Team:KIT-Kyoto/Protocol
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+ | <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>H2O</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>H2O</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>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H2Oを加える</td><td> </td><td>↓ Add 31.5 μl of 2-propanol and 7 μl of H2O.</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> | ||
Revision as of 08:57, 24 October 2010
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