https://eln.excellgen.com/index.php?action=history&feed=atom&title=1796-pT7A3-MBP-%283C-TEV%29-NRF2-6His 1796-pT7A3-MBP-(3C-TEV)-NRF2-6His - Revision history 2026-10-18T06:36:02Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1796-pT7A3-MBP-(3C-TEV)-NRF2-6His&diff=10023&oldid=prev Paul.xu at 15:29, 13 August 2013 2013-08-13T15:29:41Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1796-pT7A3-MBP-(3C-TEV)-NRF2-6His&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1796-pT7A3-MBP-(3C-TEV)-NRF2-6His <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2013-7-16--- <br /> <br /> &#039;&#039;&#039;Notebook:0&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Page:&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Clone ID:1796&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; yingying <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === cloning ===<br /> <br /> subclone MBP-NRF2 fragment from 1779 pTCold-MBP-(3C-TEV)-NRF2 to 1719 pT7A3-MBP-(E3C)-NRF2 using Bsa1 + EcoR1&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> [[Image:1796clone1 7-16-2013.jpg]]&lt;br&gt; <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot; width=&quot;366&quot;<br /> |-<br /> | lane&lt;br&gt; <br /> | 1 insert&lt;br&gt; <br /> | 2 vector&lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | 1779 pTCold-MBP-(3C-TEV)-NRF2&lt;br&gt; <br /> | 1719 pT7A3-MBP-(E3C)-NRF2&lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | colspan=&quot;2&quot; | &amp;nbsp;&amp;nbsp; EcoR1&lt;br&gt;<br /> |}<br /> <br /> &lt;br&gt; <br /> <br /> lanes above&amp;nbsp; do gel extraction and digest again with Bsa1 at 50C for 1hour and do ligation&lt;br&gt; <br /> <br /> [[Image:1796clone2 7-16-2013.jpg]]&lt;br&gt; <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot; width=&quot;350&quot;<br /> |-<br /> | lane&lt;br&gt; <br /> | 1 insert&lt;br&gt; <br /> | 2 vector&lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | 1779 pTCold-MBP-(3C-TEV)-NRF2&lt;br&gt; <br /> | 1719 pT7A3-MBP-(E3C)-NRF2&lt;br&gt;<br /> |-<br /> | enzymes&lt;br&gt; <br /> | colspan=&quot;2&quot; | &amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Bsa1&lt;br&gt;<br /> |}<br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2013-7-17 <br /> <br /> Make miniprep and digest with Pst1 <br /> <br /> [[Image:1796digest1 7-17-2013.JPG]] <br /> <br /> &lt;br&gt; <br /> <br /> Date: 2013-7-18 <br /> <br /> Make miniprep and digest with Xho1 + EcoR1&lt;br&gt; <br /> <br /> [[Image:1796 digest2 7-18-2013.JPG]] <br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Result intepretation/Summary&#039;&#039;&#039; ==<br /> <br /> == &#039;&#039;&#039;Conclusion&#039;&#039;&#039; ==<br /> <br /> &lt;br&gt; <br /> <br /> == protein expression result of NRF2 clones &lt;br&gt; ==<br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot; width=&quot;569&quot;<br /> |-<br /> | clone ID&lt;br&gt; <br /> | expression&lt;br&gt; <br /> | gel reslut&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1626, pET28-cold-NRF2&lt;br&gt; <br /> | no&lt;br&gt; <br /> | [[1625 1626 1656]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1661,pT7A3-MBP-(3c)-NRF2&lt;br&gt; <br /> | yes,&lt;br&gt; <br /> | [[1661 1674 1683]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1674,pCold-GST-(3c)-NRF2&lt;br&gt; <br /> | need to use GST beads&lt;br&gt; <br /> | [[1661 1674 1683]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1682,pCold-6His-GST-(E)-(3C)-NRF2&lt;br&gt; <br /> | yes&lt;br&gt; <br /> | [[1661 1682 1683 1719 1748 1752 1771 1779]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1683,pCold-MBP-(3C)-NRF2&lt;br&gt; <br /> | yes, &lt;br&gt; <br /> | [[1661 1674 1683]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1698,pT7A3-NRF2&lt;br&gt; <br /> | no&lt;br&gt; <br /> | [[1676 1759 1698 1769 1747 1769 1757 1682p]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1719,pT7A3-MBP-(E3C)-NRF2&lt;br&gt; <br /> | yes&lt;br&gt; <br /> | [[631 1497 1542 1719 1748 1765 1780 1781 1782 1797]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1748,pCold-8His-MBP-NRF2-6His&lt;br&gt; <br /> | yes&lt;br&gt; <br /> | [[631 1497 1542 1719 1748 1765 1780 1781 1782 1797]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1752,pCold-TF-(E3C)-NRF2&lt;br&gt; <br /> | yes&lt;br&gt; <br /> | [[1661 1682 1683 1719 1748 1752 1771 1779]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1769,pET28b-6His-NRF2&lt;br&gt; <br /> | no&lt;br&gt; <br /> | [[1676 1759 1698 1769 1747 1769 1757 1682p]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1796,pT7A3-MBP-(3C-TEV)-NRF2-6His&lt;br&gt; <br /> | not sure&lt;br&gt; <br /> | [[1720 1757 1759 1796 1800 1803 1804 1809 1759 MBP-Keap1 1719]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1779,pTCold-MBP-(3C-TEV)-NRF2&lt;br&gt; <br /> | yes&lt;br&gt; <br /> | [[1661 1682 1683 1719 1748 1752 1771 1779]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |}<br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> [[Category:Cloning]]</div> Paul.xu