GeneArt™ Site-Directed Mutagenesis 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

High glucose Dulbecco’s modified Eagle medium (DMEM) was purchased from GE Healthcare (Piscataway, NJ, USA), fetal bovine serum (FBS) and TrypLE-express from Gibco (Carlsbad, CA, USA). Live cell imaging solution was purchased from Life Technologies (Eugene, OR, USA). Glass bottom μ-dishes for live cell imaging were from ibidi (Madison, WI, USA). 8-(4-Chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate acetoxymethyl ester (8-pCPT-AM) was purchased from BioLog Life Science Institute (Bremen, Germany). The primary antibodies: Epac1 (for WB), importin β1, and Rap1 were from Cell Signaling (Danvers, MA, USA); Epac1 A-5 (for IP of endogenous Epac1) and H-70 (for immunogold labeling) were from Santa Cruz (Dallas, TX, USA), GFP from Clontech (Mountain View, CA, USA); βIII-tubulin from Abcam (Cambridge, MA, USA). The HRP-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA), Alexa Fluor 594 goat anti-rabbit was from Invitrogen (Carlsbad, CA, USA). FluorSave™ Reagent was from Calbiochem (Temecula, CA, USA).The chemiluminescence detection reagent was purchased from GE Healthcare Life Sciences (Pittsburgh, PA, USA). The transfection reagents jetPRIME® was from Polyplud transfection (New York, NY, USA), TransIT-Neural®,and TransIT®-LT1 were from Mirus (Madison, WI, USA). GFP-Trap®_A beads were from Allele Biotechnology (San Diego, CA, USA) and Protein G-sepharose and protein A-sepharose were from GE Healthcare Life Sciences (Pittsburgh, PA, USA). Ral GDS-RBD precoupled agarose beads (Millipore, Temecula, CA, USA). Membrane Lipid Arrays were from Echelon Biosciences (Salt Lake City, UT, USA). All other reagents were from (Sigma-Aldrich, St. Louis, MO, USA) unless specified otherwise.

All Epac1 mutant constructs were generated using either GENEART® Site-Directed Mutagenesis System from Invitrogen (Carlsbad, CA, USA) or Q5® Site-Directed Mutagenesis Kit from NEB and validated by DNA sequencing. Importin β1 si-RNA (FlexiTube Mm_Kpnb1_5) and (FlexiTube Hs_KPNB1_1), and control si-RNA (AllStars Neg. Control) were purchased from Qiagen (Cambridge, MA, USA).

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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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