https://eln.excellgen.com/index.php?action=history&feed=atom&title=353_pET28b-TaqMuts 2026-10-18T06:32:05Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=353_pET28b-TaqMuts&diff=1548&oldid=prev 2011-08-21T23:07:16Z

<p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of TaqMuts to pET28b&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; SubCloning of TaqMuts from pET30a to the pET28b (353). <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2011-8-19 to 2011-8-23 <br /> <br /> &#039;&#039;&#039;Notebook:&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Page:&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Clone ID:&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; Paul&lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === Digestion Analysis&lt;br&gt; ===<br /> <br /> Date: 2011-8-19 &lt;br&gt; <br /> <br /> [[Image:PET3A Taq Muts digestion 8 18 2011 1.jpg]]&lt;br&gt; <br /> <br /> [[Image:PET3A Taq Muts digestion 8 19 2011 2.jpg]]&lt;br&gt; <br /> <br /> {| width=&quot;200&quot; cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot;<br /> |-<br /> | Lane&lt;br&gt; <br /> | Sample&lt;br&gt; <br /> | Digestion&lt;br&gt;<br /> |-<br /> | 1&lt;br&gt; <br /> | pET3a-Muts&lt;br&gt; <br /> | Nde1+EcoR1&lt;br&gt;<br /> |-<br /> | 2&lt;br&gt; <br /> | pET3a-Muts&lt;br&gt; <br /> | NdeI+HindIII&lt;br&gt;<br /> |-<br /> | 3&lt;br&gt; <br /> | pET3a-Muts&lt;br&gt; <br /> | NdeI+BamH1&lt;br&gt;<br /> |-<br /> | 4&lt;br&gt; <br /> | pET3a-Muts&lt;br&gt; <br /> | BamH1&lt;br&gt;<br /> |-<br /> | 5&lt;br&gt; <br /> | pDB.His.MBP&lt;br&gt; <br /> | NdeI+EcoR1&lt;br&gt;<br /> |-<br /> | 6&lt;br&gt; <br /> | pET28b&lt;br&gt; <br /> | NdeI+EcoR1&lt;br&gt;<br /> |}<br /> <br /> Looks like there is no HindIII and EcoR1 sites in pET3a_Muts&lt;br&gt; <br /> <br /> From published paper, the TaqMuts cDNA can be cut out with NdeI+ BamH1, but there is a BamH1 site within the gene.&lt;br&gt; <br /> <br /> === PCR ===<br /> <br /> Date: 2011-8-19 <br /> <br /> Primer F: Taq-Muts-F cata cat atggaaggcat gctgaagggc&lt;br&gt;Primer-R: Taq-Muts-R cgatgaattctcaccccttcatggtatccaag&lt;br&gt; <br /> <br /> [[Image:MutS PCR 8 19 2011.jpg]]&lt;br&gt; <br /> <br /> Cut out TaqMuts bands, purify with spin column. <br /> <br /> Digest with NdeI + EcoR1, ligate to NdeI+ EcoR1 digested pET28b. <br /> <br /> Transform 10 ul using STBL3, grow on LB/Kan O/N&lt;br&gt;<br /> <br /> === Colony Screening ===<br /> <br /> Date: 2011-8-21&lt;br&gt; [[Image:PET28b-TaqMuts_NdeI_EcoR1_2011_8_21.jpg]]&lt;br&gt; <br /> <br /> digest 5 ul DNA with NdeI + EcoR1<br /> <br /> clone 2 &amp;amp; 6 look good.<br /> <br /> send clone 2 for sequencing T7-F and T7-R<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>

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