GeneArt™ Site-Directed Mutagenesis PLUS System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Use 20–25 ng of the target plasmid per 50 µL of mutagenesis reaction as a starting point.	- Create a fresh dilution of 25X SAM from the kit-supplied 200X SAM in sterile, distilled water each time you perform the mutagenesis procedure - 25X SAM is not stable, and loses activity within a few hours after preparation

Upstream tips
- Use 20–25 ng of the target plasmid per 50 µL of mutagenesis reaction as a starting point.
Protocol tips
- Create a fresh dilution of 25X SAM from the kit-supplied 200X SAM in sterile, distilled water each time you perform the mutagenesis procedure - 25X SAM is not stable, and loses activity within a few hours after preparation

Publication protocol

Gene overexpression plasmids and luciferase reporter vectors
The genes for the overexpression of NF-κB1, c-Rel, ELK1, and TAB1 were inserted into the pBI plasmid (Clontech). All the restriction enzymes used in this study were fast-digest enzymes. Specifically, the whole CDS region of the NF-κB1, c-Rel, ELK1, and TAB1 genes was cloned using primers shown in Supplementary Table 2. The PCR products were cleaved with Mlu I and Hind III, and inserted into the linearized pBI plasmid to obtain the overexpression plasmid constructs, designated as pBI- NF-κB1, pBI- c-Rel, pBI- ELK1, and pBI- TAB1.

We divided the upstream sequence of pre-miR-134 into five regions (R1, R2, R3, R4, and R5). EMSA and ChIP assays were used to confirm that NF-κB1 binds to the R3 region, c-Rel binds to the R5 region and ELK1 binds to the R1 region. Therefore, we cloned the R1, R3, and R5 regions into the pGL3-promoter vector (Promega, Madison, WI, USA) containing the luciferase reporter gene to obtain the pGL3-promoter-R1, pGL3-promoter-R3, and pGL3-promoter-R5 reporter constructs. The R1, R3, and R5 regions of the sequence upstream of pre-miR-134 were generated by PCR using genomic DNA from SKOV3-TR30 cells as a template (primers are listed in the Supplementary Table 2). The PCR product was ethanol precipitated, digested, and then cloned into the Mlu I and Xho I restriction sites on the pGL3-promoter vector. A site-directed gene mutagenesis kit (ThermoFisher Scientific, Waltham, MA, USA) was then utilized to create the mutant counterparts of the luciferase reporter vectors containing the R1, R3, and R5 regions. The mutant primers for the NF-κB1, c-Rel, and ELK1 binding sites in the R3, R5 and R1 regions, respectively, are also shown in Supplementary Table 2. The mutant plasmids were designated as pGL3-promoter-R1-mut, pGL3-promoter-R3-mut, and pGL3-promoter-R5-mut, respectively.

In order to construct the miRNA 3′-UTR luciferase reporter vectors, the wild-type 3′-UTR of TAB1, containing the putative miR-134 binding sites, was amplified by PCR using the genomic DNA from SKOV3-TR30 cells as a template. The PCR products were cleaved with restriction enzymes, Spe I and Hind III, prior to insertion into the linearized pMIR reporter vector (Ambion, Carlsbad, CA, USA) to obtain a luciferase reporter construct. The mutant counterparts were constructed using the kit, as described above. All the constructs were verified by sequencing. SKOV3-TR30 cells were utilized in the luciferase activity assays.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for GeneArt™ Site-Directed Mutagenesis PLUS System below.

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