき、37 °Cで一晩培養する</td><td> </td><td>↓ Streak <I>E.coli</I> (DH5 Alpha) to LB plate and cultivate overnight in 37 °C.</td></tr> | ||
+ | <tr><td>↓ シングルコロニーをピックアップして、2 mlのSOB培地で37 °Cで6時間培養する</td><td> </td><td>↓ Pick up a single colony from plate and cultivate 2 ml of SOB medium for 6 hours in 37 °C.</td></tr> | ||
+ | <tr><td>↓ 培養液を250 mlのSOB培地に加える</td><td> </td><td>↓ Add 2 ml of the culture 250 ml of SOB medium.</td></tr> | ||
+ | <tr><td>↓ 遠心しながら、37 °Cで吸光度(OD600)が0.4-0.8になるまで培養する</td><td> </td><td>↓ Cultivate at 37°C with agitation until the Optical Density (OD600) reaches 0.4-0.8.</td></tr> | ||
+ | <tr><td>↓ 培養液を50 mlの遠心チューブに移し、氷上で10分間冷やす</td><td> </td><td>↓ Transfer the culture into 50 ml centrifuge tube and incubate the culture on ice for 10 minutes.</td></tr> | ||
+ | <tr><td>↓ 4,500 rpm、4 °Cで10分間遠心し、上清を捨てる</td><td> </td><td>↓ Centrifuge for 10 minutes at 4,500 rpm in 4 °C and discard the supernatant.</td></tr> | ||
+ | <tr><td>↓ 84 mlの氷冷した[http://openwetware.org/wiki/TB_buffer TB Buffer]を加えて、懸濁する</td><td> </td><td>↓ Add 84 ml of refrigerated [http://openwetware.org/wiki/TB_buffer TB Buffer] and resuspend.</td></tr> | ||
+ | <tr><td>↓ 氷上で10分間冷やす</td><td> </td><td>↓ Incubate on ice for 10 minutes.</td></tr> | ||
+ | <tr><td>↓ 4,500 rpm、4 °Cで10分間遠心し、上清を捨てる</td><td> </td><td>↓ Centrifuge for 10 minutes at 4,500 rpm in 4 °C and discard the supernatant.</td></tr> | ||
+ | <tr><td>↓ 20 mlの氷冷した[http://openwetware.org/wiki/TB_buffer TB Buffer]を加えて、懸濁する</td><td> </td><td>↓ Add 20 ml of refrigerated [http://openwetware.org/wiki/TB_buffer TB Buffer] and resuspend.</td></tr> | ||
+ | <tr><td>↓ 1.4 mlの7% DMSO(1.4 mlのDMSOに18.6 mlのTB Bufferを加えたもの)を加える</td><td> </td><td>↓ Add 1.4 ml of 7% DMSO(1.4 ml of DMSO to 18.6 ml of TB).</td></tr> | ||
+ | <tr><td>↓ 氷上で10分間冷やす</td><td> </td><td>↓ Incubate on ice for 10 minutes.</td></tr> | ||
+ | <tr><td>↓ 300 μlずつ分注する</td><td> </td><td>↓ Aliquot 300 μl of the cells.</td></tr> | ||
+ | <tr><td>↓ 液体窒素で冷凍する</td><td> </td><td>↓ Freeze the cells in liquid nitrogen.</td></tr> | ||
+ | <tr><td>↓ -80 °Cで保存する</td><td> </td><td>↓ Store at -80 °C.</td></tr> | ||
+ | </table> | ||
+ | </td></tr> | ||
+ | <tr><td> | ||
+ | <div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div> | ||
+ | </td></tr> | ||
+ | <tr><td> | ||
+ | <table width="700px" align=center> | ||
+ | <tr><td width="340px"> | ||
+ | <span id="seq">'''シークエンス'''</span></td><td width="20px"> </td><td width="340px"><span id="seqeng">'''Sequencing'''</span></td></tr> | ||
+ | <tr><td>PCR</td><td> </td><td>PCR</td></tr> | ||
+ | <tr><td>↓ 下記の組成に従って試薬を混ぜる</td><td> </td><td>↓ Mix the reagent according to the following components.</td></tr> | ||
+ | <tr><td><table border=1 width="300px"><tr><td width="100px" align=center>H<sub>2</sub>O</td><td width="200px">全量10 μlになるように調整する</td></tr><tr><td align=center>Premix</td><td>2 μl</td></tr><tr><td align=center>5 x sequence buffer</td><td>1 μl</td></tr><tr><td align=center>Primer(1 μM)</td><td>1.6 μl</td></tr><tr><td align=center>プラスミド DNA</td><td>DNA量が100-250 ngになるように調整する</td></tr><tr><td> </td><td>全量 10 μl</td></tr></table></td><td> </td><td><table border=1 width="300px"><tr><td width="100px" align=center>H<sub>2</sub>O</td><td width="200px">Adjust to become total 10 μl</td></tr><tr><td align=center>Premix</td><td>2 μl</td></tr><tr><td align=center>5 x sequence buffer</td><td>1 μl</td></tr> | ||
+ | <tr><td align=center>Primer(1 μM)</td><td>1.6 μl</td></tr><tr><td align=center>Plasmid DNA</td><td>Adjust for the amount of DNA to become about 100-250 ng.</td></tr><tr><td> </td><td>10 μl system</td></tr></table></td></tr> | ||
