https://eln.excellgen.com/index.php?action=history&feed=atom&title=3230_pEPD-DP47-DPL16 3230 pEPD-DP47-DPL16 - Revision history 2026-10-18T11:16:16Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=3230_pEPD-DP47-DPL16&diff=18577&oldid=prev Paul.xu at 13:30, 3 July 2018 2018-07-03T13:30:06Z <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: 3230&#039;&#039;&#039;<br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039;&amp;nbsp;Paul&amp;nbsp;<br /> <br /> == &#039;&#039;&#039;Results&#039;&#039;&#039; ==<br /> <br /> === &#039;&#039;&#039;PCR&#039;&#039;&#039; ===<br /> <br /> Date: 6/21/2018<br /> <br /> By: Paul<br /> <br /> template:<br /> <br /> primers:<br /> <br /> Gel:<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> &#039;&#039;&#039;Cloning&#039;&#039;&#039;<br /> <br /> By: Paul<br /> <br /> Vector:&amp;nbsp;&amp;nbsp;<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 /> ~&amp;nbsp;<br /> <br /> <br /> <br /> <br /> <br /> &#039;&#039;&#039;Restriction Digestion&#039;&#039;&#039;<br /> <br /> Date: 6/~/2018<br /> <br /> By: Paul<br /> <br /> http://www.excellgen.net/eln/images/8/8c/3208_3231_3230_digestion.JPG<br /> <br /> 3230 and 3231 in pMiniT2, digest with EcoR1<br /> <br /> [[File:3232 pMiniT2 EcoR1.JPG|RTENOTITLE]]<br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> &#039;&#039;&#039;Sequencing&#039;&#039;&#039;<br /> <br /> &#039;<br /> <br /> == Fix 3030 ==<br /> <br /> [[File:3230 fix.JPG|RTENOTITLE]]<br /> <br /> Insert: 3230 in pEPD, vector is 3208&amp;nbsp;pEPD-FW, yield 3230 Fix in pEPD<br /> <br /> ligate and transform<br /> <br /> === Restriction Digestion ===<br /> <br /> BamH1 + Xma1<br /> <br /> [[File:3230 fix restriction digestion.JPG|RTENOTITLE]]<br /> <br /> both clone # 2 and 3 has 1 C missing, so the clone from 3230 in pEPD is not good<br /> <br /> 6/25/18<br /> <br /> [[File:3230 3231 digestion.JPG|RTENOTITLE]]<br /> <br /> [[File:3230 fix BamH1 Xma1.JPG|RTENOTITLE]]<br /> <br /> [[File:3032 BamH1 ApaI.JPG|RTENOTITLE]]<br /> <br /> 6/26/2018<br /> <br /> [[File:3230 3231 digestion2.JPG|RTENOTITLE]]<br /> <br /> 3230 fix #4 is parental (3208)--result also confirmed by sequencing, it could not be cut by ApaI (no&amp;nbsp;GGGCCC site) .&amp;nbsp;<br /> <br /> sequencing: both 3230 fix #2 and&amp;nbsp;3230 fix #3&amp;nbsp; has 1 C missing<br /> <br /> == Redo fixing ==<br /> <br /> [[File:3230 fix2.JPG|RTENOTITLE]]<br /> <br /> [[File:New 3230 pEPD digestion.JPG|RTENOTITLE]]<br /> <br /> [[File:3230 3231 EcoR1.JPG|RTENOTITLE]]<br /> <br /> [[File:3230 EocR1 Xho1.JPG|RTENOTITLE]]<br /> <br /> [[File:3230 plasmid PCR Screening.JPG]]<br /> <br /> == &#039;&#039;&#039;Result intepretation/Summary&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;sequencing result: new 3230 clone #9 is good&#039;&#039;&#039;<br /> <br /> == &#039;&#039;&#039;Conclusion&#039;&#039;&#039; ==<br /> [[Category:Cloning]]</div> Paul.xu