https://eln.excellgen.com/index.php?action=history&feed=atom&title=1336_pCMV5-Fc%283C%29-Dest-WPRE 1336 pCMV5-Fc(3C)-Dest-WPRE - Revision history 2026-10-18T12:52:01Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1336_pCMV5-Fc(3C)-Dest-WPRE&diff=5846&oldid=prev Paul.xu at 17:51, 29 August 2012 2012-08-29T17:51:56Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1336 pCMV5-Fc(3C)-Dest-WPRE&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of&amp;nbsp;WPRE into vector&amp;nbsp; pCMV5-Fc(3C)-Dest to make 1336 pCMV5-Fc(3C)-Dest-WPRE&lt;br&gt; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2012-8-17 to&amp;nbsp; &lt;br&gt; <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: 1328&#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 /> Date:2012-8-17 <br /> <br /> PCR WPRE from 90 pLVX-puro-HA-AKR1C4 &lt;br&gt;WPRE-Not1-F atcgatGCGGCCGC AATCAACCTCTGGATTACAAAATTTG&lt;br&gt;WPRE-Xba1-R ttagatTCTAGACCAGGCGGGGAGGCGGCCCAAAGGGAGAT <br /> <br /> Cut out band, purify and digest with Not1 + Xba1&lt;br&gt;&lt;br&gt; <br /> <br /> [[Image:1336 WPRE PCR 2012 8 17.jpg]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> digest vector 1328&amp;nbsp; pCMV5-Fc(3C)-Dest with Not1 + Xba1, ligate and transform ccdB survival&lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> === Gateway reaction&lt;br&gt; ===<br /> <br /> &lt;br&gt; <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 /> == &#039;&#039;&#039;Cloning of 1336 pCMV5-Fc(3C)-Dest-WPRE&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of&amp;nbsp;WPRE into vector&amp;nbsp; pCMV5-Fc(3C)-Dest to make 1336 pCMV5-Fc(3C)-Dest-WPRE&lt;br&gt; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2012-8-20 to&amp;nbsp; &lt;br&gt; <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: 1336&#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 /> Date:2012-8-20 <br /> <br /> PCR WPRE from 90 pLVX-puro-HA-AKR1C4 &lt;br&gt;WPRE-Not1-F atcgatGCGGCCGC AATCAACCTCTGGATTACAAAATTTG&lt;br&gt;WPRE-Xba1-R ttagatTCTAGACCAGGCGGGGAGGCGGCCCAAAGGGAGAT <br /> <br /> Cut out band, purify and digest with Not1 + Xba1 <br /> <br /> [[Image:1272+1336PCR 8-20-2012.JPG]] <br /> <br /> [[Image:1272+1336 clone 8-20-2012.JPG]]&lt;br&gt; <br /> <br /> digest vector 1328&amp;nbsp; pCMV5-Fc(3C)-Dest with Not1 + Xba1, ligate and transform ccdB survival&lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2012-8-22&lt;br&gt; <br /> <br /> digest&amp;nbsp;: Not1 + Xba1&lt;br&gt; <br /> <br /> [[Image:1272+1336 digest detect 8-22-2012.JPG]] <br /> <br /> clone 3,4 may be right <br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> === Gateway reaction&lt;br&gt; ===<br /> <br /> 2012-8-29&lt;br&gt; <br /> <br /> miniprep and digest with Kpn1+Xho1<br /> <br /> 1--8:393+1336-3/4; 9:1336-3/4<br /> <br /> [[Image:393+1336-3_8-29-2012.JPG]][[Image:393+1336-4_8-29-2012.JPG]]<br /> <br /> need to transfect to test EGFP expression&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 /> [[Category:Cloning]]</div> Paul.xu