ONE-Glo™ Luciferase Assay System

Reporter gene assay luciferase - BHK-21 baby hamster kidney cells

Experiment
Reporter gene assay luciferase - BHK-21 baby hamster kidney cells
Product
ONE-Glo™ Luciferase Assay System from Promega
Manufacturer
Promega

Protocol tips

Upstream tips
10^4 cells into 96-well plates for 24 hr
Protocol tips
Transfecte with 0.2 μg Tax expression plasmid.

Performed luciferase by using One Glo system assay after 48 h of transfection.

Add 100µl of reagent to the cells grown in 100µl of medium.

Downstream tips
Wait at least 3 minutes to allow complete cell lysis and measure in a luminometer

Publication protocol

BHK-EGFP cells were plated and stably transfected with 5 µg linearized pGL4LTRLuc plasmid using the same procedure for the first transfection. Two days after transfection, the cells were cultured in complete medium containing 0.2 mg/ml hygromycin and 1 mg/ml geniticin (G418) (Invivogen, USA) for 3-4 weeks. Stable colonies were isolated by culturing them at limited dilution in wells of a 96-well plate for three cycles. Subsequently several single clones of the G418-resistant colonies were taken under luciferase assay with following procedure. After seeding 104 cells into 96-well plates for 24 hr, BHK-EGFP-HTLV-1Luc cells were transfected with 0.2 μg Tax expression plasmid using Polyfect reagent (Qiagen, 493277090A012 Germany). Transfection was normalaized as previously described. After additional 48 hr incubation, we per-formed luciferase by using One Glo system assay (Promega-Inc.) according to the manufacturer’s instructions onto a Synergy4 luminometer (Biotek, USA). The cells with high constitutive luciferase expression were removed. Furthermore 27 colonies were selected for the lowest background and high Luc expression upon co-culture with 104 Hut-102 cells for 48 hr.

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Discussion

Discussion

2 years ago

Author: J.Han China

Which transfection reagent would work better on stem cells?

I am planning on running reporter assays on stem cells and I would like to know which transfection reagent would work better?

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Papers

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Manufacturer protocol

Download the product protocol from Promega for ONE-Glo™ Luciferase Assay System below.

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