https://eln.excellgen.com/index.php?action=history&feed=atom&title=3208_pEPD-FW 3208 pEPD-FW - Revision history 2026-10-18T10:26:09Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=3208_pEPD-FW&diff=18440&oldid=prev Paul.xu at 12:32, 26 June 2018 2018-06-26T12:32:05Z <p></p> <p><b>New page</b></p><div>== &#039;&#039;&#039;Cloning of&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Purpose:&#039;&#039;&#039;&amp;nbsp;&amp;nbsp;<br /> <br /> &#039;&#039;&#039;Date: &#039;&#039;&#039;2018-6-22<br /> <br /> &#039;&#039;&#039;Notebook:&amp;nbsp;&#039;&#039;&#039;<br /> <br /> &#039;&#039;&#039;Page:&#039;&#039;&#039;<br /> <br /> &#039;&#039;&#039;Clone ID:3208&#039;&#039;&#039;<br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039;&amp;nbsp;Yijia, Paul&amp;nbsp;<br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === &#039;&#039;&#039;PCR&#039;&#039;&#039; ===<br /> <br /> Date:&amp;nbsp;Date: 6/20/2018<br /> <br /> By: Yijia<br /> <br /> template:3152 pCEH0-Flag-VH-mIgG2a &amp;nbsp;;3010 pCMV5-Flag-kappa as template<br /> <br /> primers:<br /> <br /> Fragment 1: PCR from 3152 pCEH0-Flag-VH-mIgG2a as template<br /> <br /> 3208-F1 ACTCGCGGCCCAGCCGGCCATGgctgtacactccGAGGTTCAGCTGCAGCAGTC<br /> <br /> 3208-R1 aGATCCGCCACCGCCGGATCCACCTCCGCCTGAACCGCCTCCACCGCTAGCAGAGGACACGGTCAGTGTG (CACACTGACCGTGTCCTCTGCTAGCGGTGGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCt)<br /> <br /> Fragment 2: PCR from 3010 pCMV5-Flag-kappa as template<br /> <br /> 3208-F2 GATCCGGCGGTGGCGGATCt aga tct GCGCGC TGTGACGTGCTGATGACACAGGCTC<br /> <br /> 3208-R2 atggtgttggcctcccgggccTAGGACCtcgAGcttGgtAcctccgccaaaggTG<br /> <br /> <br /> <br /> Gel:<br /> <br /> &#039;&#039;&#039;1st PCR of 3208&#039;&#039;&#039;<br /> <br /> [[File:IMG 0915.JPG|RTENOTITLE]]<br /> <br /> &#039;&#039;&#039;2nd PCR of 3208&#039;&#039;&#039;<br /> <br /> [[File:IMG 0922.JPG|RTENOTITLE]]<br /> <br /> <br /> <br /> &#039;&#039;&#039;Cloning&#039;&#039;&#039;<br /> <br /> By: Paul<br /> <br /> Vector: 3200 pEPD digested with SfiI<br /> <br /> Ligation: 30 min with T4 DNA ligase<br /> <br /> Competent cells: NEBTurbo<br /> <br /> <br /> <br /> &#039;&#039;&#039;Colony PCR Screening&amp;nbsp;&#039;&#039;&#039;<br /> <br /> <br /> <br /> pADL-F and 3208-R2<br /> <br /> &#039;&#039;&#039;[[File:3208 colony screening.JPG|RTENOTITLE]]&#039;&#039;&#039;<br /> <br /> <br /> <br /> &#039;&#039;&#039;Restriction Digestion&#039;&#039;&#039;<br /> <br /> Date: 6/21/2018<br /> <br /> By: Paul<br /> <br /> Nco1 + Xho1<br /> <br /> [[File:3208 digestion1.JPG|RTENOTITLE]]<br /> <br /> [[File:3208 3231 3230 digestion.JPG|RTENOTITLE]]<br /> <br /> &#039;&#039;&#039;Sequencing&#039;&#039;&#039;<br /> <br /> 6/22/18, clone #1, no good, missing 1C<br /> <br /> pick more clones, make moiniprep.<br /> <br /> [[File:3208 digestion2.JPG|RTENOTITLE]]<br /> <br /> send #8 to sequencing<br /> <br /> sequencing: clone #8 is good<br /> <br /> == &#039;&#039;&#039;Result intepretation/Summary&#039;&#039;&#039; ==<br /> <br /> == &#039;&#039;&#039;Conclusion&#039;&#039;&#039; ==<br /> [[Category:Cloning]]</div> Paul.xu