Platinum pcr supermix high fidelity

Plasmids were constructed using the pcDNA3.1 (Life Technologies) mammalian expression vector. The Epac2-camps FRET-based cAMP biosensor was modified by site-directed mutagenesis to add targeting sequences in order to record changes in cAMP levels associated with specific membrane microdomains (figure 1A). Epac2-MyrPalm was designed to report cAMP changes in membrane raft domains. This plasmid contains an N-terminal acylation sequence (GCINSKRKD) from Lyn kinase, which results in myristoylation and palmitoylation that targets proteins to lipid raft/caveolar membrane fractions [31] (link). Epac2-CAAX was designed to report cAMP changes in non-raft membrane microdomains. This plasmid contains the CAAX box sequence (KKKKSKTKCVIM) from Rho GTPase attached to carboxyl terminus of Epac2-camps (figure 1A), which results in specific targeting of proteins to non-lipid raft domains of the plasma membrane [31] (link). The DNA fragments encoding Epac2-CAAX and Epac2-MyrPalm were amplified from pcDNA constructs using Platinum PCR Supermix High Fidelity (Agilent Technologies, CA). Amplified sequences were then directionally cloned into pShuttle-CMV vector in MCS using HindIII(5′) and EcoRV(3′) restriction sites. Adenovirus constructs of these plasmids were generated using AdEasy XL adenoviral vector system (Agilent Technologies, CA) following the manufacturer’s protocol.