https://eln.excellgen.com/index.php?action=history&feed=atom&title=1198_pCDNA3.2-ssRFP-Fc-%283c%29 1198 pCDNA3.2-ssRFP-Fc-(3c) - Revision history 2026-10-18T10:42:18Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=1198_pCDNA3.2-ssRFP-Fc-(3c)&diff=4329&oldid=prev Paul.xu at 17:11, 29 May 2012 2012-05-29T17:11:14Z <p></p> <p><b>New page</b></p><div>= &#039;&#039;&#039;pCDNA3.2-ssRFP-Fc-(3c) (1198)&#039;&#039;&#039;&lt;br&gt; =<br /> <br /> &#039;&#039;&#039;Date: 2012-05-26&#039;&#039;&#039; <br /> <br /> &#039;&#039;&#039;Notebook:&#039;&#039;&#039;&amp;nbsp; 4480&lt;br&gt; <br /> <br /> &#039;&#039;&#039;&amp;nbsp;Page&#039;&#039;&#039;: 4482 <br /> <br /> &#039;&#039;&#039;Clone ID:&#039;&#039;&#039; 1198 <br /> <br /> &#039;&#039;&#039;Experimenters:&#039;&#039;&#039; &lt;span style=&quot;font-weight: bold;&quot;&gt;Paul&lt;/span&gt;&lt;br&gt; <br /> <br /> == Cloning ==<br /> <br /> PCR using these primers <br /> <br /> Sal1 BamH1 Kozak&lt;br&gt;RFP-Fc-Sal1-F1 accgtcGTCGACGGATCCGCCACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACC&lt;br&gt;RFP-Fc-Sal1-F2 CTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGTGTCTAAGGGCGAAGAGCTG&lt;br&gt;ssRFP accgtcGTCGACGGATCCGCCACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGTGTCTAAGGGCGAAGAGCTG&lt;br&gt; RFP linker Fc &lt;br&gt;RFP-Fc fusion CTAGCAAACTGGGGCACAAACT GGC AGC GGC GACAAAACTCACACATGCCCAC&lt;br&gt;RFP-Fc-F3 CTAGCAAACTGGGGCACAAACT GGC AGC GGC GACAAAACTCACACATGCCCAC&lt;br&gt;RFP-Fc-R1 GTGGGCATGTGTGAGTTTTGTCGCCGCTGCCAGTTTGTGCCCCAGTTTGCTAG <br /> <br /> Fc TEV Kpn1 &lt;br&gt;ctctccctgtctccgggtaaa GGC AGC GGC GAG AAC CTG TAC TTC CAG GGC ggt acc&lt;br&gt; G S G E N L Y F Q G&lt;br&gt;Fc-TEV-Kpn1-R4 &lt;br&gt;(ctctccctgtctccgggtaaa GGC AGC GGC GAG AAC CTG TAC TTC CAG GGC ggt acc ctagta)&lt;br&gt;tactagggtaccGCCCTGGAAGTACAGGTTCTCGCCGCTGCCtttacccggagacagggagag <br /> <br /> &lt;br&gt; <br /> <br /> Fc-HRV3C-Kpn1-R5 &lt;br&gt; Fc human rhinovirus (HRV) type 14 3C protease&lt;br&gt;(ctctccctgtctccgggtaaa ctg gag gtg ctg ttc cag&amp;nbsp;! ggc ccc ggt acc ctagta)&lt;br&gt; L E V L F Q G P G T &lt;br&gt; &lt;br&gt;tactagggtaccggggccctggaacagcacctccagtttacccggagacagggagag&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> How to make&lt;br&gt;1. PCR using RFP-Fc-Sal1-F1, 10% RFP-Fc-Sal1-F2 + RFP-Fc-R1 using 114 pET28b-TagRFP as template --&amp;gt; to amplify RFP&lt;br&gt;2. PCR using RFP-Fc-F3 + Fc-HRV3C-Kpn1-R5 &lt;strike&gt;and 380 pCMVK-IL2-intFc-IRES-TagGFP&lt;/strike&gt;&amp;nbsp;1101&amp;nbsp;pFUSE-hIgG1-Fc2-IAB&amp;nbsp;as template, to amplify Fc domain.&lt;br&gt;3. Join two fragment using Gibson mix, gel purify&lt;br&gt;4. Digest with BamH1 + Kpn1.&lt;br&gt;5. Ligate to BamH1 + Kpn1 digested 1192 pCDNA3.2. <br /> <br /> &lt;br&gt; <br /> <br /> [[Image:1193 1198 1199 PCR 5 26 2012.jpg]] <br /> <br /> [[Image:1193 1198 1199 Gibson PCR 5 27 2012.jpg]]&lt;br&gt; <br /> <br /> {| width=&quot;200&quot; cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot;<br /> |-<br /> | &lt;br&gt; <br /> | 1&lt;br&gt; <br /> | 2&lt;br&gt; <br /> | 3&lt;br&gt; <br /> | 4&lt;br&gt; <br /> | 5&lt;br&gt; <br /> | 6&lt;br&gt; <br /> | 7&lt;br&gt; <br /> | 8&lt;br&gt; <br /> | 9&lt;br&gt; <br /> | 10&lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | 1199 Gibson&lt;br&gt; <br /> | 1199 PCR&lt;br&gt; <br /> | 1199 PCR&lt;br&gt; <br /> | 1199 Gibson&lt;br&gt; <br /> | <br /> 1199 Gibson <br /> <br /> | 1198 Gibson&lt;br&gt; <br /> | 1198 Gibson&lt;br&gt; <br /> | 1198 Gibson&lt;br&gt; <br /> | 1198 PCR&lt;br&gt; <br /> | 1198 PCR&lt;br&gt;<br /> |-<br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt; <br /> | &lt;br&gt;<br /> |}<br /> <br /> 5/27/2012&lt;br&gt; <br /> <br /> digest PCR product and vector with BamH1 + Kpn1&lt;br&gt; <br /> <br /> [[Image:1193 1198 1199 cloning PCR frag 5 27 2012.jpg]]&lt;br&gt; <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot; style=&quot;width: 298px; height: 106px;&quot;<br /> |-<br /> | 1&lt;br&gt; <br /> | 2&lt;br&gt; <br /> | 3&lt;br&gt; <br /> | 4&lt;br&gt;<br /> |-<br /> | 1192 pCDNA3.2&lt;br&gt; <br /> | <br /> 1198 <br /> <br /> ssRFP-Fc-(3c)&lt;br&gt; <br /> <br /> | <br /> 1193 <br /> <br /> Fc&lt;br&gt; <br /> <br /> | <br /> 1199 <br /> <br /> ssRFP-Fc-(tev)&lt;br&gt; <br /> <br /> |}<br /> <br /> gel purified, and ligate, transform DH5alpha.&lt;br&gt; &lt;br&gt; <br /> <br /> == Colony Screening by Restriction Digestion&lt;br&gt; ==<br /> <br /> 5-29-2012&lt;br&gt; <br /> <br /> [[Image:1198_digest_detect_5-29-2012.JPG]]<br /> <br /> clone 5 may be right, need to sequence<br /> <br /> [[Category:Cloning]]</div> Paul.xu