https://eln.excellgen.com/index.php?action=history&feed=atom&title=2301_pR-wtSM2-NucA 2301 pR-wtSM2-NucA - Revision history 2026-10-18T15:05:56Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=2301_pR-wtSM2-NucA&diff=12986&oldid=prev Excellgen at 19:57, 28 August 2014 2014-08-28T19:57:34Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 2301 pR-wtSM2-NucA&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2301 pR-wtSM2-NucA&#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:2301&#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 /> gene synthesis <br /> <br /> &lt;br&gt; <br /> <br /> [[Image:2197-2273 2301pcr.JPG]]&lt;br&gt; <br /> <br /> 2nd fragment&lt;br&gt; <br /> <br /> PCR sm2 gene from 1680 pCold-GST-wtSM2 using this pair of oligos <br /> <br /> &lt;br&gt; <br /> <br /> 2301-SM2-NucA-Nde1-F GCCgac catatgacgctcgaatccatcgac&lt;br&gt;old oligo&lt;br&gt;1 ul of 10 uM WtSM2-Xho1-R&lt;br&gt;(gagttgatgggctgcaaaaac taa ctcgag ctagca)&lt;br&gt;tgctagctcgagttagtttttgcagcccatcaactc <br /> <br /> &lt;br&gt; <br /> <br /> [[Image:2273-2 2301-2 2302pcr.JPG]] <br /> <br /> join 2 fragment&lt;br&gt;2301-SM2-NucA-1 TAGCACAGATCTACGTGTGGCCTGGCTGTACCATGACTGACACATTCACAACATG <br /> <br /> &lt;br&gt;1 ul of 10 uM WtSM2-Xho1-R&lt;br&gt;(gagttgatgggctgcaaaaac taa ctcgag ctagca)&lt;br&gt;tgctagctcgagttagtttttgcagcccatcaactc&lt;br&gt;[[Image:2273 2301pcr.JPG]]&lt;br&gt;&lt;br&gt; digest with BglII + Xho1&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 /> cloning, grow at 30 oC to suppress expression.&lt;br&gt;expression, grow at 30 oC, induce at 37 ~ 42 oC.&lt;br&gt; <br /> <br /> Make miniprep, use BglII + Xho1 to find positive clone&lt;br&gt; <br /> <br /> [[Image:2197 2302 2312 2301diget.JPG]] <br /> <br /> [[Image:2301 2273digest.JPG]]&lt;br&gt; <br /> <br /> send 2301-2 to sequence, it is the correct clone&lt;br&gt; <br /> <br /> Transform Ecoli K12 JM101, grow at 37oc overnight, the next morning, inoculate sevral colonies into 5ml LB, grow at&amp;nbsp; 37oC overnight.<br /> <br /> add 20 ul culture medium to 2ug plasmids DNA, incubate 30min,run gel<br /> <br /> [[Image:Benzonase_activity.JPG]]<br /> <br /> <br /> <br /> <br /> <br /> &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