https://eln.excellgen.com/index.php?action=history&feed=atom&title=2321_pFastBacDual-VSVG-CAG-Flp 2026-10-18T15:05:54Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=2321_pFastBacDual-VSVG-CAG-Flp&diff=13704&oldid=prev 2014-12-03T16:33:41Z

<p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 2321 pFastBacDual-VSVG-CAG-Flp&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2321 pFastBacDual-VSVG-CAG-Flp-&#039;&#039;&#039;==&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2014-9---- <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:2321&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039;&amp;nbsp; shanyi <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === CLONING ===<br /> <br /> Use 503 psPAX2 as template, PCR CAG promoter &lt;br&gt; <br /> <br /> 2321-CAG-Nhe1-R (ctgtctcatcattttggcaaa gaattc ggatcc gcc acc atg gct agc tagca) &lt;br&gt; <br /> <br /> tgctagctagccatggtggcggatccgaattctttgccaaaatgatgagacag <br /> <br /> 2321-CAG-BglII-F tactaAGATCTgtcgacattgattattgactag <br /> <br /> [[Image:1504 2199 2321pcr.JPG]]&lt;br&gt; <br /> <br /> Digest with BglII + Nhe1. <br /> <br /> Digest vector with BamH1 + Nhe1. <br /> <br /> [[Image:Digest for 1504 2321.JPG]][[Image:2219 2320 1981DIGEST.JPG]]&lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2014_9&lt;br&gt; <br /> <br /> Make miniprep, use&amp;nbsp; Sal1 + EcoR1 to find positive clone&lt;br&gt; <br /> <br /> [[Image:1038 1916 2321 2297digest.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Cloning of 2321 pFastBacDual-VSVG-CAG-Flp&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2321 pFastBacDual-VSVG-CAG-Flp-&#039;&#039;&#039;==&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2014-9---- <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:2321&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039;&amp;nbsp; shanyi <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === CLONING ===<br /> <br /> PCR CAG-pH-T7 promoters from 298 pTriEx-HTNC&lt;br&gt;2364-CAG-Xho1-F atagcactcgagTATATGGAGTTCCGCGTTAC&lt;br&gt;2364-CAG-BamH1-R tgtcatggatccTATATCTCCTTTGATTGTAAATAAAATG <br /> <br /> &lt;br&gt; <br /> <br /> [[Image:2321 2374PCR.JPG]]&lt;br&gt; <br /> <br /> Digest with Xho1 + BamH1. <br /> <br /> Digest vector with Xho1 + BamH1. <br /> <br /> [[Image:DIGEST FOR 2321 2374.JPG]]&lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2014_9&lt;br&gt; <br /> <br /> Make miniprep, use&amp;nbsp;Xho1 + BamH1 to find positive clone&lt;br&gt; <br /> <br /> [[Image:2321 2374digest.JPG]]&lt;br&gt; <br /> <br /> digest with NdeI+XhoI should get four bands,digest with XbaI should get two bands <br /> <br /> [[Image:2321DIGEST.JPG]] &amp;nbsp; &amp;nbsp;[[Image:2321DIGEST21.JPG]] &amp;nbsp; &amp;nbsp;[[Image:2321DIGEST3.JPG]] <br /> <br /> send 2321-2 and 2321-3 to sequence with &#039;&#039;&#039;NLS-HindIII-R&#039;&#039;&#039;(NLS-HindIII-R accttcctcttcttcttcaagcttacatac), poor quality or no priming&lt;br&gt; <br /> <br /> use primers to do pcr screening&lt;br&gt; <br /> <br /> 2364-CAG-Xho1-F&amp;nbsp;&amp;nbsp; atagcactcgagTATATGGAGTTCCGCGTTAC&lt;br&gt; <br /> <br /> hIgG-Lc-BamH1-Nhe1-B&amp;nbsp; ctagcggccatggccggctgggccgcgagtaataacaatccagcggctgccgtaggcaataggtatttcatggtggcg&lt;br&gt; <br /> <br /> [[Image:2321pcr screening.JPG]]&lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Result intepretation/Summary&#039;&#039;&#039; ==<br /> <br /> *&lt;br&gt;<br /> <br /> == &#039;&#039;&#039;Conclusion&#039;&#039;&#039; ==<br /> <br /> [[Category:Cloning]]</div>

Excellgen