To determine the frequency of 89K excision, real-time qPCR assays using SYBR Premix Ex Taq (TaKaRa) were developed to measure the percentage of cells in a culture that contained the 89K attP sequence. Amplification products were designed to be of similar length to facilitate quantitative comparison. The amount of attP DNA in each sample was normalized to that of the chromosomal hyd gene located immediately downstream of attB. The amplification specificity of each PCR was confirmed by determining melting curves and by electrophoresis of the final PCR products, and efficiency was determined using linearized pMDph plasmid DNA, which contains a single copy of attP (157 bp) and an internal region of hyd (156 bp) as a template. qPCR reactions and data analysis were performed as described by He et al. (2007) and Ramsay et al. (2006) respectively.