+ | <tr><td>↓ PCRのプログラム設定</td><td> </td><td>↓ PCR program</td></tr> | ||
+ | <tr><td><table border=1 width="300px"><tr><td width="50%" align=center>Start</td><td>96 °C、1分</td></tr> | ||
+ | <tr><td align=center rowspan=3>Cycle x 30</td><td>96 °C、10秒</td></tr><tr><td>50 °C、5秒</td></tr> | ||
+ | <tr><td>60 °C、4分</td></tr><tr><td align=center>End</td><td>4 °Cで保つ</td></tr></table></td><td> </td><td><table border=1 width="300px"><tr><td align=center width="100px">Start</td><td width="200px">96 °C for 1 minute.</td></tr><tr><td align=center rowspan=3>Cycle x 30</td><td width=>96 °C for 10 seconds. </td></tr><tr><td>50 °C for 5 seconds.</td></tr><tr><td>60 °C for 4 minutes.</td></tr><tr><td align=center>End</td><td>Keep at 4 °C forever. | ||
+ | </td></tr></table></td></tr> | ||
+ | <tr><td>エタノール沈殿</td><td> </td><td>EtOH precipitation</td></tr> | ||
+ | <tr><td>↓ PCRの後、1.5 mlチューブに移す</td><td> </td><td>↓ Remove 1.5 ml tube after PCR.</td></tr> | ||
+ | <tr><td>↓ 1.5 μlの3 M 酢酸ナトリウムを加える</td><td> </td><td>↓ Add 1.5 μl of 3 M CH3COONa.</td></tr> | ||
+ | <tr><td>↓ 氷上で15分間冷やす</td><td> </td><td>↓ Incubate on ice for 15 minutes.</td></tr> | ||
+ | <tr><td>↓ 31.5 μlの2-プロパノールと7 μlのH<sub>2</sub>Oを加える</td><td> </td><td>↓ Add 31.5 μl of 2-propanol and 7 μl of H<sub>2</sub>O.</td></tr> | ||
+ | <tr><td>↓ 15,000 rpm、4 °Cで20分間遠心し、上清を捨てる</td><td> </td><td>↓ Centrifuge for 20 minutes at 15,000 rpm in 4 °C and discard the supernatant.</td></tr> | ||
+ | <tr><td>↓ 50 μlの70% エタノールを加える.</td><td> </td><td>↓ Add 50 μl of 70% EtOH.</td></tr> | ||
+ | <tr><td>↓ 15,000 rpm、4 °Cで20分間遠心し、上清を捨てる</td><td> </td><td>↓ Centrifuge for 20 minutes at 15,000 rpm in 4 °C and discard the supernatant.</td></tr> | ||
+ | <tr><td>↓ 乾燥させる(よく乾かす)</td><td> </td><td>↓ Dry up.(Dry it well.)</td></tr> | ||
+ | <tr><td>↓ 15 μlのHi-Di formamideに溶かす</td><td> </td><td>↓ Dissolve in 15 μl of Hi-Di formamide.</td></tr> | ||
+ | <tr><td>↓ 95 °Cで2分加熱する</td><td> </td><td>↓ Incubate for 2 minutes at 95 °C.</td></tr> | ||
+ | <tr><td>↓ 氷上で冷やす(急冷)</td><td> </td><td>↓ Incubate on ice(rapid cooling).</td></tr> | ||
+ | <tr><td>↓ 0.5 mlチューブの蓋を切り取る</td><td> </td><td>↓ Cut out the cap of the 0.5 ml tube.</td></tr> | ||
+ | <tr><td>↓ サンプルを1.5 mlチューブから0.5 mlチューブに移す</td><td> </td><td>↓ Remove the sample in the 1.5 ml tube to 0.5 ml tube.</td></tr> | ||
+ | <tr><td>↓ シークエンス用の蓋をする</td><td> </td><td>↓ Do the cap for the sequencing.</td></tr> | ||
+ | <tr><td>↓ シークエンサーに入れる</td><td> </td><td>↓ Take the sample in to sequencer.</td></tr> | ||
+ | </table> | ||
+ | </td></tr> | ||
+ | <tr><td> | ||
+ | <div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div> | ||
+ | </td></tr> | ||
+ | <tr><td> | ||
+ | <table width="700px" align=center> | ||
+ | <tr><td width="340px"><span id="gly">'''グリセロールストック'''</span></td><td width="20px"> </td><td width="340px"> | ||
+ | <span id="glyeng">'''Glycerol stock'''</span></td></tr> | ||
+ | <tr><td>↓ <I>E.coli</I> (DH5 Alpha)をLBプレートにまき、37 °Cで一晩培養する</td><td> </td><td>↓ Streak <I>E.coli</I> (DH5 Alpha) to LB plate and cultivate overnight in 37 °C.</td></tr> | ||
+ | <tr><td>↓ シングルコロニーをピックアップして、2 mlのLB培地で37 °Cで一晩培養する</td><td> </td><td>↓ Pick up a single colony from plate and cultivate 2 ml of LB medium overnight in 37 °C.</td></tr> | ||
+ | <tr><td>↓ 1.5 mlチューブ内で500 μlの培養液を500 μLの50%グリセロールに加える</td><td> </td><td>↓ Add 500 μl of an overnight culture to 500 μl of 50% glycerol in a 2 mL screw top tube.</td></tr> | ||
