https://eln.excellgen.com/index.php?action=history&feed=atom&title=2022_pEC-ExcellScript 2022 pEC-ExcellScript - Revision history 2026-10-18T16:36:55Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=2022_pEC-ExcellScript&diff=11020&oldid=prev Paul.xu: Created page with "== '''Cloning of 2022 pEC-ExcellScript''' == '''Purpose:''' Cloning of2022 pEC-ExcellScript'''==''' '''Date: '''2014-1---- '''Notebook:0''' '''Page:''' '''Clone ID:2022..." 2014-01-17T19:33:29Z <p>Created page with &quot;== &#039;&#039;&#039;Cloning of 2022 pEC-ExcellScript&#039;&#039;&#039; == &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2022 pEC-ExcellScript&#039;&#039;&#039;==&#039;&#039;&#039; &#039;&#039;&#039;Date: &#039;&#039;&#039;2014-1---- &#039;&#039;&#039;Notebook:0&#039;&#039;&#039; &#039;&#039;&#039;Page:&#039;&#039;&#039; &#039;&#039;&#039;Clone ID:2022...&quot;</p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of 2022 pEC-ExcellScript&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039; Cloning of2022 pEC-ExcellScript&#039;&#039;&#039;==&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2014-1---- <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:2022&#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 <br /> <br /> 1. first PCR 7 fragments, do not amply more than 24 cycles, usually 20 cycles should be enough<br /> <br /> ssIII-Nde1-F1&lt;br&gt;ctagcCATATGACCCTAAATATAGAAGATGAGCATCGGCTAC&lt;br&gt;ssIII-L52P-R1&lt;br&gt;(CTCCTCTGATCATACCT CcG AAAGCAACC TCTACCCCCGTGTCC)&lt;br&gt;GGACACGGGGGTAGAGGTTGCTTTCgGAGGTATGATCAGAGGAG<br /> <br /> ssIII-Y64S-F2&lt;br&gt;G AAAGCAACC TCTACCCCCGTGTCC ATAAAACAA Tct CCg ATGTCACAAGAAG&lt;br&gt;ssIII-K152M-R2&lt;br&gt;(GTGCTTGATTTA AtG GATGCCTTT TTC)&lt;br&gt;GAAAAAGGCATCCaTTAAATCAAGCAC<br /> <br /> ssIII-K152M-F3&lt;br&gt;GTGCTTGATTTA AtG GATGCCTTT TTC&lt;br&gt;ssIII-H204R-R3&lt;br&gt;(GAGGCACTG CgC AGA GACCTAGCAGACTTC CGGATCCAGCACCCA GAC)&lt;br&gt;GTCTGGGTGCTGGATCCGGAAGTCTGCTAGGTCTCTGcGCAGTGCCTC<br /> <br /> &lt;br&gt;ssIII-V223H-F4&lt;br&gt;CAGACTTCCGGATCCAGCACCCAGACTTGATCCTGCTACAGTACcatGATGACTTACTGCTG&lt;br&gt;ssIII-M289L-R4&lt;br&gt;(GAGGCCAGAAAA GAGACTGTG cTG GGG CAGCCTACTCCGAAG ACCCCTCGACAACTAAG)&lt;br&gt;CTTAGTTGTCGAGGGGTCTTCGGAGTAGGCTGCCCCAgCACAGTCTCTTTTCTGGCCTC<br /> <br /> ssIII-T306K-F309N-F5&lt;br&gt;CTCCGAAG ACCCCTCGACAACTA AGGGAGTTCCTAGGG Aaa GCAGGC aaC TGT CGCCTCTGGATCCCT G&lt;br&gt;ssIII-D524G-R5&lt;br&gt;(CACC TGGTACACG GgT GGA AGCAGTCTCTTAC)&lt;br&gt;GTAAGAGACTGCTTCCAcCCGTGTACCAGGTG<br /> <br /> ssIII-D524G-F6&lt;br&gt;CACG GgT GGA AGCAGTCTCTTAC&lt;br&gt;ssIII-E562Q-R6&lt;br&gt;(CTCAGCGG GCT cag CTGATAGCA CTCACCCAGGCCCTA AAGATGGCAGAAG)&lt;br&gt;CTTCTGCCATCTTTAGGGCCTGGGTGAGTGCTATCAGctgAGCCCGCTGAG<br /> <br /> ssIII-D583N-F7&lt;br&gt;CAGGCCCTA AAGATGGCAGAAGGT AAGAAGCTAAATGTT TATACT aAT AGCCGT TATGCTTTTG&lt;br&gt;ssIII-Xho1-R7&lt;br&gt;(ctaccctcctcatagaaaattcatca ccg aattcccgcttaattaat ctcgag taacgc)&lt;br&gt;gcgttactcgagattaattaagcgggaattcggtgatgaattttctatgaggagggtag<br /> <br /> &lt;br&gt;<br /> <br /> [[Image:2022pcr1.JPG]]&lt;br&gt;2. Purify these fragments (mix gel slices, add QG, dissolve, purify using one spin column). &lt;br&gt;Use purify DNA mix as template, do PCR:<br /> <br /> &lt;br&gt;ssIII-Nde1-F1&lt;br&gt;ctagcCATATGACCCTAAATATAGAAGATGAGCATCGGCTAC<br /> <br /> ssIII-Xho1-R7&lt;br&gt;(ctaccctcctcatagaaaattcatca ccg aattcccgcttaattaat ctcgag taacgc)&lt;br&gt;gcgttactcgagattaattaagcgggaattcggtgatgaattttctatgaggagggtag<br /> <br /> [[Image:2022pcr2.JPG]]&lt;br&gt;digest full length PCR product with Nde1 + Xho1, then ligate to Nde1 + Xho1 digested vector&lt;br&gt;[[Image:DIGEST_FOR_2022_2023_1992.JPG]][[Image:2022PCRDIGEST_1274_1326_2018PCR.JPG]]&lt;br&gt;<br /> <br /> === Colony Screening ===<br /> <br /> Date: 2013-10-23 <br /> <br /> Make miniprep, digest with Nde1 + Xho1+BamHI&lt;br&gt;[[Image:2022_2023DIGEST.JPG]]&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> &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