The pLS-SOX9 plasmid containing the SOX9 3′-UTR fused to the renilla luciferase reporter gene was purchased from Active motif (Active motif, La Hulpe, Belgium). In reporter assays, 100 ng pLS-vector were co-transfected with 50 nM miRNA using the DharmaFect Duo transfection reagent (GE Dharmacon, Lafayette, CO) according to the manufacturer’s protocol into cells seeded in 96-well cell culture plates on day prior to transfection. One day after transfection, luciferase signals were measured using the Lightswitch Luciferase Assay kit (Active Motif, La Hulpe, Belgium) according to the manufacturer´s protocol. Luminescence signal was assessed with CLARIOstar Plus reader (BMG LABTECH, Ortenberg, Germany). To verify direct binding, mutation of the miRNA binding site within the SOX9 3′-UTR was performed using the Quick-change mutagenesis II kit (Qiagen, Hilden, Germany) as outlined in the manufacturer’s protocol. Primers used for mutagenesis are listed in Table S1.
Quick change mutagenesis 2 kit
Manufactured by Qiagen
Sourced in Germany
The Quick-change mutagenesis II kit is a laboratory tool designed for introducing site-specific mutations into double-stranded plasmid DNA. It provides a simple and efficient method for generating targeted DNA sequence changes.
Automatically generated - may contain errors
2 protocols using quick change mutagenesis 2 kit
1
Validating miRNA regulation of SOX9
2
Reporter Assay for miRNA Targeting SOX9 3'-UTR
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The pLS-SOX9 plasmid containing the SOX9 3'-UTR fused to renilla luciferase reporter gene was purchased from Active motif (Active motif, La Hulpe, Belgium). In reporter assays, 100 ng pLS-vector were co-transfected with 50 nM miRNA using the DharmaFect Duo transfection reagent (GE Dharmacon, Lafayette, CO) according to the manufacturer's protocol into cells seeded in 96-well cell culture plates on day prior to transfection. One day after transfection, luciferase signals were measured using Lightswitch Luciferase Assay kit (Active Motif, La Hulpe, Belgium) according to the manufacturer´s protocol.
Luminescence signal was assessed with CLARIOstar Plus reader (BMG LABTECH, Ortenberg, Germany). To verify direct binding, mutation of miRNA binding site within the SOX9 3'-UTR was performed. Therefore, the Quick-change mutagenesis II kit (Qiagen, Hilden, Germany) was used according to the manufacturer's protocol. Primers used for mutagenesis are listed in Supplementary Table 1.
Luminescence signal was assessed with CLARIOstar Plus reader (BMG LABTECH, Ortenberg, Germany). To verify direct binding, mutation of miRNA binding site within the SOX9 3'-UTR was performed. Therefore, the Quick-change mutagenesis II kit (Qiagen, Hilden, Germany) was used according to the manufacturer's protocol. Primers used for mutagenesis are listed in Supplementary Table 1.
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