https://eln.excellgen.com/index.php?action=history&feed=atom&title=1748-pCold-8His-MBP-NRF2-6His 1748-pCold-8His-MBP-NRF2-6His - Revision history 2026-10-18T05:50:12Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1748-pCold-8His-MBP-NRF2-6His&diff=10019&oldid=prev Paul.xu at 15:23, 13 August 2013 2013-08-13T15:23:50Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1748-pCold-8His-MBP-NRF2-6His&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1748-pCold-8His-MBP-NRF2-6His <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2013-5-7--2013-5-10 <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:1748&#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 /> [[Image:1619+1739+1742+1748 clone 5-7-2013.JPG]] <br /> <br /> Which one is vector? which one is NRF2 donor? <br /> <br /> lane4: 1719 pT7A3-MBP-(E3C)-NRF2&lt;br&gt; <br /> <br /> lane5: 1611 pCold-MBP-(3C)&lt;br&gt; <br /> <br /> enzyme: Nde1 + Xho1 &lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2013-5-10 <br /> <br /> Make miniprep and digest with Nde1 + Xho1 <br /> <br /> [[Image:1748 digst 5-10-2013.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> Which clone&amp;nbsp;? #6? <br /> <br /> clone6 for stock <br /> <br /> == &#039;&#039;&#039;Result intepretation/Summary&#039;&#039;&#039; ==<br /> <br /> == &#039;&#039;&#039;Conclusion&#039;&#039;&#039; ==<br /> <br /> == Protein Expression ==<br /> <br /> = 2013-5-20&lt;br&gt;&lt;br&gt; =<br /> <br /> [[Image:1676+1726+1708+1748 protein purification 5-20-2013.jpg]]&lt;br&gt; <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot; width=&quot;474&quot;<br /> |-<br /> | lane&lt;br&gt; <br /> | clone ID&lt;br&gt; <br /> | host&lt;br&gt; <br /> | induction&lt;br&gt; <br /> | purification&lt;br&gt; <br /> | MW/pI&lt;br&gt; <br /> | Note<br /> |-<br /> | 1-8&lt;br&gt; <br /> | 1748,pCold-8His-MBP-NRF2-6His&lt;br&gt; <br /> | BL21(DE3)pRare2pLysS&lt;br&gt; <br /> | &lt;br&gt; <br /> | gel filtration fractions&lt;br&gt; <br /> | &lt;br&gt; <br /> | NRF2 not expressed<br /> |-<br /> | 9&lt;br&gt; <br /> | 1676,pCold-MBP-(3C)-KEAP1 &lt;br&gt; <br /> | BL21(DE3)pRare2pLysS&lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 10&lt;br&gt; <br /> | 1726,pCMV5-hIgG-Lc-PKARIIb&lt;br&gt; <br /> | 293T&lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 11&lt;br&gt; <br /> | 1708, pT7A3-HTNCre&lt;br&gt; <br /> | NiCO21(DE3)pLysN&lt;br&gt; <br /> | &lt;br&gt; <br /> | <br /> SP-FF,MEP, <br /> <br /> &amp;nbsp;&lt;br&gt; <br /> <br /> | &lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 12 <br /> | Fermentas Page-Ruler Plus <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt;<br /> |}<br /> <br /> == ==<br /> <br /> == &#039;&#039;&#039;Cloning of 1748-pCold-8His-MBP-NRF2-6His&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1748-pCold-8His-MBP-NRF2-6His <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2013-7-10--2013- <br /> <br /> &#039;&#039;&#039;Notebook:0&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Page:006201&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Clone ID:1748&#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 /> [[Image:1719PCR 7-10-2013.JPG]]&lt;br&gt; <br /> <br /> PCR NRF2 from 1622 pLX304-NRF2&lt;br&gt;NRF2-Nde1-F&lt;br&gt;NRF2-Xho1-R&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> [[Image:1719+1748clone 7-10-2013.JPG]]&lt;br&gt; <br /> <br /> lane2: pcr product&lt;br&gt; <br /> <br /> lane4:1611 pCold-MBP-(3C)&lt;br&gt; <br /> <br /> enzymes: Nde1+Xho1 <br /> <br /> &lt;br&gt; <br /> <br /> === screening ===<br /> <br /> 2013-7-11 <br /> <br /> Nde1+Xho1 <br /> <br /> [[Image:1719+1748digest 7-11-2013.jpg]] <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