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2321 pFastBacDual-VSVG-CAG-Flp
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== '''Cloning of 2321 pFastBacDual-VSVG-CAG-Flp''' == '''Purpose:''' Cloning of2321 pFastBacDual-VSVG-CAG-Flp-'''==''' '''Date: '''2014-9---- '''Notebook:0''' '''Page:''' '''Clone ID:2321''' '''Experimenters:''' shanyi == '''Results''' == === CLONING === Use 503 psPAX2 as template, PCR CAG promoter <br> 2321-CAG-Nhe1-R (ctgtctcatcattttggcaaa gaattc ggatcc gcc acc atg gct agc tagca) <br> tgctagctagccatggtggcggatccgaattctttgccaaaatgatgagacag 2321-CAG-BglII-F tactaAGATCTgtcgacattgattattgactag [[Image:1504 2199 2321pcr.JPG]]<br> Digest with BglII + Nhe1. Digest vector with BamH1 + Nhe1. [[Image:Digest for 1504 2321.JPG]][[Image:2219 2320 1981DIGEST.JPG]]<br> === Colony Screening === Date: 2014_9<br> Make miniprep, use Sal1 + EcoR1 to find positive clone<br> [[Image:1038 1916 2321 2297digest.JPG]]<br> <br> == '''Cloning of 2321 pFastBacDual-VSVG-CAG-Flp''' == '''Purpose:''' Cloning of2321 pFastBacDual-VSVG-CAG-Flp-'''==''' '''Date: '''2014-9---- '''Notebook:0''' '''Page:''' '''Clone ID:2321''' '''Experimenters:''' shanyi == '''Results''' == === CLONING === PCR CAG-pH-T7 promoters from 298 pTriEx-HTNC<br>2364-CAG-Xho1-F atagcactcgagTATATGGAGTTCCGCGTTAC<br>2364-CAG-BamH1-R tgtcatggatccTATATCTCCTTTGATTGTAAATAAAATG <br> [[Image:2321 2374PCR.JPG]]<br> Digest with Xho1 + BamH1. Digest vector with Xho1 + BamH1. [[Image:DIGEST FOR 2321 2374.JPG]]<br> === Colony Screening === Date: 2014_9<br> Make miniprep, use Xho1 + BamH1 to find positive clone<br> [[Image:2321 2374digest.JPG]]<br> digest with NdeI+XhoI should get four bands,digest with XbaI should get two bands [[Image:2321DIGEST.JPG]] [[Image:2321DIGEST21.JPG]] [[Image:2321DIGEST3.JPG]] send 2321-2 and 2321-3 to sequence with '''NLS-HindIII-R'''(NLS-HindIII-R accttcctcttcttcttcaagcttacatac), poor quality or no priming<br> use primers to do pcr screening<br> 2364-CAG-Xho1-F atagcactcgagTATATGGAGTTCCGCGTTAC<br> hIgG-Lc-BamH1-Nhe1-B ctagcggccatggccggctgggccgcgagtaataacaatccagcggctgccgtaggcaataggtatttcatggtggcg<br> [[Image:2321pcr screening.JPG]]<br> == '''Result intepretation/Summary''' == *<br> == '''Conclusion''' == [[Category:Cloning]]
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