GeneArt™ Site-Directed Mutagenesis System

Protocol tips

Upstream tips	Protocol tips	Downstream 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

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

Plasmids and Reagents
The IL-33-pGL3 reporters (−1050 to +49, −662 to +49, −520 to +49, −460 to +49, −300 to +49, −160 to +49) were generated by cloning the PCR products from THP-1 cell genomic DNA into the pGL3-basic vector (Promega). The IRF7 binding site mutants (Mut.A, Mut.B and Mut.AB) were generated by GeneArt® Site-Directed Mutagenesis System from Life Technologies. Human IRF7 sequence was amplified by PCR with total cDNA from THP-1 cells, which was cloned into pcDNA3.0-HA vector. All primers used were shown in the Supporting Information Table 1. Recombinant human SAA was obtained from PeproTech (Rocky Hill, NJ). cycloheximide (CHX) and actinomycin D (ActD) was purchased from Sigma-Aldrich (St Louis, MO). The TRAF6 plasmid, Pam3CSK4, polyI: C, TLR2 neutralizing antibody and its isotype control IgA were obtained from InVivoGen (San Diego, CA). Anti-IL-33 (ab118503) and anti-IRF7 were obtained from Abcam (Cambridge, MA). Anti-pSer/Thr was obstained from BD Biosciences (San Jose, CA). Antibodies for caspase-3, β-actin, HDAC1, HRP-conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG were obtained from Cell Signaling Technology (Danvers, MA).

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Manufacturer protocol

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

Download manufacturer protocol

Videos

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