https://eln.excellgen.com/index.php?action=history&feed=atom&title=2062_pEC-Mxe-gyrA 2026-10-18T16:58:47Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=2062_pEC-Mxe-gyrA&diff=11551&oldid=prev 2014-03-25T15:00:27Z

<p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 2062 pEC-Mxe-gyrA&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2062 pEC-Mxe-gyrA&#039;&#039;&#039;==&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2014-2---- <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:2062&#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 /> 1. Gene synthesis using&lt;br&gt;Mxe_gyrA_F1 (1 ul of 10 uM)&lt;br&gt;tcgacAAGCTTGCGGCCGCActcgagggctcttcctgcatcTGTggagatgcactGgttgccctGccGgagggcgagt&lt;br&gt;Mxe_gyrA_F2 (1 ul of 1 uM)&lt;br&gt;gttgccctGccGgagggcgagtcggtacgcatcgccgacatcgtgccgggtgcgcggccGaacagtgacaacgcca <br /> <br /> Mxe_gyrA_R1 (1 ul of 10 uM)&lt;br&gt;(G aacagtgacaacgccatcgacctgaaagtccttgaccggcatggcaatccG gtgctcgc cgaccggc)&lt;br&gt;gccggtcggcgagcacCggattgccatgccggtcaaggactttcaggtcgatggcgttgtcactgttC <br /> <br /> 2. PCR fragments using 1126 pTXB1 as template&lt;br&gt;Fragment 1&lt;br&gt;Mxe_gyrA_F3&lt;br&gt;tccG gtgctcgc cgaccggctgttccac tGc ggcgagca <br /> <br /> Mxe_gyrA_R2&lt;br&gt;(gaaaa ccG gaatttgcg ccG a caacctacacagtcggcgt)&lt;br&gt;acgccgactgtgtaggttgtCggcgcaaattcCggttttc <br /> <br /> Fragment 2&lt;br&gt;Mxe_gyrA_F4&lt;br&gt;G a caacctacacagtcggcgtccctggactggtgcgtttcttggaagcacac cac cgTgac <br /> <br /> Mxe_gyrA_R3&lt;br&gt;(caccggtctgaactcaggcctcggtacc tacgtac)&lt;br&gt;gtacgtaggtaccgaggcctgagttcagaccggtg <br /> <br /> [[Image:2028 2062firstpcr 2068pcr.JPG]]&lt;br&gt; <br /> <br /> 3. Gel purify, join 3 fragments by overlap extension PCR <br /> <br /> Mxe_gyrA_F1 + Mxe_gyrA_R3 <br /> <br /> [[Image:2062 2028pcr.JPG]]&lt;br&gt; <br /> <br /> 4.digest with Kpn1 + HindIII, clone into Kpn1 + HindIII digested vector pEC <br /> <br /> [[Image:DIGEST FOR 2062.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2014_2_14 <br /> <br /> Make miniprep, digestion with EcoR1 + HindIII &lt;br&gt; <br /> <br /> [[Image:2062 2063DIGEST.JPG]] <br /> <br /> sent 2062-1 to sequence,one extra G&lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Cloning of fixing 2062 pEC-Mxe-gyrA&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2062 pEC-Mxe-gyrA&#039;&#039;&#039;==&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2014-2---- <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:2062&#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 /> Fix mutation &amp;gt;2062 clone #1 <br /> <br /> Mxe-gyrA-fix-F&lt;br&gt;gtcgactgtgca ggt ttC gcc cgT ggC aaa ccg gaa ttt gcgccgac&lt;br&gt;Mxe-gyrA-fix-R&lt;br&gt;gtcggcgcaaattccggtttGccAcgggcGaaacctgcacagtcgac <br /> <br /> [[Image:2062fixpcr1st.JPG]] <br /> <br /> join two fragments by pcr <br /> <br /> [[Image:1560fixpcr 2062fixpcr2.JPG]]&lt;br&gt;&lt;br&gt; <br /> <br /> .digest with Kpn1 + HindIII, clone into Kpn1 + HindIII digested vector pEC <br /> <br /> [[Image:Digest for 1560fix 2062fix.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2014_2_ <br /> <br /> Make miniprep, digestion with EcoR1 + HindIII &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> [[Image:1560fix 2062fix 2070digest.JPG]]&lt;br&gt; <br /> <br /> sent 2062fix-1to sequence, one mutation&lt;br&gt; <br /> <br /> == &#039;&#039;&#039;Cloning of fixing 2062fix pEC-Mxe-gyrA&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2062 pEC-Mxe-gyrA&#039;&#039;&#039;==&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2014-3---- <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:2062&#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 /> Fix mutation &amp;gt;2062 fixclone #1 <br /> <br /> Mxe_gyrA_F1 (1 ul of 10 uM)&lt;br&gt;tcgacAAGCTTGCGGCCGCActcgagggctcttcctgcatcTGTggagatgcactGgttgccctGccGgagggcgagt <br /> <br /> Mxe_gyrA_R2&lt;br&gt;(gaaaa ccG gaatttgcg ccG a caacctacacagtcggcgt)&lt;br&gt;acgccgactgtgtaggttgtCggcgcaaattcCggttttc <br /> <br /> Fragment 2&lt;br&gt;Mxe_gyrA_F4&lt;br&gt;G a caacctacacagtcggcgtccctggactggtgcgtttcttggaagcacac cac cgTgac <br /> <br /> Mxe_gyrA_R3&lt;br&gt;(caccggtctgaactcaggcctcggtacc tacgtac)&lt;br&gt;gtacgtaggtaccgaggcctgagttcagaccggtg&lt;br&gt;[[Image:2062fix2pcr1.JPG]]&lt;br&gt; <br /> <br /> join two fragments by pcr <br /> <br /> [[Image:2132 2062fix2.JPG]]&lt;br&gt;&lt;br&gt; <br /> <br /> .digest with Kpn1 + HindIII, clone into Kpn1 + HindIII digested vector pEC <br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> === Colony Screening ===<br /> <br /> Date: 2014_3_ <br /> <br /> Make miniprep, digestion with EcoR1 + HindIII &lt;br&gt; <br /> <br /> [[Image:2062fix2 2132digest.JPG]]&lt;br&gt; <br /> <br /> sent 2062fix2-2&amp;nbsp; to sequence, good&lt;br&gt;&lt;br&gt; <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>

Paul.xu