https://eln.excellgen.com/index.php?action=history&feed=atom&title=1624-pCold-6His-GST-%28E%29-%283C%29 1624-pCold-6His-GST-(E)-(3C) - Revision history 2026-10-18T05:04:06Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1624-pCold-6His-GST-(E)-(3C)&diff=9070&oldid=prev Paul.xu at 20:14, 3 May 2013 2013-05-03T20:14:02Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1624-pCold-6His-GST-(E)-(3C)&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1624-pCold-6His-GST-(E)-(3C) <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2013-2-5----2013-2-7 <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:1624&#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:1584+1656+1624 clone 2-5-2013.JPG]]&lt;br&gt; <br /> <br /> Digest 1612 pCold-GST-E with Nde1 + Nco1--&amp;gt; vector&lt;br&gt;ligate to annealed oligos&lt;br&gt;Nde1-3C-Nco1-T+Nde1-3C-Nco1-B <br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2013-2-7 <br /> <br /> Make miniprep and digest with Pst1 + BglII <br /> <br /> [[Image:1584+1624 digest detect 2-7-2013.JPG]] <br /> <br /> &lt;br&gt; <br /> <br /> 2013-2-27&lt;br&gt; <br /> <br /> [[Image:1624 1digest detect 2-27-2013.JPG]][[Image:1624 2digest detect 2-27-2013.JPG]] <br /> <br /> 2013-3-4 <br /> <br /> [[Image:1624 digest detect 3-4-2013.JPG]] <br /> <br /> two Nhe1 sites <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 purification&#039;&#039;&#039; ==<br /> <br /> [[Image:626+1717+1719+1720 protein screening 5-2-2013.JPG]] <br /> <br /> {| border=&quot;1&quot; width=&quot;200&quot; cellspacing=&quot;1&quot; cellpadding=&quot;1&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 /> [[Category:Cloning]]</div> Paul.xu