Author: Qi Liu; Amita Gupta; Ayse Okesli-Armlovich; Wenjie Qiao; Curt R. Fischer; Mark Smith; Jan E. Carette; Michael C. Bassik; Chaitan Khosla
Title: Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism Document date: 2020_3_25
ID: 1zk64gsg_44
Snippet: The plasmid pDENV-Luc replicon containing a Renilla luciferase expressing DENV subgenomic replicon was described previously (Marceau et al., 2016) . For in vitro transcription, 10 ug of plasmid DNA was linearized using XbaI restriction enzyme. Replicon RNA was generated using the MEGAscript T7 Transcription Kit in the presence of 5mM m7G(5′)ppp(5′)G RNA Cap Structure Analogue. Two million cells were washed twice with PBS and re-suspended in e.....
Document: The plasmid pDENV-Luc replicon containing a Renilla luciferase expressing DENV subgenomic replicon was described previously (Marceau et al., 2016) . For in vitro transcription, 10 ug of plasmid DNA was linearized using XbaI restriction enzyme. Replicon RNA was generated using the MEGAscript T7 Transcription Kit in the presence of 5mM m7G(5′)ppp(5′)G RNA Cap Structure Analogue. Two million cells were washed twice with PBS and re-suspended in electroporation buffer (Teknova, E0399), mixed with 4 ug of purified replicon RNA, and electroporated using Bio-Rad Gene Pulser Xcell electroporator using square wave protocol. Electroporated cells were resuspended in pre-warmed culture medium without antibiotics and seeded as required. Luciferase expression was measured using Renilla Luciferase Assay system (Promega, E2820) following the manufacturers' instructions.
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