https://eln.excellgen.com/index.php?action=history&feed=atom&title=2277_pET28b-Cas9-Nickase 2277 pET28b-Cas9-Nickase - Revision history 2026-10-18T10:45:12Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=2277_pET28b-Cas9-Nickase&diff=12967&oldid=prev Excellgen at 17:15, 26 August 2014 2014-08-26T17:15:23Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 2277 pET28b-Cas9-Nickase&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2277 pET28b-Cas9-Nickase&#039;&#039;&#039;==&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2014-7---- <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:22767&#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 from 2275 CAS9P <br /> <br /> hCas9-D10A-Nco1-F ctagcccatggacaagaagtactccattgggctcgCtatcggcac GACAAGAAGTACTCCATTGGGCTCGATATCGGC <br /> <br /> hCas9-Not1-R (CTCAGCTCGGTGgagacagcagggctgaccccaagaagaagaggaaggtgGCGGCCGCtagcta) tagctaGCGGCCGCcaccttcctcttcttcttggggtcagccctgctgtctcCACCGAGCTGAG <br /> <br /> [[Image:2276 2277 2278pcr.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> digest with Nco1 + Not1, clone tov12 pET28b&lt;br&gt; <br /> <br /> [[Image:Digest for 2276 2277 2278.JPG]]&lt;br&gt;&lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2014_7 <br /> <br /> Make miniprep, use NdeI+BamHI&amp;nbsp; to find positive clone&lt;br&gt; <br /> <br /> [[Image:2276 2277 2278DIGEST.JPG]] <br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; &lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Cloning of 2276 pET28b-Cas9&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2276 pET28b-Cas9&#039;&#039;&#039;==&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2014-8---- <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:2276&#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 from 2275 CAS9P&lt;br&gt;Fragment 1&lt;br&gt;hCas9-D10A-Nco1-F&lt;br&gt;ctagcccatggacaagaagtactccattgggctcgCtatcggcac&lt;br&gt;&lt;br&gt;Cas9-R2 B8 ctgaggcacgatATGgtccacatcgtagtc&lt;br&gt; <br /> <br /> [[Image:2276 2277 2278fragment1 212pcr.JPG]] <br /> <br /> Fragment 2&lt;br&gt;Cas9-F2 B7 gactacgatgtggac CAT atcgtgcctcag &lt;br&gt;1 ul of 1 uM of Cas9-R4 &lt;br&gt;(gacctgtctcagctgggagacagcagggctgacccc)&lt;br&gt; D L S Q L G D S R A D P &lt;br&gt;ggggtcagccctgctgtctcccagctgagacaggtc <br /> <br /> hCas9-Not1-R&lt;br&gt;(gagacagcagggctgaccccaagaagaagaggaaggtgcaccaccaccaccaccactgctaaGCGGCCGCtagcta)&lt;br&gt;tagctaGCGGCCGCttagcagtggtggtggtggtggtgcaccttcctcttcttcttggggtcagccctgctgtctc&lt;br&gt;[[Image:2276 2277 2278fragment2 2300 2198 22291stpcr.JPG]]&lt;br&gt;hCas9-D10A-Nco1-F&lt;br&gt;ctagcccatggacaagaagtactccattgggctcgCtatcggcac&lt;br&gt;&lt;br&gt;hCas9-Not1-R&lt;br&gt;(CTCAGCTCGGTGgagacagcagggctgaccccaagaagaagaggaaggtgGCGGCCGCtagcta)&lt;br&gt;tagctaGCGGCCGCcaccttcctcttcttcttggggtcagccctgctgtctcCACCGAGCTGAG&lt;br&gt;[[Image:2276 2277 2278pcr2N.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> digest with Nco1 + Not1, clone tov12 pET28b&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> [[Image:DIGEST FOR 2276 2277 2278.JPG]]&lt;br&gt;&lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2014_8_ <br /> <br /> Make miniprep, use&amp;nbsp; Nco1 + Not1 to find positive clone&lt;br&gt; <br /> <br /> [[Image:2276 2277 2278 2234digest.JPG]] <br /> <br /> digest with EcoRV+BamHi&lt;br&gt; <br /> <br /> [[Image:212 2198 2215-1 2276 2277 2278digest.JPG]] <br /> <br /> &lt;br&gt; <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; &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