+ | <tr><td>↓ -80 °Cでグリセロールストックを冷凍する</td><td> </td><td>↓ Freeze the glycerol stock tube at -80 °C.</td></tr> | ||
+ | </table> | ||
+ | </td></tr> | ||
+ | <tr><td> | ||
+ | <div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div> | ||
+ | |||
+ | </td></tr> | ||
+ | |||
+ | <tr><td> | ||
+ | <table width="700px" align=center> | ||
+ | <tr><td width="340px"><span id="lb">'''LB培地'''</span></td><td width="20px"> </td><td width="340px"> | ||
+ | <span id="lbeng">'''LB culture medium'''</span></td></tr> | ||
+ | <tr><td>↓ 下記の組成に従って試薬を混ぜる</td><td> </td><td> ↓Mix the reagent according to the following components.</td></tr> | ||
+ | <tr><td><table border=1 width="300px"><tr><td width="200px" align=center>Bacto | ||
+ | Tryptone</td><td align=right>10 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>5 g</td></tr> | ||
+ | <tr><td align=center>NaCl</td><td align=right>1 g</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td><td> </td><td><table border=1 width="300px"><tr><td width="200px" align=center>Bacto Tryptone</td><td align=right>10 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>5 g</td></tr> | ||
+ | <tr><td align=center>NaCl</td><td align=right>1 g</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td></tr> | ||
+ | <tr><td>↓ 121 °Cで20分間オートクレーブする</td><td> </td><td>↓ Autoclave for 20 minutes at 121 °C.</td></tr> | ||
+ | <tr><td>LBプレートを作るときは、20 gの寒天を加え、オートクレーブ後、20 mlずつプレートに温かい培地を注ぐ</td><td> </td><td>If you make LB plates, add 20 g of agar and after autoclave pour 20 ml of warm media into each plate.</td></tr> | ||
+ | <tr><td>抗生物質入りのプレートを作るときは、オートクレーブ後、冷めた培地に抗生物質を加える</td><td> </td><td>If you make LB plates with the antibiotic, after autoclave add the antibiotic to the medium cooled.</td></tr> | ||
+ | </table> | ||
+ | </td></tr> | ||
+ | <tr><td> | ||
+ | |||
+ | <div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div> | ||
+ | |||
+ | </td></tr> | ||
+ | |||
+ | <tr><td> | ||
+ | <table width="700px" align=center> | ||
+ | <tr><td width="340px"><span id="yt">'''2 x YT培地'''</span></td><td width="20px"> </td><td width="340px"> | ||
+ | <span id="yteng">'''2 x YT culture medium'''</span></td></tr> | ||
+ | <tr><td>↓ 下記の組成に従って試薬を混ぜる</td><td> </td><td>↓ Mix the reagent according to the following components.</td></tr> | ||
+ | <tr><td><table border=1 width="300px"><tr><td width="200px" align=center>Bacto Tryptone</td><td align=right>16 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>10 g</td></tr><tr><td align=center>NaCl</td><td align=right>5 g</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td><td> </td><td><table border=1 width="300px"><tr><td width="200px" align=center>Bacto Tryptone</td><td align=right>16 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>10 g</td></tr> | ||
+ | <tr><td align=center>NaCl</td><td align=right>5 g</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td></tr> | ||
+ | <tr><td>↓ 121 °Cで20分間オートクレーブする</td><td> </td><td>↓ Autoclave for 20 minutes at 121 °C.</td></tr> | ||
+ | </table> | ||
+ | </td></tr> | ||
+ | <tr><td> | ||
+ | |||
+ | <div align="right"><span style="font-ize:10pt;">[[#Contents|>>back to Contents]]</span></div> | ||
+ | |||
+ | </td></tr> | ||
+ | |||
+ | |||
+ | <tr><td> | ||
+ | <table width="700px" align=center> | ||
+ | <tr><td width="340px"><span id="sob">'''SOB培地'''</span></td><td width="20px"> </td><td width="340px"> | ||
+ | <span id="sobeng">'''SOB culture medium'''</span></td></tr> | ||
