Danijela Maric Blog April10
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GenY HSA:iRFP670 Maxi Prep Diagnostic Digest • Maxi prep DNA was Made my Mike and purified with Cesium Chloride (1.95μg/μl conc.) • Digestion with Ecor5 and Sal1 is suppose to drop down the IRES_HSA:iRFP670 insert band of 1,800bp • We used this DNA to make VSVg virus to transduce 293T cells and monitor luciferase and iRFP670 expression • Gen2 iRFP670 construct was used to make control VSVg virus for 2kb 1,800bp 1.5kb 1kb 3kb
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Danijela Blog April 10, 2015
Transcript of Danijela Maric Blog April10
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GenY HSA:iRFP670 Maxi Prep Diagnostic Digest
Maxi prep DNA was Made my Mike and purified with Cesium Chloride (1.95g/l conc.)Digestion with Ecor5 and Sal1 is suppose to drop down the IRES_HSA:iRFP670 insert band of 1,800bpWe used this DNA to make VSVg virus to transduce 293T cells and monitor luciferase and iRFP670 expressionGen2 iRFP670 construct was used to make control VSVg virus for comparison2kb1,800bp1.5kb1kb3kb
Luciferase:FITCiRFP670 (direct)Merge293T Transduced Cells with Gen2 iRFP670 VSVg VirusConclusion: Cells transduced with Gen2 iRFP670 virus express both Luciferase and iRFP670 proteins robustly.
Luciferase:FITCiRFP670 (direct)Merge293T Transduced Cells with GenY HSA:iRFP670 VSVg VirusConclusion: Cells transduced with GenY HSA:iRFP670 virus express Luciferase robustly but no iRFP670.293T Transduced Cells with GenY HSA:iRFP670 VSVg Virus
Luciferase:FITCiRFP670 (direct)MergeConclusion: Cells transduced with GenY HSA:iRFP670 virus express Luciferase robustly but no iRFP670. Same conclusion as on the prior slide.293T Transduced Cells with GenY HSA:iRFP670 VSVg Virus
HSA:FITCConclusion: Cells transduced with GenY HSA:iRFP670 virus also express HSA robustly. So in overall conclusion Luciferase and HSA are expressed while iRFP670 is not. Gen2_iRFP670 vs. GenY_HSA:iRFP670 Sequence Comparison
Gen2_iRFP670 SequenceGenY_HSA:iRFP670 SequenceACCGGT: Age1 SiteGTCGAC: Sal1 SiteiRFP670 sequence (start and stop sites present)ACCGGT: Age1 SitesGTCGAC: Sal1 SiteGCCGCCACC: Kozak SequenceHSA Sequence (start site present, stop site deleted)iRFP670 sequence (start site deleted, stop site present)
