https://eln.excellgen.com/index.php?action=history&feed=atom&title=1758_pCMV5-hIgG-Lc-FN3K 1758 pCMV5-hIgG-Lc-FN3K - Revision history 2026-10-18T22:08:32Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1758_pCMV5-hIgG-Lc-FN3K&diff=9514&oldid=prev Paul.xu at 15:15, 20 June 2013 2013-06-20T15:15:09Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1758 pCMV5-hIgG-Lc-FN3K&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of1758 pCMV5-hIgG-Lc-FN3K <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2013-6--7- <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:1758&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; Shanyi <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === cloning ===<br /> <br /> PCR using&lt;br&gt;Harvard Kinase Plate HRG007900, 79&lt;br&gt;Well: G10&lt;br&gt;donor vector: pDNR-DUAL <br /> <br /> Primers&lt;br&gt;FN3K-Kpn1-R&lt;br&gt;TGA TCG GGT ACC CTT GAG CAG CCT TCG CAT GGT G&lt;br&gt;FN3K-Nhe1-F&lt;br&gt;ATC GAC GCT AGC ATG GAG CAG CTG CTG CGC GCC GA <br /> <br /> 72oC 1min, 24cycle, Q5 HOT START POLYMERASE&lt;br&gt; <br /> <br /> [[Image:1758pcr.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> Digest vector 13951395 pCMV5-hIgG-Lc-TagGFP-Fc(3c)-WPRE-OriP with Nhe1 + Kpn1&lt;br&gt;Digest purified PCR product with Nhe1 + Kpn1&lt;br&gt; <br /> <br /> [[Image:Digest for1758 1755.JPG]]&lt;br&gt; <br /> <br /> ligate and transform NEB turbo&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2013-6-7 <br /> <br /> Make miniprep and digest with&amp;nbsp;Nhe1 + Kpn1 <br /> <br /> [[Image:1758digest.JPG]] <br /> <br /> send 1758-1tosequence, it&#039;s not we want.&lt;br&gt; <br /> <br /> === cloning ===<br /> <br /> PCR using&lt;br&gt;Addgene broad kinase plate#3,Well:G12&lt;br&gt;<br /> <br /> Primers&lt;br&gt;FN3K-Kpn1-R&lt;br&gt;TGA TCG GGT ACC CTT GAG CAG CCT TCG CAT GGT G&lt;br&gt;FN3K-Nhe1-F&lt;br&gt;ATC GAC GCT AGC ATG GAG CAG CTG CTG CGC GCC GA <br /> <br /> 72oC 1min, 25cycle, phusion HOT START POLYMERASE&lt;br&gt; <br /> <br /> [[Image:589_1758pcr.JPG‎ ]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> Digest vector 1395 pCMV5-hIgG-Lc-TagGFP-Fc(3c)-WPRE-OriP with Nhe1 + Kpn1&lt;br&gt;Digest purified PCR product with Nhe1 + Kpn1&lt;br&gt; <br /> <br /> [[Image:Digest_for_1758.JPG‎ ]]<br /> <br /> &lt;br&gt; <br /> <br /> ligate and transform NEB turbo&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2013-6-7 <br /> <br /> Make miniprep and digest with&amp;nbsp;Nhe1 + Kpn1 <br /> <br /> [[Image:1758DIGEST2.JPG‎ ]][[Image:1758DIGEST3.JPG‎]]&lt;br&gt; <br /> <br /> send 1758-1tosequence&lt;br&gt;&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 /> [[Category:Cloning]]</div> Paul.xu