https://eln.excellgen.com/index.php?action=history&feed=atom&title=2015-pEC2 2015-pEC2 - Revision history 2026-10-18T18:29:56Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=2015-pEC2&diff=11359&oldid=prev Paul.xu at 22:33, 4 March 2014 2014-03-04T22:33:56Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 2015-pEC2&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 2015-pEC2 <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2014-2-23---2014-2-28 <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:2015&#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:2015 2076pcr 2-21-2014.JPG]][[Image:2015pcr3 2-21-2014.JPG]] <br /> <br /> pcr1&lt;br&gt;Fragment 1 pUC19 as template&lt;br&gt;M13-F-pUC19-F+pUC19-HindIII-R&lt;br&gt; <br /> <br /> pcr2&lt;br&gt; <br /> <br /> Fragment 2 pUC19 as template&lt;br&gt;pUC19-HindIII-F+pUC19-BglII-R&lt;br&gt; <br /> <br /> pcr3&lt;br&gt; <br /> <br /> gene synthesis <br /> <br /> 1 ul of 10 uM BglII-T7-Term&lt;br&gt;1 ul of 1 uM of the following oligos&lt;br&gt;pEC2-1 &lt;br&gt;pEC2-2 &lt;br&gt;pEC2-3 &lt;br&gt;pEC2-4 &lt;br&gt; <br /> <br /> 1 ul of 10 uM pFastBacF-Nco1-6H-Nde1-R&lt;br&gt; <br /> <br /> pcr4&lt;br&gt; <br /> <br /> pEC as template <br /> <br /> pFastBacF-Nco1-6H-Nde1-F+M13-F-pUC19-R&lt;br&gt; <br /> <br /> [[Image:2012 2014 2015 2076 2078PCR 2-24-2014.JPG]]&lt;br&gt; <br /> <br /> PCR:Gel purify these 4 fragments&lt;br&gt;mix, and PCR again using&lt;br&gt;BglII-T7-Term+pUC19-BglII-R&lt;br&gt; <br /> <br /> [[Image:2012 2015 2076 2082clone 2-25-2014.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 /> | 3&lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | PCR&lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | BglII&lt;br&gt;<br /> |}<br /> <br /> the final PCR product should be 2.5 kb.&lt;br&gt;digest with BglII, purify and ligate.&lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2014-2-28 <br /> <br /> Make miniprep and digest with <br /> <br /> [[Image:2015digest 2-28-2014.JPG]]&lt;br&gt; <br /> <br /> Digest with HindIII alone, run on EB gel:&amp;nbsp; should get 1kb and 1.5 kb fragments<br /> <br /> [[Image:2015 HindIII cut.JPG]][[Image:Lambda EcoR1 HindIII.jpg]]&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