Quantifying Bacterial Invertible Elements

qPCR was used to quantify the percent of the population with each of the seven invertible elements in a given orientation as described previously [28 (link)]. Rough and smooth colonies were collected from a BHIS plate, and genomic DNA was purified. qPCR was performed with SensiMix SYBR (BioLine). Twenty-microliter reactions with 100 ng of genomic DNA and 100 nM primers were used. Reactions were run on Lightcycler 96 system (Roche) with the following three-step cycling conditions: 98 °C for 2 minutes, followed by 40 cycles of 98 °C for 30 seconds, 60 °C for 1 minute, and 72 °C for 30 seconds. Quantification was done using the ΔΔCt method, with rpoA as the reference gene and the indicated reference condition. All primers used in this and other experiments are listed in S2 Table.