Site-Directed Mutagenesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
In case of using much amount of plasmid DNA, sometimes Mutazyme™ Enzyme couldn’t reaction with sample. So we suggest you to follow the procedure correctly for good mutation efficiency.

Upstream tips
In case of using much amount of plasmid DNA, sometimes Mutazyme™ Enzyme couldn’t reaction with sample. So we suggest you to follow the procedure correctly for good mutation efficiency.

Publication protocol

Dual-Luciferase reporter assay
The 3′-UTR sequences of human REGγ mRNA, containing the putative miR-195-5p binding site, were amplified by PCR and cloned into the XhoI /BglII site of the pGL3 vector (Promega, Madison, WI, USA) in order to construct the wild-type REGγ 3′-UTR plasmid (wt 3′-UTR). The mutant REGγ 3′-UTR plasmid (mut 3′-UTR) was generated using the Site-Directed Mutagenesis Kit (SBS Genetech, Beijing, China). The cultured 786-O cells were co-transfected with wt or mut REGγ 3′-UTR plasmid, miR-195-5p mimics (miR-195-5p), negative control miR (miR-NC) and a control Renilla luciferase pRL-TK vector (Promega) using Lipofectamine 2000 reagent (Invitrogen). Following 48 h of incubation, the cells were harvested and lysed. The luciferase activity was analyzed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. The firefly luciferase fluorescence was normalized to Renilla luminescence from the same vector. Experiments were independently repeated 3 times.



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Manufacturer protocol

Download the product protocol from SBS Genetech for Site-Directed Mutagenesis Kit below.

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