https://eln.excellgen.com/index.php?action=history&feed=atom&title=1346-pET30b-A%2FWSN%2F33_M15%2F88E 1346-pET30b-A/WSN/33 M15/88E - Revision history 2026-10-18T06:34:43Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1346-pET30b-A/WSN/33_M15/88E&diff=6014&oldid=prev Paul.xu at 13:05, 12 September 2012 2012-09-12T13:05:54Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1346-pET30b-A/WSN/33 M15/88E&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1346-pET30b-A/WSN/33 M15/88E <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2012-8-21---2012-8-24 <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:1346&#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:1346 PCR 8-21-2012.JPG]]&lt;br&gt; &lt;br&gt; <br /> <br /> [[Image:1288+1346 clone 8-22-2012.JPG]] <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: 2012-08-24 <br /> <br /> Make miniprep and digest with Nde1 + Xho1 &lt;br&gt; <br /> <br /> [[Image:1346 Screening.JPG]] <br /> <br /> 4 and 6 may be correct <br /> <br /> &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 /> &amp;amp;amp;lt;br&amp;amp;amp;gt; <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot;<br /> |-<br /> | &lt;br&gt;<br /> |}<br /> <br /> &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 /> == &#039;&#039;&#039;Cloning of 1346-pET30b-A/WSN/33 M15/88E&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of 1346-pET30b-A/WSN/33 M15/88E <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2012-8-31---2012-9-6 <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:1346&#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:1346 PCR 8-21-2012.JPG]]&lt;br&gt; &lt;br&gt; <br /> <br /> [[Image:1298+1324+1346+1347+1350+1356 clone 8-31-2012.JPG]]&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: 2012-9-4 <br /> <br /> Make miniprep and digest with Nde1 + Xho1 <br /> <br /> [[Image:1346+1347+1350 digest detct 9-4-2012.JPG]]&lt;br&gt; <br /> <br /> clone 1,4 may be right <br /> <br /> &#039;&#039;&#039;pET30b vector is contaminated&#039;&#039;&#039; <br /> <br /> &lt;br&gt; <br /> <br /> 9/7/2012: <br /> <br /> PCR&lt;br&gt; <br /> <br /> [[Image:1368 1366 1348 1346 9 7 2012.jpg]] <br /> <br /> &lt;br&gt; <br /> <br /> &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 <br /> <br /> <br /> <br /> === &#039;&#039;&#039;colony screen&#039;&#039;&#039; ===<br /> <br /> 2012-9-11<br /> <br /> mini prep and digest <br /> <br /> [[Image:1346_digest_detect_9-12-2012.JPG]]<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