https://eln.excellgen.com/index.php?action=history&feed=atom&title=2199_pET30b-Ecoli-Muts 2199 pET30b-Ecoli-Muts - Revision history 2026-10-18T12:08:07Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=2199_pET30b-Ecoli-Muts&diff=13950&oldid=prev Paul.xu at 17:27, 19 February 2015 2015-02-19T17:27:44Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 2199 pET30b-Ecoli-Muts&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2199 pET30b-Ecoli-Muts&#039;&#039;&#039;==&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2014-9-15--- <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:2199&#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 Escherichia coli str. K-12 substr. MG1655 genomic DNA <br /> <br /> Do not amplify more than 20 cycles for the fist PCR <br /> <br /> 1st fragment <br /> <br /> Ecoli-Muts-Nde1-F tacgacatatgagtgcaatagaaaatttc <br /> <br /> 2199-Ecoli-Muts-R2 (gaaacgctggctg caC atgccagtg) cactggcatGtgcagccagcgtttc <br /> <br /> [[Image:1916 2326 2327 2328 2199pcr.JPG]]&lt;br&gt; 2nd fragment <br /> <br /> 2199-Ecoli-Muts-F2 ctggctg caC atgccagtgcgcgatac <br /> <br /> Ecoli-Muts-Xho1-R (gatttatcgcttgaagagcctggtg ctcgag tagct) <br /> <br /> agctactcgagcaccaggctcttcaagcgataaatc <br /> <br /> [[Image:2328 1916 2199 2325 1504 2321 2345PCR.JPG]]&lt;br&gt; join two fragments by PCR again Ecoli-Muts-Nde1-F+ Ecoli-Muts-Xho1-R <br /> <br /> [[Image:1504 2199 2321pcr.JPG]]&lt;br&gt; <br /> <br /> Digest with Nde1 + Xho1&lt;br&gt; <br /> <br /> [[Image:Digest for 1916 2199 2346.JPG]]&lt;br&gt; <br /> <br /> [[Image:DIGEST FOR 2297 1504 1319 2240 2241 2246 2199.JPG]] <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2014_9&lt;br&gt; <br /> <br /> Make miniprep, use Nde1 + Xho1 to find positive clone&lt;br&gt; <br /> <br /> [[Image:1319 1504 2241 2346digest.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt;<br /> <br /> == Cloning ==<br /> <br /> PCR from Escherichia coli str. K-12 substr. MG1655 genomic DNA &lt;br&gt; <br /> <br /> Oligos:&lt;br&gt; <br /> <br /> Ecoli-Muts-Nde1-F (5_14_2014, A05)&lt;br&gt;Sequence TACGACATATGAGTGCAATAGAAAATTTC&lt;br&gt; <br /> <br /> 2199-Ecoli-Muts-R2 (9_8_2014 E09)&lt;br&gt;(gaaacgctggctg caC atgccagtg)&lt;br&gt;cactggcatGtgcagccagcgtttc <br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> 2nd fragment <br /> <br /> 2199-Ecoli-Muts-F2: (9_8_2014, E10 )&lt;br&gt;ctggctg caC atgccagtgcgcgatac <br /> <br /> Ecoli-Muts-Xho1-R: (5_14_2014, A06 )&lt;br&gt;(gatttatcgcttgaagagcctggtg ctcgag tagct)&lt;br&gt;agctactcgagcaccaggctcttcaagcgataaatc <br /> <br /> join two fragments by PCR again&lt;br&gt;Ecoli-Muts-Nde1-F&lt;br&gt;Ecoli-Muts-Xho1-R <br /> <br /> clone into vector pET30b&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> Paul.xu