https://eln.excellgen.com/index.php?action=history&feed=atom&title=1719-pT7A3-MBP-%28E3C%29-NRF2 1719-pT7A3-MBP-(E3C)-NRF2 - Revision history 2026-10-18T03:13:56Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1719-pT7A3-MBP-(E3C)-NRF2&diff=10016&oldid=prev Paul.xu at 15:22, 13 August 2013 2013-08-13T15:22:37Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1719-pT7A3-MBP-(E3C)-NRF2&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1719-pT7A3-MBP-(E3C)-NRF2 <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2013-4-18---2013-4--22 <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:1719&#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:1719+1723 PCR 4-11-2013.JPG]] <br /> <br /> &lt;br&gt; <br /> <br /> [[Image:1619+1704+1719 clone 4-19-2013.JPG]]&lt;br&gt; <br /> <br /> lane5:pcr product <br /> <br /> lane6:1699 pT7A3-MBP-(E3C)&lt;br&gt; <br /> <br /> enzyme:Nde1+Xho1&lt;br&gt; <br /> <br /> == Colony Screening ==<br /> <br /> Date: 2013-4-8 <br /> <br /> Make miniprep and digest with Nde1+Xho1&lt;br&gt; <br /> <br /> [[Image:1719 digest detect 4-22-2013.JPG]]&lt;br&gt; <br /> <br /> Which clone is used for making glycerol stock?&lt;br&gt; <br /> <br /> clone3 for stock <br /> <br /> == &#039;&#039;&#039;Result intepretation/Summary&#039;&#039;&#039; ==<br /> <br /> *&amp;lt;br&amp;gt;<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 /> == ==<br /> <br /> == &#039;&#039;&#039;Cloning of 1719-pT7A3-MBP-(E3C)-NRF2&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1719-pT7A3-MBP-(E3C)-NRF2 <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2013-7-10---2013-7-11 <br /> <br /> &#039;&#039;&#039;Notebook:006026&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Page:006202&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Clone ID:1719&#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 /> [[Image:1719+1748clone 7-10-2013.JPG]]&lt;br&gt; <br /> <br /> lane1: pcr product&lt;br&gt; <br /> <br /> lane3: vector 1721 pT7A3-MBP-(E3C)-SOD&lt;br&gt; <br /> <br /> enzymes: Nde1+Xho1 <br /> <br /> digest for 1hour and 30mins,then ligate <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 /> == &#039;&#039;&#039;Cloning of 1719-pT7A3-MBP-(E3C)-NRF2&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1719-pT7A3-MBP-(E3C)-NRF2 <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2013-7-11---2013- <br /> <br /> &#039;&#039;&#039;Notebook:006026&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Page:006202&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Clone ID:1719&#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 7-11-2013.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 /> [[Image:1719clone 7-11-2013.jpg]]&lt;br&gt; <br /> <br /> lane3: pcr product&lt;br&gt; <br /> <br /> lane4: vector 1699pT7A3-MBP-(E3C)&lt;br&gt; <br /> <br /> enzymes: Nde1+Xho1 <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