FuGENE® HD Transfection Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Always bring FuGENE HD to room temperature and mix well prior to use.

Upstream tips
Always bring FuGENE HD to room temperature and mix well prior to use.

Publication protocol

Transfection and luciferase assays.
For promoter studies using the Gaussia Luciferase (GLuc) and Secreted Alkaline Phosphatase (SEAP) system, a human SphK1 promoter reporter clone was purchased from GeneCopoeia (Rockville, MD). Activities of GLuc/SEAP were analyzed with the Secrete-Pair Dual Luminescence Assay Kit (GeneCopoeia) per manufacturer's guidelines, using a GloMax luminometer (Promega). For promoter deletion studies, fragments of the SPHK1 promoter were amplified by PCR, purified, and ligated into pGL4.10[luc2] promoterless luciferase reporter vectors purchased from Promega (Madison, WI). The hRluc Renilla luciferase reporter vector pGL4.74[hRluc/TK] (Promega) was used as a transfection normalization control in these studies. Luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega). FuGENE HD transfection reagent (Promega) was used for all vector transfections in hPASMCs per manufacturer's guidelines. For siRNA experiments, ON-TARGETplus siRNAs specific for EGR1 and nontargeting control were purchased from GE Dharmacon (Lafayette, CO). Transfection of siRNAs (50 nM) was completed using Lipofectamine RNAiMAX reagent per manufacturer's instructions (Thermo Fisher Scientific). Results are representative of at least three independent experiments.

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Reviews

Discussion

Papers

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Manufacturer protocol

Download the product protocol from Promega for FuGENE® HD Transfection Reagent below.

Download manufacturer protocol

Videos

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