https://eln.excellgen.com/index.php?action=history&feed=atom&title=202-pET28b-DNase1 202-pET28b-DNase1 - Revision history 2026-10-18T15:08:14Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=202-pET28b-DNase1&diff=2137&oldid=prev Luyingying: Created page with "= '''pCMVK-StrepII (193)'''<br/> = == '''Make&nbsp; pCMVK-StrepII''' == '''Date: 2011-09-02''' '''Notebook:''' '''Page:''' '''Clone ID:''' 193 '''Experimenters:''' <span st..." 2011-11-02T19:50:46Z <p>Created page with &quot;= &#039;&#039;&#039;pCMVK-StrepII (193)&#039;&#039;&#039;&lt;br/&gt; = == &#039;&#039;&#039;Make  pCMVK-StrepII&#039;&#039;&#039; == &#039;&#039;&#039;Date: 2011-09-02&#039;&#039;&#039; &#039;&#039;&#039;Notebook:&#039;&#039;&#039; &#039;&#039;&#039;Page:&#039;&#039;&#039; &#039;&#039;&#039;Clone ID:&#039;&#039;&#039; 193 &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; &lt;span st...&quot;</p> <p><b>New page</b></p><div>= &#039;&#039;&#039;pCMVK-StrepII (193)&#039;&#039;&#039;&lt;br/&gt; =<br /> <br /> == &#039;&#039;&#039;Make&amp;nbsp; pCMVK-StrepII&#039;&#039;&#039; ==<br /> <br /> &#039;&#039;&#039;Date: 2011-09-02&#039;&#039;&#039;<br /> <br /> &#039;&#039;&#039;Notebook:&#039;&#039;&#039;<br /> <br /> &#039;&#039;&#039;Page:&#039;&#039;&#039;<br /> <br /> &#039;&#039;&#039;Clone ID:&#039;&#039;&#039; 193<br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; &lt;span style=&quot;font-weight: bold&quot;&gt;Paul&lt;/span&gt;<br /> <br /> === Annealing ===<br /> <br /> Date:&lt;br/&gt;Anneal 8685-T and 8685-B at 95&lt;sub&gt;&lt;sup&gt;0&lt;/sup&gt;&lt;/sub&gt;C for 2 minutes.&lt;br/&gt;Digest the vector pCMVK-GFP(#194) with BgIII+ Sal1<br /> <br /> Run gel and cut out vector,&amp;nbsp; purify using spin column<br /> <br /> Ligate with annealed oligos for 2 hours<br /> <br /> Primer-F 8685-T tc gac CACGTG GAATTC TCTAGA TGG AGC CAC CCG CAG TTC GAA AAG TAA CTC GAG GGATCC A&lt;br/&gt;Primer-R 8685-B GATCTGGATCCCTCGAGTTACTTTTCGAACTGCGGGTGGCTCCATCTAGAGAATTCcacgtgg<br /> <br /> and transform Top10<br /> <br /> [[File:PCMVK-StrepII-08-30-2011-1.JPG|RTENOTITLE]]<br /> <br /> Lane 1 shows the digestion of vector (pCMVK-GFP) with BgIII+ Sal1;<br /> <br /> Lane 2: the plasmid F2 from # 7896 Kinase, digested with Sal1 + HindIII<br /> <br /> Lane 3: the plasmid F02 from # 7898 Kinase, digested with Sal1 + HindIII<br /> <br /> Lane 4:digestion of #348<br /> <br /> Lane 5: PlentiGP-app, run DNA Argrose gel after miniprep<br /> <br /> <br /> <br /> == Colony Screening by Restriction Digestion&lt;br/&gt; ==<br /> <br /> 1, 2,: Digest with BglII + KpnI<br /> <br /> 3, 4, 5: 194 digest with BglII + Kpn1<br /> <br /> 6~10: 193 digestion with BglII + Kpn1<br /> <br /> 11, 12, 13: 194 digest with Kpn1+ Xho1<br /> <br /> 14~18: 193 digest with Kpn1 + Xho1<br /> <br /> [[File:192 193 194 digestion 94 2011.jpg|RTENOTITLE]]<br /> <br /> [[File:192 193 194 digestion 9 4 2011 2.jpg|RTENOTITLE]]<br /> <br /> <br /> <br /> 193: digest with Kpn1+ EcoR1<br /> <br /> [[File:193 Kpn1 EcoR1 9 4 2011.jpg|RTENOTITLE]]<br /> <br /> [[File:193 Kpn1 EcoR1 9 4 2011 2.jpg|RTENOTITLE]]<br /> <br /> <br /> <br /> [[File:193 Kpn1 Xho1 KpnI BamH1.jpg|RTENOTITLE]]<br /> <br /> digest with Kpn1 + Sal1<br /> <br /> [[File:193 Kpn1 Sal1 TaqMuts.jpg|RTENOTITLE]]<br /> <br /> Looks like clone #4 is good.<br /> <br /> --[[User:Paul.xu|Paul.xu]] 15:44, 4 September 2011 (EST)<br /> <br /> &lt;br/&gt;&lt;br/&gt;<br /> <br /> [[Category:Cloning]] &lt;br/&gt;</div> Luyingying