https://eln.excellgen.com/index.php?action=history&feed=atom&title=1465_pT7A3-MBP-3C 2026-10-18T15:05:29Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1465_pT7A3-MBP-3C&diff=8278&oldid=prev 2013-02-19T16:12:43Z

<p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1465 pT7A3-MBP-3C&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1465 pT7A3-MBP-3C, native 3C, not&amp;nbsp; E coli optimized&lt;br&gt; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2012-11-21-- &#039;&#039;&#039;Notebook:0&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Page:&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Clone ID:1465&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; SHANYI <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === cloning ===<br /> <br /> [[Image:1454 1037 1279 1420 1429 digest11 21 2012.JPG]]&lt;br&gt; <br /> <br /> subclone from 1420 using Nde1 + Xho1 to 1429 <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2012-11- <br /> <br /> digest with Nde1 + Xho1 <br /> <br /> [[Image:1459 1464 1465 DIGEST.JPG]] &lt;br&gt; <br /> <br /> = &lt;br&gt; =<br /> <br /> == Protein Expression&lt;br&gt; ==<br /> <br /> 2/18/2013 <br /> <br /> &lt;br&gt; <br /> <br /> [[Image:2 18 2013 protein SDS PAGE.jpg]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> {| width=&quot;552&quot; cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot;<br /> |-<br /> | Lane&lt;br&gt; <br /> | Clone ID&lt;br&gt; <br /> | Name&lt;br&gt; <br /> | Gene Name&lt;br&gt; <br /> | Host&lt;br&gt; <br /> | Induction <br /> | Purification&lt;br&gt; <br /> | Note&lt;br&gt;<br /> |-<br /> | M&lt;br&gt; <br /> | Invitrogen&lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1&lt;br&gt; <br /> | 1465&lt;br&gt; <br /> | pT7A3-MBP-3C&lt;br&gt; <br /> | MBP-Native 3C&lt;br&gt; <br /> | 3 ml&lt;br&gt; <br /> | &lt;br&gt; <br /> | Ni, &lt;br&gt; <br /> | Only MBP Expressed&lt;br&gt;<br /> |-<br /> | 2&lt;br&gt; <br /> | 1661&lt;br&gt; <br /> | pT7A3-MBP-(3c)-NRF2&lt;br&gt; <br /> | MBP-NRF2&lt;br&gt; <br /> | 3 ml, pRare2&lt;br&gt; <br /> | &lt;br&gt; <br /> | Ni&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 3&lt;br&gt; <br /> | 1661&lt;br&gt; <br /> | pT7A3-MBP-(3c)-NRF2&lt;br&gt; <br /> | MBP-NRF2&lt;br&gt; <br /> | 3 ml, pRare2PLysS&lt;br&gt; <br /> | &lt;br&gt; <br /> | Ni&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 4-8&lt;br&gt; <br /> | 1613&lt;br&gt; <br /> | pET28-cold-GST-3C&lt;br&gt; <br /> | GST-Native 3C&lt;br&gt; <br /> | 8L&lt;br&gt; <br /> | &lt;br&gt; <br /> | After Ni and Glutathione column&lt;br&gt; <br /> | aggregate after sitting at 4 oC for 7 days, probably due to oxidation, need to add b-ME&lt;br&gt;<br /> |-<br /> | 9-11&lt;br&gt; <br /> | 1613&lt;br&gt; <br /> | pET28-cold-GST-3C&lt;br&gt; <br /> | GST-Native 3C&lt;br&gt; <br /> | 8L&lt;br&gt; <br /> | &lt;br&gt; <br /> | After Ni&lt;br&gt; <br /> | extract protein from cell pellet of 4~8 (above)&lt;br&gt;<br /> |}<br /> <br /> Note: 9~11: Add 300 ml of buffer A to cell pellets from previous week purification, homogenize,&amp;nbsp; spin and load supernatant to Ni column. Wash with Buffer A and elute with Buffer B (300 mM Imidazole). Got a lot of protein from pellet. &lt;br&gt; <br /> <br /> [[Category:Cloning]]</div>

Paul.xu