https://eln.excellgen.com/index.php?action=history&feed=atom&title=1208_pCDNA3.3 1208 pCDNA3.3 - Revision history 2026-10-18T10:25:57Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1208_pCDNA3.3&diff=4432&oldid=prev Jennifer.truong at 02:09, 6 June 2012 2012-06-06T02:09:45Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 1208&amp;nbsp;pCDNA3.3&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039;ake pCDNA3.3 vector&lt;br&gt; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2012-6-1 <br /> <br /> &#039;&#039;&#039;Notebook:&amp;nbsp;&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Page:&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Clone ID: &#039;&#039;&#039;1208 <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; Paul <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === cloning ===<br /> <br /> 6-1-2012&lt;br&gt; <br /> <br /> digest pCDNA3-YFP-IRES-SVLT 413 with Nhe1 + EcoR1 + Xba1, gel purified vector <br /> <br /> digestion: Nhe1 + EcoR1 + Xba1<br /> <br /> &lt;br&gt; [[Image:1208 vector digestion 6 1 2012.jpg]]&lt;br&gt; <br /> <br /> ligate to annealed pCDNA3.1-T + pCDNA3.1-B <br /> <br /> 5&#039; CTAGC GGATCC GAATTC ggtacc AAGCTT TTAATTAA Ctcgag GCGGCCGC GATATC T 3&#039;&lt;br&gt;3&#039; g CCTAGG CTTAAG ccatgg TTCGAA AATTAATT Gagctc CGCCGGCG CTATAG AGATCT 5&#039; <br /> <br /> pCDNA3.1-T&lt;br&gt;CTAGCGGATCCGAATTCggtaccAAGCTTTTAATTAACtcgagGCGGCCGCGATATCT&lt;br&gt;pCDNA3.1-B&lt;br&gt;TCTAGAGATATCGCGGCCGCctcgaGTTAATTAAAAGCTTggtaccGAATTCGGATCCg&lt;br&gt; <br /> <br /> transform DH5alpha <br /> <br /> 6/3/2012&lt;br&gt; <br /> <br /> colony screening by miniprep: nothing&lt;br&gt; <br /> <br /> 6/3/2012&lt;br&gt; <br /> <br /> Digest 1192 pCDNA3.2&amp;nbsp; (Lane 1) with Nde1 + Xba1--&amp;gt;insert&lt;br&gt; <br /> <br /> Digest 413 pCDNA3-YFP-IRES-SVLT (Lane 2) with Nde1 + Xba1--&amp;gt;vector&lt;br&gt; <br /> <br /> ligate <br /> <br /> [[Image:1208 6 3 12012.jpg]] <br /> <br /> [[Image:1208 6 3 12012 1.jpg]] <br /> <br /> &lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 6/4/2012&lt;br&gt; <br /> <br /> [[Image:1208 PCR Screening.JPG]] <br /> <br /> Positive yield for all colonies, grew lanes 1-7 overnight in AMP<br /> <br /> Date: 6/5/2012<br /> <br /> Did MiniPrep for samples 1-7, digested samples with&amp;nbsp;PacI + Nde1 to find positive clones<br /> <br /> [[Image:1208_Digestion.JPG]]<br /> <br /> Gel shows samples 3-6 are positive<br /> <br /> Digested samples 3-6 with&amp;nbsp;Nhe1 + Nde1, Xba1 + Nde1 to confirm<br /> <br /> [[Image:1208_Digestion_2.JPG]]<br /> <br /> Lanes 2-5 were digested with Nhe1 + Nde1 and lanes 6-9 were digested with Xba1 + Nde1 (samples 3, 4, 5, 6, 3, 4, 5, 6, respectively)<br /> <br /> Plasmids were labled, and a glycerol stock was prepared from sample 5 with AMP LB<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> Jennifer.truong