https://eln.excellgen.com/index.php?action=history&feed=atom&title=423-pET30b-DeepVent-sso7d 2026-10-18T16:40:35Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=423-pET30b-DeepVent-sso7d&diff=4171&oldid=prev 2012-05-10T18:25:22Z

<p></p> <p><b>New page</b></p><div>Cloning of pET30b-DeepVent-sso7d <br /> <br /> Purpose: to clone pET30b-DeepVent-sso7dby PCR <br /> <br /> Date: 2011-12-8 <br /> <br /> Notebook:Lu yingying <br /> <br /> Page: <br /> <br /> Clone ID:423 <br /> <br /> Experimenters: lu yingying&lt;br&gt;Results&lt;br&gt; <br /> <br /> [[Image:422+423 PCR 12-9-2011.JPG]]&lt;br&gt; <br /> <br /> DVent-Nde1-F <br /> <br /> acgatc CATATG atgatacttgacgctgactac <br /> <br /> DVent-BglII-R&lt;br&gt;(ggcttaacatcaagaagaag AGATCT atcaga) <br /> <br /> tctgatAGATCTcttcttcttgatgttaagcc&lt;br&gt;NEB720 Deep Vent 385 as template&lt;br&gt; --[[User:Yingying.lu|Yingying.lu]] 13:03, 9 December 2011 (EST) <br /> <br /> &lt;br&gt; <br /> <br /> Cloning of pET30b-DeepVent-sso7d(423)&lt;br&gt;Purpose: to clone pET30b-DeepVent-sso7d&lt;br&gt;Date: 2012-5-3 to 2012-5-&lt;br&gt;Notebook: Xiaojing Wang&lt;br&gt;Page: 004238 <br /> <br /> Clone ID: 423&lt;br&gt;Experimenters: Xiaojing Wang &lt;br&gt; <br /> <br /> Results &lt;br&gt; <br /> <br /> NEB720 Deep Vent 385 as template <br /> <br /> Fragment 1 PCR: DeepVent-Nde1-F1 + DeepVent-R1, 25 cycles&lt;br&gt;Fragment 2 PCR: DeepVent-F2 + DeepVent-BglII-R2, 25 cycles&lt;br&gt; <br /> <br /> [[Image:423 PCR 5.3.2012.JPG]]&lt;br&gt; <br /> <br /> join two fragments using Gibson Mix&lt;br&gt; <br /> <br /> [[Image:423 gibson 5.3.2012.JPG]]&lt;br&gt; <br /> <br /> digest with Nde1 + BglII, clone into Nde1 + BglII digested pET30b-sso7d-KX, 368&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> Ligate and transform DH5alpha, K+ <br /> <br /> 5/6/2012&lt;br&gt; <br /> <br /> miniprep digest with Nde1 + BglII, marker is Fermentas 1 kb ladder &lt;br&gt; <br /> <br /> 20000, 10000, 7000, &#039;&#039;&#039;5000&#039;&#039;&#039;, 4000, 3000, 2000, &#039;&#039;&#039;1500&#039;&#039;&#039;, 1000, 700, &#039;&#039;&#039;500&#039;&#039;&#039;, 400, 300, 200, 75&lt;br&gt;&lt;br&gt; <br /> <br /> [[Image:423 bglII Nde1 5 6 2012 3.jpg]]&lt;br&gt; <br /> <br /> [[Image:423 bglII Nde1 5 6 2012 2.jpg]]&lt;br&gt; <br /> <br /> [[Image:423 bglII Nde1 5 6 2012.jpg]]&lt;br&gt; <br /> <br /> [[Image:423 Nde1 HindIII Xho1 1.jpg]]&lt;br&gt;[[Image:423 Nde1 HindIII Xho1 2.jpg]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> 4 is not good, Nde1+ Xho1 cut should get 1 band (823 bp), Nde1+HindIII should get one band (1406 bp), bands are too big&lt;br&gt; <br /> <br /> [[Image:423 BglII HindIII Xho1 5 6 2012.jpg]]&lt;br&gt; <br /> <br /> digest with BglII-Xho1, HindIII-BglII. Looks like there are two Xho1 site, which is not expected <br /> <br /> &lt;br&gt; <br /> <br /> join two fragments using Gibson Mix, again 5/8/2012&lt;br&gt; <br /> <br /> [[Image:423 Gibson 5.8.2012.JPG]] <br /> <br /> digest with Nde1 + BglII, clone into Nde1 + BglII digested 423-9&lt;br&gt; <br /> <br /> [[Image:423 i v digest 5.8.2012.JPG]] <br /> <br /> Ligate and transform DH5alpha, K+ &lt;br&gt; <br /> <br /> [[Category:Cloning]]</div>

Xiaojing.Wang