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Pnl2.1 vector

Manufactured by Promega
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The PNL2.1 vector is a plasmid designed for the expression and purification of recombinant proteins in bacterial systems. It contains a T7 promoter for high-level protein expression and a polylinker with multiple cloning sites for the insertion of your gene of interest. The vector also includes an N-terminal His-tag sequence for convenient affinity purification of the expressed protein.

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Lab products found in correlation

Lipofectamine 3000 reagent, by Thermo Fisher Scientific (4 mentions) Nano glo dual luciferase reporter assay, by Promega (2 mentions) Nano glo dual luciferase assay, by Promega (1 mentions)

3 protocols using pnl2.1 vector

1

Luciferase Assay for Promoter Activity

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The MDK promoter (encompassing 1,063 bp upstream and 204 downstream of the Midkine starting site; NM_001012333; chr11+: 46358816‐46360083) and the Flt4 promoter (encompassing 1364 bp upstream and 19 downstream of the Flt4 starting site; NM_002020; chr5‐: 180009187‐180010570) were cloned into pNL2.1 vector (#N1061; Promega, MA). These reporter plasmids were transfected into SK‐Mel‐147 cells or HLEC. Cells were co‐transduced with pGL4.52‐Luc2 (#E1320; Promega) vector as transfection control. Luciferase activity was monitored 16 h thereafter (in the presence or absence of BO‐110) using the Nano‐Glo® Dual‐Luciferase assay (#N1610; Promega). Cell viability was kept over 80% in all conditions analyzed.
Olmeda D., Cerezo‐Wallis D., Mucientes C., Calvo T.G., Cañón E., Alonso‐Curbelo D., Ibarz N., Muñoz J., Rodriguez‐Peralto J.L., Ortiz‐Romero P., Ortega S, & Soengas M.S. (2021). Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics. EMBO Molecular Medicine, 13(12), e12924.
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2

BCAT1 Promoter-Driven NanoLuc Assay

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BCAT1 promoter regions were conjugated to the translation start site of the NanoLuc gene in the pNL2.1 vector (Promega). CAFs were plated in 96-well plates 12 h before transfection. The NanoLuc reporter vectors were co-transfected with promoter firefly luciferase reporter vector using Lipofectamine 3000 Reagent (Thermo) according to the manufacturer’s protocol. After 48 h of the transfection, the luminescence was quantified and normalized using Nano-Glo Dual-Luciferase Reporter Assay (Promega).
Zhu Z., Achreja A., Meurs N., Animasahun O., Owen S., Mittal A., Parikh P., Lo T.W., Franco-Barraza J., Shi J., Gunchick V., Sherman M.H., Cukierman E., Pickering A.M., Maitra A., Sahai V., Morgan M.A., Nagrath S., Lawrence T.S, & Nagrath D. (2020). Tumor-Reprogrammed Stromal BCAT1 Fuels Branched Chain Ketoacid Dependency in Stromal-Rich PDAC Tumors. Nature metabolism, 2(8), 775-792.
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3

BCAT1 Promoter-Driven NanoLuc Assay

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BCAT1 promoter regions were conjugated to the translation start site of the NanoLuc gene in the pNL2.1 vector (Promega). CAFs were plated in 96-well plates 12 h before transfection. The NanoLuc reporter vectors were co-transfected with promoter firefly luciferase reporter vector using Lipofectamine 3000 Reagent (Thermo) according to the manufacturer’s protocol. After 48 h of the transfection, the luminescence was quantified and normalized using Nano-Glo Dual-Luciferase Reporter Assay (Promega).
Zhu Z., Achreja A., Meurs N., Animasahun O., Owen S., Mittal A., Parikh P., Lo T.W., Franco-Barraza J., Shi J., Gunchick V., Sherman M.H., Cukierman E., Pickering A.M., Maitra A., Sahai V., Morgan M.A., Nagrath S., Lawrence T.S, & Nagrath D. (2020). Tumor-Reprogrammed Stromal BCAT1 Fuels Branched Chain Ketoacid Dependency in Stromal-Rich PDAC Tumors. Nature metabolism, 2(8), 775-792.
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