https://eln.excellgen.com/index.php?action=history&feed=atom&title=326_pAd5-CMV-Dest-HA 2026-10-18T10:36:15Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=326_pAd5-CMV-Dest-HA&diff=2962&oldid=prev 2012-01-05T20:17:29Z

<p></p> <p><b>New page</b></p><div>&#039;&#039;&#039;The primer-Rs of 326 and 326&#039; &amp;nbsp;are different.&#039;&#039;&#039;<br /> <br /> &lt;br&gt; &#039;&#039;&#039;1. Cloning of pAd5-CMV-Dest-HA (326)&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; to clone pAd5-CMV-Dest-HA by PCR <br /> <br /> &#039;&#039;&#039;Date:&#039;&#039;&#039; 2011-11-28 to 2011-12- <br /> <br /> &#039;&#039;&#039;Notebook:&#039;&#039;&#039; Xiaojing Wang <br /> <br /> &#039;&#039;&#039;Page: &#039;&#039;&#039;004127---004131 <br /> <br /> &#039;&#039;&#039;Clone ID:&#039;&#039;&#039; 326 <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; Xiaojing Wang <br /> <br /> &#039;&#039;&#039;Results&#039;&#039;&#039; <br /> <br /> [[Image:327 326 373 PCR 11.29.2011.JPG]]&lt;br&gt; The middle bands show PCR product of 326, <br /> <br /> Primer: <br /> <br /> ad-GW-F&lt;br&gt; Kpn1&lt;br&gt;aactc GGTACC ATCAACAAGTTTGTACAAAAAAG <br /> <br /> ad-GW-R Xho1&lt;br&gt;(TCTTGTACAAAGTGGTTGATtacccatacgatgttccagattacgcttgaCTCGAGatcga)&lt;br&gt;tcgatCTCGAGtcaagcgtaatctggaacatcgtatgggtaATCAACCACTTTGTACAAGA&lt;br&gt; <br /> <br /> Digest result: <br /> <br /> [[Image:327 326 373 PCR digest 11.29.2011 S.JPG|153x269px|327 326 373 PCR digest 11.29.2011 S.JPG]][[Image:327 326 373 vector digest 11.29.2011.JPG|356x270px|327 326 373 vector digest 11.29.2011.JPG]] <br /> <br /> &lt;br&gt; <br /> <br /> digest PCR product and vector with Kpn I+Xho I,run gel <br /> <br /> the left picture shows 326 insert, the right picture shows 326 vector (pAd5-CMV-MCS, 280)&amp;nbsp; <br /> <br /> gel purify, ligate, transform ccdb survival,AMP <br /> <br /> &lt;br&gt; <br /> <br /> Colony Screening <br /> <br /> [[Image:326 373 plasmid digest 12.1.2011.JPG]]&lt;br&gt; <br /> <br /> Select 5 clones to mini-prep, digestd by Kpn I and Xho I. 326-1,-3,-4,-5 may be correct. Control is 326-1.&lt;br&gt; <br /> <br /> pDonr221_EGFP(393) + pAd5-CMV-Dest-HA(326-1) make gateway by LR Clonase II. <br /> <br /> Transform 393+326-1 by STBL3, Amp. <br /> <br /> Select 4 clones to mini-prep(i.e.326+393 1-4 plasmids), then transfect 293T cell line. <br /> <br /> 48 hours after transfection, take pictures. &lt;br&gt; <br /> <br /> [[Image:326+393 gateway 12.8.2011.jpg]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> 326+393 1-4 plasmids are digested by BamHI. Control is 326+393-4. <br /> <br /> [[Image:326+393 digest 12.5.2011.JPG|377x479px|326+393 digest 12.5.2011.JPG]]&lt;br&gt; <br /> <br /> 326+393 -1, -2 and -4 may be correct.&lt;br&gt; <br /> <br /> &lt;br&gt; &lt;br&gt; <br /> <br /> &#039;&#039;&#039;2. Cloning of pAd5-CMV-Dest-HA (326&#039;)&#039;&#039;&#039;&lt;br&gt;Purpose: to clone pAd5-CMV-Dest-HA by PCR&lt;br&gt;Date: 2011-12-16 to 2012-1-3&lt;br&gt;Notebook: Xiaojing Wang&lt;br&gt;Page: 004127---004131&lt;br&gt;Clone ID: 326&#039;&lt;br&gt;Experimenters: Xiaojing Wang&lt;br&gt;Results <br /> <br /> Primer: <br /> <br /> ad-GW-F&lt;br&gt; Kpn1&lt;br&gt;aactc GGTACC ATCAACAAGTTTGTACAAAAAAG <br /> <br /> ad-GW-HA-R2&lt;br&gt; Nhe1 Xho1 &lt;br&gt;(cttTCTTGTACAAAGTGGTTGATtacccatacgatgttccagattacgcttga gct ag CTCGAG atcga) &lt;br&gt;tcgatCTCGAGctagctcaagcgtaatctggaacatcgtatgggtaATCAACCACTTTGTACAAGAaag <br /> <br /> [[Image:327 326&#039; 373&#039; pcr 12.16.2011 s.jpg]] <br /> <br /> &lt;br&gt; <br /> <br /> Digest result: <br /> <br /> [[Image:455 327 326&#039; 373&#039; 252 PCR digest 12.20.2011 s.JPG]] <br /> <br /> [[Image:252 327 326&#039; 373&#039; 455 vector digest 12.20.2011&#039; s.JPG]] <br /> <br /> Ligation, Transformation&lt;br&gt;Select 5 clones to mini-prep&lt;br&gt;326&#039; plasmids are digested by Kpn1 + Xho1, 326&#039;-1,-2,-4,-5 plasmids may be correct.&lt;br&gt; <br /> <br /> [[Image:326&#039; 373&#039; digest 12.23.2011 s.JPG]] <br /> <br /> pDonr221_EGFP(393) + pAd5-CMV-Dest-HA(326&#039;-1) make gateway by LR Clonase II.&lt;br&gt;Transform 393+326&#039;-1 by STBL3, Amp.&lt;br&gt;Select 4 clones to mini-prep(i.e.326&#039;+393 1-4 plasmids), then transfect 293T cell line.&lt;br&gt;48 hours after transfection, take pictures. &lt;br&gt; <br /> <br /> [[Image:326&#039;+393 1.3.2012.jpg]] <br /> <br /> 393+326&#039; 1-4 plasmids are digested by BamHI. <br /> <br /> [[Image:326&#039;+393 373&#039;+393 digest bamh1 1.3.2012.JPG ]]&lt;br&gt; <br /> <br /> 326&#039;+393 -2, -3 and -4 may be correct.&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> [[Category:Cloning]]</div>

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