https://eln.excellgen.com/index.php?action=history&feed=atom&title=1720-pT7A3-MBP-%28E3C%29-Keap1 1720-pT7A3-MBP-(E3C)-Keap1 - Revision history 2026-10-18T05:52:12Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1720-pT7A3-MBP-(E3C)-Keap1&diff=9870&oldid=prev Paul.xu at 18:25, 29 July 2013 2013-07-29T18:25:30Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1720-pT7A3-MBP-(E3C)-Keap1&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1519-pT7A3-T3-DNA-Ligase <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2013-4-13---2013-4-16 <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:1720&#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:1675+1719+1720+1723+1727 clone 4-11-2013.JPG]] <br /> <br /> lane5: pcr&lt;br&gt; <br /> <br /> lane6:vector:1699 pT7A3-MBP-(E3C)&lt;br&gt; <br /> <br /> Nde1 + Xho1 &lt;br&gt; <br /> <br /> == Colony Screening ==<br /> <br /> Date: 2013-4-16 <br /> <br /> Make miniprep and digest with Nde1 + Xho1 <br /> <br /> [[Image:1720 digest detect 4-16-2013.JPG]]&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 /> == &#039;&#039;&#039;protein screening&#039;&#039;&#039; ==<br /> <br /> [[Image:626+1717+1719+1720 protein screening 5-2-2013.JPG]] <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot; width=&quot;200&quot;<br /> |-<br /> | lane&lt;br&gt; <br /> | 1 <br /> | 2 <br /> | 3 <br /> | 4 <br /> | 5 <br /> | 6 <br /> | 7 <br /> | 8&lt;br&gt; <br /> | 9&lt;br&gt; <br /> | 10&lt;br&gt;<br /> |-<br /> | clone ID <br /> | &lt;br&gt;626,pT7A3-Taq_d289 <br /> | <br /> 1717,&lt;br&gt; <br /> <br /> pT7A3-6H-(TEV)-MBP-(3C) <br /> <br /> | <br /> 1719, <br /> <br /> pT7A3-MBP-(E3C)-NRF2&lt;br&gt; <br /> <br /> | 1720,pT7A3-MBP-(E3C)-Keap1&lt;br&gt; <br /> | 1719, <br /> pT7A3-MBP-(E3C)-NRF2,&lt;br&gt; <br /> <br /> fraction 38&lt;br&gt; <br /> <br /> | &lt;br&gt;1719, <br /> pT7A3-MBP-(E3C)-NRF2, <br /> <br /> fraction 23&lt;br&gt; <br /> <br /> | <br /> cre, <br /> <br /> 3.9ug/ul&lt;br&gt; <br /> <br /> | cre,4/2/2013&lt;br&gt; <br /> | 1624,pCold-6His-GST-(E)-(3C)&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | host&lt;br&gt; <br /> | &lt;br&gt;Nico21DE3plysN&lt;br&gt; <br /> | Nico21DE3plysN&lt;br&gt; <br /> | BL21DE3pRare2pLysS&lt;br&gt; <br /> | BL21DE3pRare2pLysS&lt;br&gt; <br /> | BL21DE3pRare2pLysS&lt;br&gt; <br /> | &lt;br&gt;BL21DE3pRare2pLysS&lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | Nico21DE3plysN&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | induction&lt;br&gt; <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | 0.2 mM IPTG 25 oC o/n&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | purification&lt;br&gt; <br /> | Ni beads&lt;br&gt; <br /> | Ni beads&lt;br&gt; <br /> | Ni beads&lt;br&gt; <br /> | Ni beads&lt;br&gt; <br /> | 5ml HisTrap HP&lt;br&gt; <br /> | 5ml HisTrap HP&lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | Ni beads&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | MW&lt;br&gt; <br /> | 61KD/5.97&lt;br&gt; <br /> | 49KD/5.46&lt;br&gt; <br /> | 112KD/4.82&lt;br&gt; <br /> | 115KD/5.57&lt;br&gt; <br /> | 112KD/4.82&lt;br&gt; <br /> | 112KD/4.82&lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | 33KD/6.0&lt;br&gt; <br /> | &lt;br&gt;<br /> |}<br /> <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 /> == &#039;&#039;&#039;Cloning of 1720-pT7A3-MBP-(E3C)-Keap1&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1519-pT7A3-T3-DNA-Ligase <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2013-7-26--- <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:1720&#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:1720clone 7-26-2013.JPG]]&lt;br&gt; <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot; width=&quot;415&quot;<br /> |-<br /> | lane&lt;br&gt; <br /> | 1&lt;br&gt; <br /> | 2&lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | 1720,pT7A3-MBP-(E3C)-Keap1(old clone)&lt;br&gt; <br /> | 1718 pT7A3-MBP-(3C)-MCS-(TEV)-6H&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; Nde1 + Xho1&lt;br&gt;<br /> |}<br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2013-7-28<br /> <br /> Make miniprep <br /> <br /> digest with BglII <br /> <br /> [[Image:1720 BglII.JPG]] <br /> <br /> transform BL21star(DE3)pRare2pLysS with clone #4 <br /> <br /> &lt;br&gt;<br /> <br /> &lt;br&gt;<br /> <br /> Date: 2013-7-29<br /> <br /> digest with Nde1 + Xho1&lt;br&gt;<br /> <br /> [[Image:1720digest_7-29-2013.JPG]]<br /> <br /> &lt;br&gt;<br /> <br /> &lt;br&gt;<br /> <br /> [[Category:Cloning]]</div> Paul.xu