Gentra Puregene Cell Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Frozen samples should be thawed and equilibrated to room temperature (15–25°C) before beginning the procedure.

Upstream tips
Frozen samples should be thawed and equilibrated to room temperature (15–25°C) before beginning the procedure.

Publication protocol

Human genomic DNA was isolated from SH-SY5Y
cells with a PUREGENE DNA Isolation Kit (Gentra
SYSTEMS, Minneapolis, MN, USA). The 5′-flanking
and non-coding regions of the human VGF gene were
amplified by PCR using KOD -Plus- (TOYOBO, Osaka)
and cloned into the NanoLuc vector pNL1.2 (Promega,
Madison, WI, USA). The 2.3-kb 5′-flanking and noncoding regions (12) were cloned into the EcoRV/HindIII site of pNL1.2. A series of 5′-deletion constructs were made by cleavage with restriction enzymes or amplification by PCR. All constructs were subjected to sequencing analyses to verify the orientation and fidelity of the insert. NanoLuc plasmids (0.4 mg/well) and the firefly luciferase vector pGL4.23 (0.1 mg/well; Promega) were co-transfected into SH-SY5Y cells with TransIT-LT1 Reagent (Mirus, Madison, WI, USA). These cells were cultured for 24 h, and then stimulated with 20 mM clioquinol for 24 h. This concentration of clioquinol was previously shown to induce significant cytotoxicity (10). NanoLuc activity in cell lysates was determined and normalized with firefly luciferase activity.

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Manufacturer protocol

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