https://eln.excellgen.com/index.php?action=history&feed=atom&title=1835-pT7A3-MBP-Taq-Muts-6His 1835-pT7A3-MBP-Taq-Muts-6His - Revision history 2026-10-18T16:06:36Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1835-pT7A3-MBP-Taq-Muts-6His&diff=10364&oldid=prev Paul.xu at 23:10, 1 October 2013 2013-10-01T23:10:52Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1835-pT7A3-MBP-Taq-Muts-6His&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1835-pT7A3-MBP-Taq-Muts-6His <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2013-9-17---2013=9-20 <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:1835&#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:1835PCR 9-4-2013.JPG]]&lt;br&gt; <br /> <br /> PCR from 352 pET3a-Muts &lt;br&gt; <br /> <br /> Taq-Muts-F+Taq-Muts-HindIII-R &lt;br&gt; <br /> <br /> [[Image:556+673+1835clone 9-18-2013.JPG]]&lt;br&gt; <br /> <br /> {| width=&quot;416&quot; border=&quot;1&quot; cellspacing=&quot;1&quot; cellpadding=&quot;1&quot;<br /> |-<br /> | lane&lt;br&gt; <br /> | 1&lt;br&gt; <br /> | 2&lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | PCR product&lt;br&gt; <br /> | 1699 pT7A3-MBP-(E3C)&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; Nde1 + HindIII &lt;br&gt;<br /> |}<br /> <br /> === Colony Screening ===<br /> <br /> Date: 2013-9-20 <br /> <br /> Make miniprep and digest with Nde1 + HindIII <br /> <br /> [[Image:1835digest 9-20-2013.JPG]] <br /> <br /> &lt;br&gt; <br /> <br /> &amp;lt;br&amp;gt; <br /> <br /> == Protein Purification ==<br /> <br /> &lt;br&gt; 9/29/2013&lt;br&gt; <br /> <br /> Purification of Nde1 using 25 ml heparin HyperD column (XK16).&lt;br&gt; <br /> <br /> Dilute partial purified Nde1 with 50 mM Tris-HCl. Load to heparin column.&lt;br&gt; <br /> <br /> Elute with 250 ml linear gradient of 0.1 M to 0.6 M NaCl. Nde1 is eluted at 0.4 M NaCl.&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> [[Image:Nde1 purification.jpg]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> {| width=&quot;305&quot; border=&quot;1&quot; cellspacing=&quot;1&quot; cellpadding=&quot;1&quot;<br /> |-<br /> | Lane&lt;br&gt; <br /> | Clone ID&lt;br&gt; <br /> | Desc&lt;br&gt;<br /> |-<br /> | 1&lt;br&gt; <br /> | 1710&lt;br&gt; <br /> | Nde1, Eluted from Heparin column&lt;br&gt;<br /> |-<br /> | 2&lt;br&gt; <br /> | 1710&lt;br&gt; <br /> | Nde1, Eluted from Heparin column&lt;br&gt;<br /> |-<br /> | 3&lt;br&gt; <br /> | 1710&lt;br&gt; <br /> | Nde1, Eluted from Heparin column&lt;br&gt;<br /> |-<br /> | 4&lt;br&gt; <br /> | 1813&lt;br&gt; <br /> | FLP, 50 mM I wash from Ni column &lt;br&gt;<br /> |-<br /> | 5&lt;br&gt; <br /> | 1813&lt;br&gt; <br /> | FLP, 300 mM I eluted from Ni, diluted&lt;br&gt;<br /> |-<br /> | 6&lt;br&gt; <br /> | 1710&lt;br&gt; <br /> | Nde1, Eluted from Heparin column&lt;br&gt;<br /> |-<br /> | 7&lt;br&gt; <br /> | 1813&lt;br&gt; <br /> | FLP, 300 mM I eluted from Ni, diluted&lt;br&gt;<br /> |-<br /> | 8&lt;br&gt; <br /> | 1813&lt;br&gt; <br /> | FLP, heparin column FT&lt;br&gt;<br /> |-<br /> | 9&lt;br&gt; <br /> | 1813&lt;br&gt; <br /> | FLP, 50 mM I wash from Ni column&lt;br&gt;<br /> |-<br /> | 10&lt;br&gt; <br /> | 1835&lt;br&gt; <br /> | MBP-Taq-Muts-6XHis, partial purified from Ni column&lt;br&gt;<br /> |-<br /> | 11&lt;br&gt; <br /> | 1813&lt;br&gt; <br /> | FLP heparin column elute&lt;br&gt;<br /> |-<br /> | M&lt;br&gt; <br /> | &lt;br&gt; <br /> | Fermentas PageRuler Plus&lt;br&gt;<br /> |-<br /> | 12&lt;br&gt; <br /> | 1813&lt;br&gt; <br /> | FLP heparin column elute&lt;br&gt;<br /> |-<br /> | 13&lt;br&gt; <br /> | 1813&lt;br&gt; <br /> | FLP heparin column elute&lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt;<br /> |}<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 /> &lt;br&gt; <br /> <br /> [[Category:Cloning]]</div> Paul.xu