Pnl1.1 tk nluc tk

Manufactured by Promega

Sourced in United States

PNL1.1.TK [Nluc/TK] is a laboratory equipment product from Promega. It consists of a reporter construct that expresses the Nano-Luciferase (Nluc) and thymidine kinase (TK) fusion protein. The core function of this product is to provide a bioluminescent reporter system for gene expression and cell-based assays.

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Lab products found in correlation

Quickchange site directed mutagenesis, by Agilent Technologies (1 mentions) Ifn beta pgl3, by Addgene (1 mentions) Pgl4.32 nf κb luc, by Promega (1 mentions) Supremerun, by Eurofins (1 mentions) Pgl3 promoter, by Promega (1 mentions) Lightrun, by Eurofins (1 mentions) Nano glo dual luciferase reporter assay system, by Promega (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)

4 protocols using pnl1.1 tk nluc tk

Generation and Transfection of SARS-CoV-2 Plasmids

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The following pLVX- SARS-CoV-2-Strep plasmids obtained from Addgene were used in this study: NSP1 (No 141367), NSP2 (No 141368), NSP4 (No 141369), NSP5 (No 141371), NSP7 (No 141373), NSP8 (No 141374), NSP9 (No 141375), NSP10 (No 141376), NSP11 (No 141377), NSP12 (No 141378), NSP13 (No 141379), NSP14 (No 141380), and NSP15 (No 141381) [28 ]. Additional plasmids include: pLVX-EGFP-2xStrep (Addgene No 141395), pHAGE-N-FLAG-HA TBK1 (Addgene No 131791) IFN-Beta_pGL3 (Addgene No 102597), pNL1.1.TK [Nluc/TK] (Promega), p.GL4.45 ISRE-Luc (Promega), and p.GL4.32 NFκB-Luc (Promega). pLVX-nsp1-2xStrep containing either the K164A/H165A (NSP1^KH) or the R125A/K126A (NSP1^RK) mutations were generated using QuickChange site directed mutagenesis (Agilent) with the primers 5’-CTCGCGAGTAACGCCACTGCTAGCTGCCGTATTCCAGTTTTCCTGGAAA-3’ and 5’-TTTCCAGGAAAACTGGAATACGGCAGCTAGCAGTGGCGTTACTCGCGAG-3’ (KH) and 5’-CCTGCGCCCTTGTTCCCATTTGCTGCCAACAGCACCTTCCGATATGC-3’ and 5’-GCATATCGGAAGGTGCTGTTGGCAGCAAATGGGAACAAGGGCGCAGG-3’ (RK).
The sequences of all plasmids were verified by DNA sequence (Genewiz) and are available upon request. Transfections were performed using Polyjet transfection reagent (SignaGen Laboratories) according to manufacturer’s instructions.

Vazquez C., Swanson S.E., Negatu S.G., Dittmar M., Miller J., Ramage H.R., Cherry S, & Jurado K.A. (2021). SARS-CoV-2 viral proteins NSP1 and NSP13 inhibit interferon activation through distinct mechanisms. PLoS ONE, 16(6), e0253089.

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TRIP6 Promoter Cloning and Mutagenesis

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pGL3-Promoter, pGL4.10[luc2], pGL4.24[luc2P/minP], and pNL1.1TK[Nluc/TK] vectors were purchased from Promega (Madison, WI, USA). 5′ flanking sequence of the TRIP6 was taken from Ensembl genome browser (Homo sapiens GRCh38.p12) for TRIP6-201 transcript (ENST00000200457.9). Sequence −936/+111 (where +1 means TRIP6 transcription start) was amplified from MCF-7/PacR genomic DNA by PCR and cloned into pGL3-Promoter vector via KpnI and NcoI sites. The inserted sequence was subcloned by PCR into pGL4.10[luc2] and pGL4.24 vectors (Figure S5). Mutagenesis of the CRE motif was achieved by cleavage with AatII-HF enzyme followed by 3′ overhangs removal (Large Klenow Fragment, NEB). The construction of plasmids, primers and PCR conditions are summarized in Figure S5 and Tables S4–S8. The constructs have been verified by restriction endonuclease cleavage and insert sequencing (LightRun, SupremeRun, Eurofins Genomics, Ebersberg, Germany).

Daniel P., Balušíková K., Václavíková R., Šeborová K., Ransdorfová Š., Valeriánová M., Wei L., Jelínek M., Tlapáková T., Fleischer T., Kristensen V.N., Souček P., Ojima I, & Kovář J. (2023). ABCB1 Amplicon Contains Cyclic AMP Response Element-Driven TRIP6 Gene in Taxane-Resistant MCF-7 Breast Cancer Sublines. Genes, 14(2), 296.

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Studying NS3 Protease Regulation Using CHO-K1 Cells

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CHO-K1 cells stably expressing Gal4_DB-NS3-VP64 were transfected with DNA encoding a UAS regulated firefly luciferase reporter construct (5xGAL4-TATA-luciferase). A constitutively transcribed NanoLuciferase construct (pNL1.1.TK[Nluc/TK], Promega) was used as a Co-transfection control. Approximately 16 hours after transfection, cells were treated with either BILN-2061, or Grazoprevir (both at 3 uM). Following a 12 hour period of drug treatment, a time-series was initiated during which the drug-containing media was removed from individual wells and replaced with drug-free media over the course of a 48 hour period. At the end of the series (approximately 56 hours after the initial drug exposure, and 72 hours after transfection), the amount of luciferase and NanoLuciferase present in cells was quantified using the Nano-Glo Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. The CHO-K1 cell line used in these analyses also contained a stably integrated UAS H2B-Citrine reporter construct, and fluorescence imaging confirmed the activation of the Gal4-dependent H2B-citrine gene in all drug treated-wells.

Tague E.P., Dotson H.L., Tunney S.N., Sloas D.C, & Ngo J.T. (2018). Chemogenetic control of gene expression and cell signaling with antiviral drugs. Nature methods, 15(7), 519-522.

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Luciferase Reporter Gene Assay

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GL4.36[luc2P/MMTV/Hygro], pGL4.54[luc2/TK], and pNL1.1.TK[Nluc/TK] were from Promega (Madison, WI, USA). Lipofectamine 3000 was from Life Technologies (Grand Island, NY, USA).

Cartledge D.M., Robbins K.M., Drake K.M., Sternberg R., Stabley D.L., Gripp K.W., Kolb E.A., Sol-Church K, & Napper A.D. (2017). Cytotoxicity of Zardaverine in Embryonal Rhabdomyosarcoma from a Costello Syndrome Patient. Frontiers in Oncology, 7, 42.

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