https://eln.excellgen.com/index.php?action=history&feed=atom&title=1676_pCold-MBP-%283C%29-KEAP1 1676 pCold-MBP-(3C)-KEAP1 - Revision history 2026-10-18T11:23:53Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1676_pCold-MBP-(3C)-KEAP1&diff=9301&oldid=prev Paul.xu at 19:49, 29 May 2013 2013-05-29T19:49:45Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Subcloning of KEAP1 into pCold-MBP-(3C)&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Subcloning of KEAP1 from 1625 pT7A3-MBP-(3c)-KEAP1 to 1611 pCold-MBP-(3C)&lt;br&gt; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2013-2-26---2013-2-28 <br /> <br /> &#039;&#039;&#039;Notebook:&amp;nbsp;&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Page:&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Clone ID: 1676&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039;&amp;nbsp; Yingying&lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === Subcloning ===<br /> <br /> &#039;&#039;&#039;Date&#039;&#039;&#039;: <br /> <br /> &#039;&#039;&#039;Vector&#039;&#039;&#039;: 1611 pCold-MBP-(3C) <br /> <br /> &#039;&#039;&#039;Parent:&#039;&#039;&#039; 1625 pT7A3-MBP-(3c)-KEAP1&lt;br&gt; <br /> <br /> &#039;&#039;&#039;Enzymes&#039;&#039;&#039;: Nde1 + Xho1&lt;br&gt; <br /> <br /> [[Image:1541+1585+1676 clone 2-26-2013.JPG]]&lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2013-2-28&lt;br&gt; <br /> <br /> Nde1 + Xho1&lt;br&gt; <br /> <br /> [[Image:1541+1585+1676 digest detect 2-28-2013.JPG]] <br /> <br /> == Protein Expression ==<br /> <br /> 2013/3/5&lt;br&gt; <br /> <br /> [[Image:1661 1674 1683 1676 3 6 2013.jpg]]&lt;br&gt; <br /> <br /> {| width=&quot;684&quot; cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot;<br /> |-<br /> | Lane&lt;br&gt; <br /> | Name&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 /> |-<br /> | M&lt;br&gt; <br /> | Fermentas PageRuler Plus&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 /> | pT7A3-MBP-(3c)-NRF2 &lt;br&gt; <br /> | <br /> 1661 <br /> <br /> clone 1&lt;br&gt; <br /> <br /> | <br /> 4 ml,&lt;br&gt; <br /> <br /> BL21(DE3)Star, pRare2-pLysS <br /> <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | Ni beads&lt;br&gt;<br /> |-<br /> | 2&lt;br&gt; <br /> | pT7A3-MBP-(3c)-KEAP1&lt;br&gt; <br /> | <br /> 1661 <br /> <br /> Clone 2&lt;br&gt; <br /> <br /> | <br /> 4 ml, <br /> <br /> BL21(DE3)Star, pRare2-pLysS &lt;br&gt; <br /> <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | Ni beads&lt;br&gt;<br /> |-<br /> | 3&lt;br&gt; <br /> | pCold-GST-(3c)-NRF2&lt;br&gt; <br /> | 1674&lt;br&gt; <br /> | <br /> 4 ml, <br /> <br /> BL21(DE3)Star, pRare2-pLysS &lt;br&gt; <br /> <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | Ni beads (wrong, should use Glutathione agarose beads, no his tag)&lt;br&gt;<br /> |-<br /> | 4&lt;br&gt; <br /> | pCold-GST-(3c)-NRF2&lt;br&gt; <br /> | 1674&lt;br&gt; <br /> | <br /> 4 ml, <br /> <br /> BL21(DE3)Star, pRare2-pLysS &lt;br&gt; <br /> <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | Ni beads (wrong, should use Glutathione agarose beads, no his tag)&lt;br&gt;<br /> |-<br /> | 5&lt;br&gt; <br /> | pCold-MBP-(3C)-NRF2&lt;br&gt; <br /> | 1683&lt;br&gt; <br /> | <br /> 4 ml, <br /> <br /> BL21(DE3)Star, pRare2-pLysS &lt;br&gt; <br /> <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | Ni beads&lt;br&gt;<br /> |-<br /> | 6&lt;br&gt; <br /> | pCold-MBP-(3C)-NRF2&lt;br&gt; <br /> | 1683&lt;br&gt; <br /> | 4 ml,BL21(DE3)Star, pRare2-pLysS &lt;br&gt; <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | Ni beads&lt;br&gt;<br /> |-<br /> | 7&lt;br&gt; <br /> | pT7A3-MBP-(3c)-KEAP1&lt;br&gt; <br /> | 1625&lt;br&gt; <br /> | 4 ml,BL21(DE3)Star, pRare2-pLysS &lt;br&gt; <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | Ni beads&lt;br&gt;<br /> |-<br /> | 8&lt;br&gt; <br /> | pCold-MBP-(3C)-KEAP1&lt;br&gt; <br /> | 1676&lt;br&gt; <br /> | 4 ml,BL21(DE3)Star, pRare2-pLysS &lt;br&gt; <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | Ni beads&lt;br&gt;<br /> |-<br /> | 9 <br /> | <br /> | <br /> | <br /> | <br /> | <br /> |-<br /> | 10 <br /> | Purified MBP <br /> | <br /> | <br /> | <br /> | <br /> |}<br /> <br /> == &#039;&#039;&#039;Result intepretation/Summary&#039;&#039;&#039; ==<br /> <br /> &lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Conclusion&#039;&#039;&#039; ==<br /> == Protein expression of KEAP1 clones&lt;br&gt; ==<br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot; width=&quot;415&quot;<br /> |-<br /> | clone ID&lt;br&gt; <br /> | expression&lt;br&gt; <br /> | ge image&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1625,pT7A3-MBP-(3c)-KEAP1&lt;br&gt; <br /> | yes&lt;br&gt; <br /> | [[1625 1626 1656]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1656,pCold-GST-(3c)-KEAP1&lt;br&gt; <br /> | need to use GST beads&lt;br&gt; <br /> | [[1625 1626 1656]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1676,pCold-MBP-(3C)-KEAP1&lt;br&gt; <br /> | yes&lt;br&gt; <br /> | [[1661 1674 1683]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1701,pMAL-c5E-KEAP1&lt;br&gt; <br /> | yes&lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | 1720,pT7A3-MBP-(E3C)-Keap1&lt;br&gt; <br /> | no&lt;br&gt; <br /> | [[626 1717 1719 1720]]&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt;<br /> |}<br /> <br /> &lt;br&gt; <br /> <br /> [[Category:Cloning]]</div> Paul.xu