https://eln.excellgen.com/index.php?action=history&feed=atom&title=2273_pHCMC04-Cre 2273 pHCMC04-Cre - Revision history 2026-10-18T16:58:32Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=2273_pHCMC04-Cre&diff=12887&oldid=prev Excellgen at 16:45, 22 August 2014 2014-08-22T16:45:12Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 2273 pHCMC04-Cre&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2273 pHCMC04-Cre&#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:2273&#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 298&lt;br&gt; <br /> <br /> 6His-BamH1-F B4 tactaggatccatgggccatcaccatcaccatcac&lt;br&gt;Cre-Xma1-R B5 tgatacccgggCTCGAGCTAATCGCCATCTTCCAGCAG&lt;br&gt;&lt;br&gt; <br /> <br /> [[Image:2264 734 2279 2273 2268 2278-2pcr.JPG]]&lt;br&gt; digest with BamH1+ Xma1, clone to 2271 pHCMC04&lt;br&gt; <br /> <br /> [[Image:Digest for 735 2268 2273.JPG]]&lt;br&gt;&lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2014_7 <br /> <br /> Make miniprep, use XhoI to find positive clone&lt;br&gt; <br /> <br /> [[Image:2273DIGESR.JPG]] <br /> <br /> &lt;br&gt; <br /> <br /> use BamH1+ Xma1&amp;nbsp; to verify&lt;br&gt; <br /> <br /> [[Image:735 2268 2273DIGEST.JPG]] <br /> <br /> &lt;br&gt; <br /> <br /> sequenced, not right , there is a BamHI site in cre sequence&lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Cloning of 2273 pHCMC04-Cre&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2273new pHCMC04-Cre&#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:2273&#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 298&lt;br&gt; <br /> <br /> 2 fragments&lt;br&gt;1st fragment( the lower band)&lt;br&gt;6His-BamH1-F tactaggatccatgggccatcaccatcaccatcac&lt;br&gt;Cre-dBamH1-R CTTTTCGGATaCCGCCGCATAAC <br /> <br /> [[Image:2287 2273-1pcr.JPG]] <br /> <br /> 2nd fragment&lt;br&gt;Cre-dBamH1-F GTTATGCGGCGGtATCCGAAAAG&lt;br&gt;Cre-Xma1-R tgatacccgggCTCGAGCTAATCGCCATCTTCCAGCAG <br /> <br /> [[Image:2273-2 2286 2254 2255pcr.JPG]] <br /> <br /> join 2 fragment&lt;br&gt;6His-BamH1-F tactaggatccatgggccatcaccatcaccatcac&lt;br&gt;Cre-Xma1-R tgatacccgggCTCGAGCTAATCGCCATCTTCCAGCAG&lt;br&gt;[[Image:2273 2287pcr.JPG]]&lt;br&gt;&lt;br&gt; digest with BamH1+ Xma1, clone to 2271 pHCMC04&lt;br&gt; <br /> <br /> [[Image:DIGEST FOR 2273 2287.JPG]]&lt;br&gt;&lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2014_7 <br /> <br /> Make miniprep, use XhoI to find positive clone&lt;br&gt; <br /> <br /> [[Image:2273 2286digest.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> use BamH1+ Xma1&amp;nbsp; to verify&lt;br&gt; <br /> <br /> [[Image:2273 2286 1395 2254 2287digest.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Cloning of 2273 pHCMC04-Cre&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2273 pHCMC04-Cre&#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:2273&#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 298&lt;br&gt; <br /> <br /> 2 fragments&lt;br&gt;1st fragment&lt;br&gt;6His-BamH1-F tactaggatccatgggccatcaccatcaccatcac&lt;br&gt;2273-Cre-dBamH1-R2 CTTTTCGGATaCGCCGCATAAC <br /> <br /> [[Image:2197-2273 2301pcr.JPG]]&lt;br&gt; <br /> <br /> 2nd fragment&lt;br&gt;2273-Cre-dBamH1-F2 GTTATGCGGCGtATCCGAAAAG <br /> <br /> Cre-Xma1-R tgatacccgggCTCGAGCTAATCGCCATCTTCCAGCAG <br /> <br /> [[Image:2273-2 2301-2 2302pcr.JPG]] <br /> <br /> join 2 fragment&lt;br&gt;6His-BamH1-F tactaggatccatgggccatcaccatcaccatcac&lt;br&gt;Cre-Xma1-R tgatacccgggCTCGAGCTAATCGCCATCTTCCAGCAG&lt;br&gt;[[Image:2273 2301pcr.JPG]]&lt;br&gt;&lt;br&gt; digest with BamH1+ Xma1, clone to 2271 pHCMC04&lt;br&gt; <br /> <br /> [[Image:Digest for 2197 2273 2301 2302 2312.JPG]]&lt;br&gt;&lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2014_8 <br /> <br /> Make miniprep, use XhoI to find positive clone&lt;br&gt; <br /> <br /> [[Image:2301 2273digest.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> use BamH1+ Xma1&amp;nbsp; to verify&lt;br&gt; <br /> <br /> [[Image:2273digest.JPG]]&lt;br&gt; <br /> <br /> &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