Team:KIT-Kyoto/Protocol
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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 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>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>インサート DNA</td><td align=right>0.5-6.5 μl</td></tr> | ||
- | <tr><td align=center> | + | <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>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>Insert DNA</td><td align=right>0.5-6.5 μl</td></tr> | ||
- | <tr><td align=center> | + | <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>↓ 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 | <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 | ||
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<tr><td>PCR</td><td> </td><td>PCR</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>↓ 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> | + | <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 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>↓ PCRのプログラム設定</td><td> </td><td>↓ PCR program</td></tr> | ||
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<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>↓ 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>↓ 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 | + | <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>↓ 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>↓ 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> | ||
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<tr><td><table border=1 width="300px"><tr><td width="200px" align=center>Bacto | <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> | 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> | + | <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> | + | <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>↓ 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>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> | ||
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<span id="yteng">'''2 x YT culture medium'''</span></td></tr> | <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>↓ 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> | + | <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> | + | <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> | <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> | </table> | ||
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<tr><td align=center>2M KCl</td><td align=right>1.25 ml</td></tr></table></td></tr> | <tr><td align=center>2M KCl</td><td align=right>1.25 ml</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>↓ 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 | + | <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> | </table> | ||
</td></tr> | </td></tr> | ||
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<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></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></table></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></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></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>↓ 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 | + | <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> | <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> | </table> |
Revision as of 10:11, 24 October 2010
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