https://eln.excellgen.com/index.php?action=history&feed=atom&title=2278_pET28b-dCas9 2278 pET28b-dCas9 - Revision history 2026-10-18T04:26:10Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=2278_pET28b-dCas9&diff=12968&oldid=prev Excellgen at 17:15, 26 August 2014 2014-08-26T17:15:42Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 2278 pET28b-dCas9&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2278 pET28b-dCas9&#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:2278&#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 /> Fragment 1 hCas9-D10A-Nco1-F ctagcccatggacaagaagtactccattgggctcgCtatcggcac <br /> <br /> Cas9-H840A-R ctggggcacgatGGCatccacgtcgtag <br /> <br /> [[Image:2278-1 735pcr.JPG]]&lt;br&gt; Fragment 2 <br /> <br /> Cas9-H840A-F ctacgacgtggat GCC atcgtgccccag <br /> <br /> hCas9-Not1-R (gagacagcagggctgaccccaagaagaagaggaaggtgcaccaccaccaccaccactgctaaGCGGCCGCtagcta) tagctaGCGGCCGCttagcagtggtggtggtggtggtgcaccttcctcttcttcttggggtcagccctgctgtctc <br /> <br /> [[Image:2264 734 2279 2273 2268 2278-2pcr.JPG]] <br /> <br /> Purify and Join two fragments hCas9-D10A-Nco1-F + hCas9-Not1-R <br /> <br /> [[Image:2276 2277 2278pcr.JPG]] <br /> <br /> &lt;br&gt; <br /> <br /> digest with Nco1 + Not1, clone to v12 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_2_14 <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<br /> <br /> <br /> <br /> Cas8-R3 D12 ctgaggcacgatGGCgtccacatcgtagtc&lt;br&gt;&lt;br&gt; <br /> <br /> [[Image:2276 2277 2278fragment1 212pcr.JPG]] <br /> <br /> Fragment 2&lt;br&gt;<br /> <br /> Cas9-F3 D11 gactacgatgtggac GCC atcgtgcctcag<br /> <br /> <br /> <br /> 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 /> &lt;br&gt;hCas9-Not1-R&lt;br&gt;(gagacagcagggctgaccccaagaagaagaggaaggtgcaccaccaccaccaccactgctaaGCGGCCGCtagcta)&lt;br&gt;tagctaGCGGCCGCttagcagtggtggtggtggtggtgcaccttcctcttcttcttggggtcagccctgctgtctc&lt;br&gt;<br /> <br /> &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 /> <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