+ | <tr><td>↓ 下記の組成に従って試薬を混ぜる</td><td> </td><td>↓ Mix the reagent according to the following components.</td></tr> | ||
+ | <tr><td><table border=1 width="300px"><tr><td align=center width="200px">Bacto Tryptone</td><td align=right width="100px">20 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>5g</td></tr><tr><td align=center>5M NaCl</td><td align=right>2ml</td></tr><tr><td align=center>2M KCl</td><td align=right>1.25 ml</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td><td> </td><td><table border=1 width="300px"><tr><td align=center width="200px">Bacto Tryptone</td><td align=right width="100px">20 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>5g</td></tr><tr><td align=center>5M NaCl</td><td align=right>2ml</td></tr> | ||
+ | <tr><td align=center>2M KCl</td><td align=right>1.25 ml</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td></tr> | ||
+ | <tr><td>↓ 121 °Cで20分間オートクレーブする</td><td> </td><td>↓ Autoclave for 20 minutes at 121 °C.</td></tr> | ||
+ | <tr><td>↓ 10 mlの2M Mg solution (1 M MgSO<sub>4</sub>・7H<sub>2</sub>O +1 M MgCl<sub>2</sub>・6H<sub>2</sub>O)を加える</td><td> </td><td>↓ Add 10 ml of 2M Mg solution (1 M MgSO<sub>4</sub>・7H<sub>2</sub>O +1 M MgCl<sub>2</sub>・6H<sub>2</sub>O).</td></tr> | ||
+ | </table> | ||
+ | </td></tr> | ||
+ | <tr><td> | ||
+ | |||
+ | <div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div> | ||
+ | |||
+ | </td></tr> | ||
+ | |||
+ | <tr><td> | ||
+ | <table width="700px" align=center> | ||
+ | <tr><td width="340px"><span id="soc">'''SOC培地'''</span></td><td width="20px"> </td><td width="340px"> | ||
+ | <span id="soceng">'''SOC culture medium'''</span></td></tr> | ||
+ | <tr><td>↓ 下記の組成に従って試薬を混ぜる</td><td> </td><td>↓ Mix the reagent according to the following components.</td></tr> | ||
+ | <tr><td><table border=1 width="300px"><tr><td align=center width="200px">Bacto Tryptone</td><td align=right width="100px">20 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>5g</td></tr><tr><td align=center>5M NaCl</td><td align=right>2ml</td></tr><tr><td align=center>2M KCl</td><td align=right>1.25 ml</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td><td> </td><td><table border=1 width="300px"><tr><td align=center width="200px">Bacto Tryptone</td><td align=right width="100px">20 g</td></tr><tr><td align=center>Extract of Yeast</td><td align=right>5g</td></tr><tr><td align=center>5M NaCl</td><td align=right>2ml</td></tr><tr><td align=center>2M KCl</td><td align=right>1.25 ml</td></tr><tr><td align=center>H<sub>2</sub>O</td><td align=right>1 L</td></tr></table></td></tr> | ||
+ | <tr><td>↓ 121 °Cで20分間オートクレーブする</td><td> </td><td>↓ Autoclave for 20 minutes at 121 °C.</td></tr> | ||
+ | <tr><td>↓ 10 mlの2M Mg solution (1 M MgSO<sub>4</sub>・7H<sub>2</sub>O +1 M MgCl<sub>2</sub>・6H<sub>2</sub>O)を加える</td><td> </td><td>↓ Add 10 ml of 2M Mg solution (1 M MgSO<sub>4</sub>・7H<sub>2</sub>O +1 M MgCl<sub>2</sub>・6H<sub>2</sub>O).</td></tr> | ||
+ | <tr><td>↓ 培地が50 °C以下に冷めた後、20 mlの滅菌済みの20%グルコース溶液を加える</td><td> </td><td>↓ After cooling medium to less than 50°C, add 20 ml filter sterilized 20% glucose solution.</td></tr> | ||
+ | </table> | ||
+ | </td></tr> | ||
+ | <tr><td> | ||
+ | |||
+ | <div align="right"><span style="font-size:10pt;">[[#Contents|>>back to Contents]]</span></div> | ||
+ | |||
+ | </td></tr> | ||
</table> | </table> | ||
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Latest revision as of 15:33, 26 October 2010
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