https://eln.excellgen.com/index.php?action=history&feed=atom&title=1262-pCMV5-Fc%283C%29 2026-10-18T02:19:12Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1262-pCMV5-Fc(3C)&diff=5431&oldid=prev 2012-08-02T22:00:55Z

<p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1262-pCMV5-Fc(3C)&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1262-pCMV5-Fc(3C) <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2012-7-6---2012-7-9 <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:1262&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; yingying <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === subcloning ===<br /> <br /> [[Image:1223+1254+1262 clone 7-6-2012.JPG]]&lt;br&gt; <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot; style=&quot;width: 330px; height: 96px;&quot;<br /> |-<br /> | &lt;br&gt; <br /> | band 5&lt;br&gt; <br /> | band 6&lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | pLex-CMV5-CuO 448&lt;br&gt; <br /> | 1219 pCDNA3.3-Fc(3C)&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; Nde1 + BamH1&lt;br&gt;<br /> |}<br /> <br /> &lt;br&gt; <br /> <br /> 1219: pCDNA3.3-Fc(3C), Nde1+ BamH1 should digest out 417 bp insert (CMV promoter, from 184 to 901). If there are three bands,&amp;nbsp; it is unlikely correct.&lt;br&gt; <br /> <br /> 448: pLex-CMV5-CuO, Has two Nde1 sites (2964, 7933), BamH1 is 3979, CMV5 promoter is&amp;nbsp; 1015 bp&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot;<br /> |-<br /> | &lt;br&gt;<br /> |}<br /> <br /> === Colony Screening ===<br /> <br /> Date: 2012-7-9 <br /> <br /> Make miniprep and digest with Nde1 + BamH1 <br /> <br /> [[Image:1223+1262 digest detect 7-9-2012.JPG]] <br /> <br /> clone 1,2,3,4,5,6 right&lt;br&gt; &lt;br&gt; <br /> <br /> Need to sequence 1262, it is unlikely good. <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 /> {|<br /> |-<br /> | &lt;br&gt;<br /> |}<br /> <br /> &lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Cloning of 1262-pCMV5-Fc(3C)&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1262-pCMV5-Fc(3C) <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2012-7-17---2012-7-19 <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:1262&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; yingying <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === subcloning ===<br /> <br /> [[Image:1262 clone 7-17-2012.JPG]][[Image:1219 digest detect 7-16-2012.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot; style=&quot;width: 330px; height: 100px;&quot;<br /> |-<br /> | &lt;br&gt; <br /> | insert&lt;br&gt; <br /> | lane 1&lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | pLex-CMV5-CuO 448&lt;br&gt; <br /> | 1219 pCDNA3.3-Fc(3C)&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; Nde1 + BamH1&lt;br&gt;<br /> |}<br /> <br /> &lt;br&gt; <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot;<br /> |-<br /> | &lt;br&gt;<br /> |}<br /> <br /> === Colony Screening ===<br /> <br /> Date: 2012-7-19 <br /> <br /> Make miniprep and digest with Nde1 + BamH1&lt;br&gt; <br /> <br /> [[Image:1262 digest detct 7-19-2012.JPG|262x272px|1262 digest detct 7-19-2012.JPG]] <br /> <br /> clone 1,2,3,5,6,7,8 may be right <br /> <br /> &lt;br&gt; <br /> <br /> digest detect again with Xho1 <br /> <br /> [[Image:1262 2digest detect 7-19-2012.JPG]] <br /> <br /> 8/2/2012; Digest 1262 with Xba1, BamH1, Nhe1<br /> <br /> {| width=&quot;279&quot; cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot; height=&quot;59&quot;<br /> |-<br /> | 1<br /> | 2<br /> | 3<br /> | 4<br /> |-<br /> | Xba1+ BamH1<br /> | Xba1<br /> | Nhe1<br /> | Nhe1+ BamH1<br /> |}<br /> <br /> <br /> <br /> [[Image:1262_2012_8_2.jpg]]<br /> <br /> [[Image:1262_2012_8_2_2.jpg]]<br /> <br /> Is it possible there are two Xba1 sites? or star activity/overdigestion?<br /> <br /> &lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Result intepretation/Summary&#039;&#039;&#039; ==<br /> <br /> *&amp;amp;lt;br&amp;amp;gt;<br /> <br /> == &#039;&#039;&#039;Conclusion&#039;&#039;&#039; ==<br /> <br /> &amp;amp;lt;br&amp;amp;gt; <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot;<br /> |-<br /> | &lt;br&gt;<br /> |}<br /> <br /> &amp;amp;lt;br&amp;amp;gt; <br /> <br /> {| cellspacing=&quot;1&quot;<br /> |-<br /> | &lt;br&gt;<br /> |}<br /> <br /> &amp;lt;br&amp;gt; <br /> <br /> {| cellspacing=&quot;1&quot;<br /> |-<br /> | &lt;br&gt;<br /> |}<br /> <br /> &lt;br&gt; <br /> <br /> {|<br /> |-<br /> | &lt;br&gt;<br /> |}<br /> <br /> [[Category:Cloning]]</div>

Paul.xu