https://eln.excellgen.com/index.php?action=history&feed=atom&title=1377_pCMV5-Fc-%283c%29-NPR1-Cyto-WPRE-oriP 2026-10-18T06:22:16Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1377_pCMV5-Fc-(3c)-NPR1-Cyto-WPRE-oriP&diff=6215&oldid=prev 2012-09-25T15:07:43Z

<p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of pCMV5-Fc-(3c)-NPR1-Cyto-WPRE-oriP&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; subcloning of gene NPR1-cyto from 1284 pCMV5-Fc(3c)-NPR1-Cyto into 1369 pCMV5-Fc-(3c)-TagRFP-WPRE-oriP with NheI and NotI&lt;br&gt; <br /> <br /> &amp;nbsp;&#039;&#039;&#039;Date:&amp;amp;nbsp;&#039;&#039;&#039; 2012-09-20 <br /> <br /> &amp;nbsp;&#039;&#039;&#039;Notebook:&amp;amp;amp;nbsp;&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Page:&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Clone ID: 1377&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Experimenters: Xiuli&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;insert size: ~ 1647bp&#039;&#039;&#039;&lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> &lt;br&gt; <br /> <br /> Cloning <br /> <br /> [[Image:9202012-1377-1381.jpg]]&lt;br&gt; <br /> <br /> 09-19-2012 <br /> <br /> A.Restriction enzyme digestion: plasmid 1284,1276 1264, 1269, 1263 and 1396 cut with NheI and NotI.&lt;br&gt; <br /> <br /> B. Gel purification and elute with 15 ul H2O <br /> <br /> c Ligation: 2xLigation buffer (12ul) +12 ul elution from above +0,5 ul T7 ligase. Incubate at RT for 1 hour&lt;br&gt; <br /> <br /> D. Transformation. <br /> <br /> 9/22/2012 <br /> <br /> make miniprep, digest with Nhe1 + Not1 <br /> <br /> [[Image:1377 78 79 80 81 2012 9 22.jpg]] <br /> <br /> [[Image:1377 78 79 80 81 2012 9 22 2.jpg]] <br /> <br /> 9/24/2012 <br /> <br /> Select 1377-2, 1378-1, 1379-2, 1380-2, 1381-1 to make maxiprep using Qiagen maxiprep kit. 1381, 1392&amp;nbsp; using invitrogen maxiprep&amp;nbsp; kit <br /> <br /> <br /> <br /> <br /> [[Image:1377-1381 maxi cut .jpg]][[Image:1377-1381 maxi-cut.jpg]] <br /> <br /> 1377-1378-1379-1380-1380-1381 Plasmid Maxiprep&lt;br&gt;<br /> <br /> &lt;br&gt;<br /> <br /> Clones for maxiprep <br /> <br /> {| width=&quot;330&quot; height=&quot;125&quot; border=&quot;1&quot; cellspacing=&quot;1&quot; cellpadding=&quot;1&quot;<br /> |-<br /> | Clone ID&lt;br&gt;<br /> | Parent&lt;br&gt;<br /> | Clone Name&lt;br&gt;<br /> | <br /> Expected Size<br /> <br /> (bp)&lt;br&gt;<br /> <br /> | Actual Size on Gel (Kb)&lt;br&gt;<br /> | Clone Identity&lt;br&gt;<br /> | DNA conc&lt;br&gt;<br /> | OD260/280&lt;br&gt;<br /> | RNA&lt;br&gt;<br /> |-<br /> | 1377-1&lt;br&gt;<br /> | 1284&lt;br&gt;<br /> | &lt;br&gt;<br /> | 1647&lt;br&gt;<br /> | 2.5&lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> |-<br /> | 1378-2&lt;br&gt;<br /> | 1276&lt;br&gt;<br /> | &lt;br&gt;<br /> | 1322&lt;br&gt;<br /> | 1.6&lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> |-<br /> | 1379-2&lt;br&gt;<br /> | 1264&lt;br&gt;<br /> | &lt;br&gt;<br /> | 3205&lt;br&gt;<br /> | 4&lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> |-<br /> | 1380-2&lt;br&gt;<br /> | 1269&lt;br&gt;<br /> | &lt;br&gt;<br /> | 2169&lt;br&gt;<br /> | 3&lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> |-<br /> | 1381-1&lt;br&gt;<br /> | 1263&lt;br&gt;<br /> | &lt;br&gt;<br /> | 1586&lt;br&gt;<br /> | 2.5&lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> | &lt;br&gt;<br /> |}<br /> <br /> <br /> <br /> <br /> 1381 and 1392 using Invitrogen maxi prep kit have too much RNA. After treating&amp;nbsp; sample 1381 and 1392 with 0.5 ul RNAse (Qiagen) at 37oC for 2-3 hours, precipitate DNA&amp;nbsp;with isoprapanol. <br /> <br /> [[Category:Cloning]]</div>

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