https://eln.excellgen.com/index.php?action=history&feed=atom&title=1392_pAAV-VP16-EBNA1c 1392 pAAV-VP16-EBNA1c - Revision history 2026-10-18T07:29:44Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1392_pAAV-VP16-EBNA1c&diff=6198&oldid=prev Paul.xu at 16:55, 24 September 2012 2012-09-24T16:55:19Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1348 pLVX-EF1-VP16-EBNA1c&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1348 pLVX-EF1-VP16-EBNA1c <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2012-9-7---2012-9-12 <br /> <br /> &#039;&#039;&#039;Notebook:&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Page:&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Clone ID: &#039;&#039;&#039;1348 <br /> <br /> &#039;&#039;&#039;Experimenters: &#039;&#039;&#039;yingying lu <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === &lt;br&gt; ===<br /> <br /> === Cloning ===<br /> <br /> Date: &lt;br&gt; <br /> <br /> Gene synthesis this VP16 fragment&lt;br&gt; <br /> <br /> [[Image:1298+1348p1+1350 PCR 8-31-2012.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> PCR out EBNA1C from 371 pCEP4 E02S CK2M EN2L, ~950 bp&lt;br&gt; <br /> <br /> [[Image:1348 PCR2 2012-9-7.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> 9/7/2012: <br /> <br /> PCR <br /> <br /> [[Image:1368 1366 1348 1346 9 7 2012.jpg]]&lt;br&gt; <br /> <br /> &lt;br&gt; &lt;u&gt;&#039;&#039;&#039;9/8/2012 Digestion for cloning&#039;&#039;&#039;&lt;/u&gt;&lt;br&gt; <br /> <br /> &#039;&#039;&#039;Lane 2, 4, 6:&#039;&#039;&#039; TagGFP amplified with TagGFP-EcoR1-BamH1-F &amp;amp; TaqGFP-R3 using 110 pLVX-TagGFP as template as template&lt;br&gt; <br /> <br /> &#039;&#039;&#039;Lane #9&#039;&#039;&#039;: TagRFP amplified with TagRFP-EcoR1-BamH1-F gtattcgaattcGCGGCCGCggatccGTGTCTAAGGGCGAAGAGCTG&lt;br&gt;ATG AGC GGG GGC GAGGAGCTGTTCGCCGGCATCG <br /> <br /> TagRFP-Xba1-R&lt;br&gt;(GCAAACTGGGGCACAAACTTAA TCTAGA CGATCAA)&lt;br&gt;TTG ATC GTC TAG ATT AAG TTT GTG CCC CAG TTT GC&lt;br&gt; <br /> <br /> &#039;&#039;&#039;Lane #10&#039;&#039;&#039;: PCR product VP16 + EBNA1c has this configuration: EcoR1 ---VP16--BamH1--EBNA1c--Xho1&lt;br&gt; <br /> <br /> &#039;&#039;&#039;Lane #12&#039;&#039;&#039;: A/WSN/33 M15/88E amplified &lt;br&gt;FluASeg-7-NdeI-F&lt;br&gt;actagc CAT ATG CAC CAT CAT CAT CAT CAT agtcttctaaccgaggtc <br /> <br /> FluASeg-7-Xho1-R&lt;br&gt;(catcggtctcataggcaaatgCTCGAG atcaga)&lt;br&gt;tctgatCTCGAGcatttgcctatgagaccgatg&lt;br&gt;using 61 pHW2000-A/WSN/33 M15/88E as template&lt;br&gt; <br /> <br /> [[Image:9 8 2012 digestion.jpg]] <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot; style=&quot;width: 598px; height: 107px;&quot;<br /> |-<br /> | &lt;br&gt; <br /> | 1&lt;br&gt; <br /> | 2&lt;br&gt; <br /> | 3&lt;br&gt; <br /> | 4&lt;br&gt; <br /> | 5&lt;br&gt; <br /> | 6&lt;br&gt; <br /> | 7&lt;br&gt; <br /> | 8&lt;br&gt; <br /> | 9&lt;br&gt; <br /> | 10&lt;br&gt; <br /> | 11&lt;br&gt; <br /> | 12&lt;br&gt; <br /> | 13&lt;br&gt;<br /> |-<br /> | Plasmid ID&lt;br&gt; <br /> | 1333&lt;br&gt; <br /> | &lt;br&gt; <br /> | 1334&lt;br&gt; <br /> | &lt;br&gt; <br /> | 1329&lt;br&gt; <br /> | &lt;br&gt; <br /> | 1330&lt;br&gt; <br /> | 1329&lt;br&gt; <br /> | 1368&lt;br&gt; <br /> | &lt;br&gt; <br /> | 254&lt;br&gt; <br /> | &lt;br&gt; <br /> | 1036&lt;br&gt;<br /> |-<br /> | Plasmid or DNA Name&lt;br&gt; <br /> | &lt;br&gt; <br /> | TagGFP&lt;br&gt; <br /> | &lt;br&gt; <br /> | TagGFP&lt;br&gt; <br /> | &lt;br&gt; <br /> | TagGFP&lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | TagRFP&lt;br&gt; <br /> | VP16 + EBNA1c&lt;br&gt; <br /> | &lt;br&gt; <br /> | A/WSN/33 M15/88E&lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> | Digestion&lt;br&gt; <br /> | EcoR1+ Xba1&lt;br&gt; <br /> | EcoR1+ Xba1&lt;br&gt; <br /> | EcoR1+ Xba1&lt;br&gt; <br /> | EcoR1+ Xba1&lt;br&gt; <br /> | EcoR1+ Xba1&lt;br&gt; <br /> | EcoR1+ Xba1&lt;br&gt; <br /> | EcoR1+ Xba1&lt;br&gt; <br /> | EcoR1+ Xba1&lt;br&gt; <br /> | EcoR1+ Xba1&lt;br&gt; <br /> | BamH1 + Xho1&lt;br&gt; <br /> | BamH1 + Xho1&lt;br&gt; <br /> | Nde1 + Xho1&lt;br&gt; <br /> | Nde1 + Xho1&lt;br&gt;<br /> |}<br /> <br /> cut out bands as indicated, purify and ligate using T7 ligase + 10X T4 ligase buffer.&lt;br&gt; <br /> <br /> transform DH5a &lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> 2012-9-11&lt;br&gt; <br /> <br /> BamH1 + Xho1&lt;br&gt; <br /> <br /> [[Image:1348-2 digest detect 9-11-2012.JPG]]&lt;br&gt; <br /> <br /> &amp;nbsp;no EBNA1c insert&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 /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> = == &#039;&#039;&#039;Cloning of &#039;&#039;&#039;1399 pAAV--EBNA1c&lt;br&gt; =<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1392 pLVX-EBNA1c <br /> <br /> &lt;br&gt; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2012-9-12 <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:1392&#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:1289+1341+1346+1348-2 clone 9-12-2012.JPG]]&lt;br&gt; <br /> <br /> lane 7: EBNA1c PCR product&amp;nbsp; <br /> <br /> lane 8: 254,pAAV.CMV-MCS, pAAV-MCS&lt;br&gt; <br /> <br /> BamH1 + Xho1&lt;br&gt; <br /> <br /> {| width=&quot;7&quot; 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: 2012-9-14 <br /> <br /> [[Image:1341+1348 digest detect 9-14-2012.JPG]]&lt;br&gt; <br /> <br /> need to sequence: EBNA1c &lt;br&gt; <br /> <br /> pick clone 2&amp;nbsp; to sequence, right <br /> <br /> &lt;br&gt; <br /> <br /> === cloning of 1392 pAAV-VP16-EBNA1c&lt;br&gt; ===<br /> <br /> === [[Image:1348 clone 9-17-2012.JPG]] ===<br /> <br /> lane1: VP16 insert&lt;br&gt; <br /> <br /> lane2: clone2-1399 <br /> <br /> EcoR1+BamH1 <br /> <br /> &lt;br&gt; <br /> <br /> == &#039;&#039;&#039;colony screen&#039;&#039;&#039;&lt;br&gt; ==<br /> <br /> 2012-9-19&lt;br&gt; <br /> <br /> mini prep and digest detect EcoR1+BamH1 / BamH1 + Xho1&lt;br&gt; <br /> <br /> [[Image:1348 digest detect 9-19-2012.JPG]]&lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Result intepretation/Summary&#039;&#039;&#039; ==<br /> <br /> *&amp;amp;amp;lt;br&amp;amp;amp;gt;<br /> <br /> == &#039;&#039;&#039;Conclusion&#039;&#039;&#039; ==<br /> <br /> Note: the cloning vector for assembling VP16-EBNA1c is 254,pAAV.CMV-MCS, pAAV-MCS, not pLVX-EF1. Therefore, need to do subcloning.&lt;br&gt; <br /> <br /> {|<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