https://eln.excellgen.com/index.php?action=history&feed=atom&title=2379_pT7A3-Cre-%28Tev%29-Tat-NLS-iFLP-6H 2026-10-18T23:08:05Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=2379_pT7A3-Cre-(Tev)-Tat-NLS-iFLP-6H&diff=13925&oldid=prev 2015-02-04T20:06:09Z

<p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 2379 pT7A3-Cre-(Tev)-Tat-NLS-iFLP-6H&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2379 pT7A3-Cre-(Tev)-Tat-NLS-iFLP-6H-&#039;&#039;&#039;==&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2014-12---- <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:2379&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039;&amp;nbsp; shanyi <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === CLONING ===<br /> <br /> subclone Flp from 2203 pCold-MBP-Tat-NLS-iFLP-6H using Nde1 + Xho1 TO 2378 pT7A3-Cre <br /> <br /> [[Image:Digest for 2364 2379 2385..JPG]]&lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2014_12&lt;br&gt; <br /> <br /> Make miniprep, useNde1 + Xho1 to find positive clone&lt;br&gt; <br /> <br /> [[Image:2364 2379 2385DIGEST.JPG]]&lt;br&gt; <br /> <br /> === Purification ===<br /> <br /> 02/02/2015-02/04/2015&lt;br&gt;<br /> <br /> [[Image:2379purification.jpg]]&lt;br&gt;<br /> <br /> sample (15ml from 1L culture)&lt;br&gt;<br /> <br /> lane1:RT 0.2mM IPTG Induce overnight,, &lt;br&gt;<br /> <br /> lane2:30oC&amp;nbsp; 0.4mM IPTG Induce overnight,,&lt;br&gt;<br /> <br /> lane3:37oC 0.3mM IPTG Induce overnight,,&lt;br&gt;<br /> <br /> lane4:pellte&lt;br&gt;<br /> <br /> load lysate to Ni column, elute, protein expression level is very low&lt;br&gt;<br /> <br /> dilute(concentration of Nacl is 30mM), load to spFF, before loading A280=0.26 , after loading A280=0.18, 250ml&lt;br&gt;<br /> <br /> 50-500 mM Nacl elute, no peak, 1M Nacl elute , small peak, run gel, it is not the cre-flp<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 /> <br /> [[Category:Cloning]]</div>

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