https://eln.excellgen.com/index.php?action=history&feed=atom&title=Multiple_clones_pAd5 Multiple clones pAd5 - Revision history 2026-10-18T11:49:12Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=Multiple_clones_pAd5&diff=1565&oldid=prev Hylei at 20:43, 26 August 2011 2011-08-26T20:43:25Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;SubCloning of fluorescent fragments to pAd5-MCS (280)&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; SubCloning of fluorescent fragments to pAd5-MCS (280). <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2011-8-23 <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; <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; Haiyan <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === Subcloning ===<br /> <br /> Date: 2011-8-23 <br /> <br /> Release fragments with Xhoi + BamH1 &lt;br&gt; <br /> <br /> Digest pAd5-MCS (clone #280) with Xhoi + BamH1 &lt;br&gt; <br /> <br /> cut out insert (1-9) and vector (10, 11), ligate and transform STBL3 &lt;br&gt; [[Image:0823.jpg]] &lt;br&gt; <br /> <br /> {| width=&quot;200&quot; cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot;<br /> |-<br /> ! scope=&quot;row&quot; | lane <br /> | 1 <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 /> | &lt;br&gt; <br /> | &lt;br&gt;<br /> |-<br /> ! scope=&quot;row&quot; | plasmid <br /> | lss-mkate2 <br /> | IFP&lt;br&gt; <br /> | RFP&lt;br&gt; <br /> | eEGFP&lt;br&gt; <br /> | NEPTUNE&lt;br&gt; <br /> | BFP&lt;br&gt; <br /> | YFP&lt;br&gt; <br /> | mkate2&lt;br&gt; <br /> | CFP&lt;br&gt; <br /> | 280&lt;br&gt; <br /> | 280&lt;br&gt; <br /> | plenti-YFP1&lt;br&gt; <br /> | plenti-YFP2&lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt;<br /> |}<br /> <br /> lane 12, 13 colonies screenign fro plenti-YFP &lt;br&gt; <br /> <br /> lane 14, 15 colonies screenign fro pAAV-YFP &lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2011-8-26&lt;br&gt; <br /> <br /> Make miniprep and digest with xhoi + BamH1&lt;br&gt; <br /> <br /> [[Image:0826-1.jpg]] <br /> <br /> Lane 1-4: pAd5-LSS-mkate2 (365) bamhi+xhoi&lt;br&gt;<br /> <br /> Lane 5-8: pAd5-IFP (234) bamhi+xhoi&lt;br&gt;<br /> <br /> Lane 9-12: pAd5-RFP (230)bamhi+xhoi&lt;br&gt;<br /> Lane 13-16: pAd5-EGFP (304)bamhi+xhoi&lt;br&gt;<br /> <br /> Pick up 1, 6, 9, 15 to freeze DNA and bacteria.&lt;br&gt;<br /> <br /> [[Image:0826-2.jpg]] <br /> <br /> Lane 17-20: pAd5- Neptune (366) bamhi+xhoi&lt;br&gt;<br /> <br /> Lane 21-24: pAd5-BFP (233) bamhi+xhoi&lt;br&gt;<br /> <br /> Lane 25-28: pAd5-YFP (231)bamhi+xhoi&lt;br&gt;<br /> Lane 29-31: pAAV-mKate2 (228)bamhi+xhoi&lt;br&gt;<br /> Lane 32: pAd5-cfp (232)bamhi+xhoi&lt;br&gt;<br /> <br /> Pick up 17, 22, 26, 30, 32 to freeze DNA and bacteria.&lt;br&gt;<br /> &lt;br&gt;<br /> <br /> <br /> <br /> == &#039;&#039;&#039;Result intepretation/Summary&#039;&#039;&#039; ==<br /> <br /> *PKAalpha 3+4 has one internal Xho1 site. GFP-Puro can be cut out with Xho1 + Mlu1. So Lane 1 has correct number of bands. <br /> *PKA alpha 3+4 with CMV promoter can be cut out with Nde1+ BamH1. Lane 5 has correct number of bands. <br /> *lane 6 maybe good (incomplete digestion) <br /> *Sequencing Result:&lt;br&gt;<br /> <br /> == &#039;&#039;&#039;Conclusion&#039;&#039;&#039; ==<br /> <br /> Restriction digestion &lt;br&gt; <br /> <br /> shows&amp;nbsp; pLentiGP clone #1 may be good, &lt;br&gt; <br /> <br /> pLVGP-PKAalpha 3+4 is good&lt;br&gt; <br /> <br /> === ===<br /> <br /> [[User:Paul.xu|Paul.xu]] 08:23, 15 June 2011 (EST)&lt;br&gt; <br /> <br /> [[Category:Cloning]]</div> Hylei