Editing 1198 pCDNA3.2-ssRFP-Fc-(3c)

= '''pCDNA3.2-ssRFP-Fc-(3c) (1198)'''<br>  =

'''Date: 2012-05-26''' 

'''Notebook:'''&nbsp; 4480<br> 

'''&nbsp;Page''': 4482 

'''Clone ID:''' 1198 

'''Experimenters:''' <span style="font-weight: bold;">Paul</span><br> 

== Cloning  ==

PCR using these primers 

Sal1 BamH1 Kozak<br>RFP-Fc-Sal1-F1 accgtcGTCGACGGATCCGCCACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACC<br>RFP-Fc-Sal1-F2 CTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGTGTCTAAGGGCGAAGAGCTG<br>ssRFP accgtcGTCGACGGATCCGCCACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGTGTCTAAGGGCGAAGAGCTG<br> RFP linker Fc <br>RFP-Fc fusion CTAGCAAACTGGGGCACAAACT GGC AGC GGC GACAAAACTCACACATGCCCAC<br>RFP-Fc-F3 CTAGCAAACTGGGGCACAAACT GGC AGC GGC GACAAAACTCACACATGCCCAC<br>RFP-Fc-R1 GTGGGCATGTGTGAGTTTTGTCGCCGCTGCCAGTTTGTGCCCCAGTTTGCTAG 

Fc TEV Kpn1 <br>ctctccctgtctccgggtaaa GGC AGC GGC GAG AAC CTG TAC TTC CAG GGC ggt acc<br> G S G E N L Y F Q G<br>Fc-TEV-Kpn1-R4 <br>(ctctccctgtctccgggtaaa GGC AGC GGC GAG AAC CTG TAC TTC CAG GGC ggt acc ctagta)<br>tactagggtaccGCCCTGGAAGTACAGGTTCTCGCCGCTGCCtttacccggagacagggagag 

<br> 

Fc-HRV3C-Kpn1-R5 <br> Fc human rhinovirus (HRV) type 14 3C protease<br>(ctctccctgtctccgggtaaa ctg gag gtg ctg ttc cag&nbsp;! ggc ccc ggt acc ctagta)<br> L E V L F Q G P G T <br> <br>tactagggtaccggggccctggaacagcacctccagtttacccggagacagggagag<br> 

<br> 

How to make<br>1. PCR using RFP-Fc-Sal1-F1, 10% RFP-Fc-Sal1-F2 + RFP-Fc-R1 using 114 pET28b-TagRFP as template --&gt; to amplify RFP<br>2. PCR using RFP-Fc-F3 + Fc-HRV3C-Kpn1-R5 <strike>and 380 pCMVK-IL2-intFc-IRES-TagGFP</strike>&nbsp;1101&nbsp;pFUSE-hIgG1-Fc2-IAB&nbsp;as template, to amplify Fc domain.<br>3. Join two fragment using Gibson mix, gel purify<br>4. Digest with BamH1 + Kpn1.<br>5. Ligate to BamH1 + Kpn1 digested 1192 pCDNA3.2. 

<br> 

[[Image:1193 1198 1199 PCR 5 26 2012.jpg]] 

[[Image:1193 1198 1199 Gibson PCR 5 27 2012.jpg]]<br> 

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5/27/2012<br> 

digest PCR product and vector with BamH1 + Kpn1<br> 

[[Image:1193 1198 1199 cloning PCR frag 5 27 2012.jpg]]<br> 

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| 1192 pCDNA3.2<br> 
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1198 

ssRFP-Fc-(3c)<br> 

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1193 

Fc<br> 

| 
1199 

ssRFP-Fc-(tev)<br> 

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gel purified, and ligate, transform DH5alpha.<br> <br> 

== Colony Screening by Restriction Digestion<br>  ==

5-29-2012<br> 

[[Image:1198_digest_detect_5-29-2012.JPG]]

clone 5 may be right, need to sequence

[[Category:Cloning]]