Team:KIT-Kyoto/Protocol
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+ | <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 cluture 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> | ||
Revision as of 07:23, 24 October 2010
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