Abstract
pOCS is a 6,969 bp plasmid construct developed by Goh et al. that contains brkA, an OmpT cleavage site, a 6x His tag, and an ampicillin resistance cassette for transformant selection. This plasmid has been used by the CURE lab to surface display heterologous proteins via the BrkA autotransporter system. For surface display, the pOCS plasmid backbone must first be linearized to enable insertion of a gene of interest through Gibson Assembly. A widely-used method for plasmid linearization is inverse PCR, in which outward-facing primers are used to amplify and generate a linearized plasmid backbone. Although this approach is well-established, technical challenges may still arise due to primer design issues and unoptimized thermocycler settings. In this paper, we present an optimized inverse PCR protocol for linearizing pOCS using the existing pOCS primer set (1ɣ_pOCS_FOR and 1ɣ_pOCS_REV), which are prone to concatemer formation. Inverse PCR was performed using freshly-extracted pOCS plasmid DNA in a 2-step protocol with Platinum Superfi I polymerase, incorporating a 290 seconds extension step and a 120 seconds final extension step at 72oC. Amplified products were analyzed by 1% agarose gel electrophoresis at 110 volts to validate linearization.