https://eln.excellgen.com/index.php?action=history&feed=atom&title=1234_pET30b-M1wt-1-165 2026-10-18T09:55:13Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1234_pET30b-M1wt-1-165&diff=9782&oldid=prev 2013-07-19T12:50:14Z

<p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1234 pET30b-M1wt-1-165&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of&amp;nbsp; ORF for 1-165 aa of wt Flu M1 <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2012-6-5 <br /> <br /> &#039;&#039;&#039;Notebook:&amp;nbsp;&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Page:&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Clone ID: 1204&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; Paul <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === cloning ===<br /> <br /> Date: 5-29-2012 <br /> <br /> PCR using Kapa HiFi.&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> cut out band and purify using gel extraction kit. digest with Nde1 + Xho1. ligate into Nde1 + Xho1 digested pET28b. <br /> <br /> transform DH5alpha <br /> <br /> &lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 6/7/12 <br /> <br /> digest miniprep with Nde1 + Xho1 <br /> <br /> [[Image:1204 1234 6 7 2012.jpg]]&lt;br&gt; <br /> <br /> send clone #6 to sequencing&lt;br&gt; <br /> <br /> sequecing: #6: no priming, #1 is MMLV (contamination) <br /> <br /> 6/10/2012 <br /> <br /> redo PCR: <br /> <br /> [[Image:1234 PCR too big 6 10 2012.jpg]] <br /> <br /> 6/11/2012&lt;br&gt; <br /> <br /> PCR <br /> <br /> 1, 2: mutant <br /> <br /> 3,4: wt <br /> <br /> [[Image:1234 6 11 2012 pcr.jpg]] <br /> <br /> Digest vector and PCR product with Nde1 + Xho1 <br /> <br /> 1: pET30b <br /> <br /> 2: PCR product <br /> <br /> [[Image:1234 6 11 2012 digestion.jpg]] <br /> <br /> digest miniprep with Nde1 + Xho1 &lt;br&gt; <br /> <br /> [[Image:1234 Screening.JPG]] <br /> <br /> &lt;br&gt; <br /> <br /> 2012-6-20 <br /> <br /> digest detect&amp;nbsp;:Nde1+Xho1 <br /> <br /> [[Image:1023+1234 digest detect 6-20-2012.JPG]] <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 /> == &#039;&#039;&#039;Cloning of 1234 pET30b-M1wt-1-165&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of&amp;nbsp; ORF for 1-165 aa of wt Flu M1 <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2013-7-17---2013-7-18<br /> <br /> &#039;&#039;&#039;Notebook: 006026&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Page:006209&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Clone ID: 1234&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; Paul <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === cloning ===<br /> <br /> template is wt M1&lt;br&gt;FluASeg-7-NdeI-F&lt;br&gt; <br /> <br /> FluASeg-7-Xho1-R&lt;br&gt; <br /> <br /> [[Image:1234PCR 7-17-2013.jpg]]&lt;br&gt; <br /> <br /> [[Image:1234clone 7-17-2013.jpg]]&lt;br&gt; <br /> <br /> {| width=&quot;200&quot; cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot;<br /> |-<br /> | lane&lt;br&gt; <br /> | 1&lt;br&gt; <br /> | 2&lt;br&gt; <br /> | 3&lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | PCR product&lt;br&gt; <br /> | pET28b&lt;br&gt; <br /> | 501,pET30b-MMLV&lt;br&gt;<br /> |-<br /> | enzymes&lt;br&gt; <br /> | colspan=&quot;3&quot; | &amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Nde1+Xho1<br /> |}<br /> <br /> &lt;br&gt; <br /> <br /> === screening ===<br /> <br /> [[Image:1234digest_7-18-2013.JPG]]<br /> <br /> [[Category:Cloning]]</div>

Paul.xu