2.4 | Biochemical analyses
Quantitative real-time PCR (qPCR) reactions were carried out using a Bio-Rad CFX96 real-time system operated by CFX Manager software (version 3.1). The standard reaction mixture consisted of 80 mM Tris-HCl pH 8.3, 100 mM KCl, 6 mM MgCl2, 300 mM trehalose, 200 mM betaine-HCl, 0.2 mg/mL bovine serum albumin, 0.2 % (v/v) Tween-20, 1 X SYBR Green I, 2 mM dNTP mix, 0.4 U/µL Taq polymerase, 5 μM each of forward and reverse primers and 1 µg/mL DNA template in a total volume reaction volume of 10 µL. The sequences of the primers used in this study is shown in Table S1. The standard PCR reaction included an initial denaturation step of 95 °C for 5 min and 40 cycles of 95 °C for 10 s and annealing and extension at 65 °C for 30 s, followed by melt curve analysis from 65 °C to 95 °C in 0.5 °C increments. Results were expressed as proportions of the copy number determined for each target within the sample, and each sample was represented as a percentage of the culture with the highest bacterial density which was assigned an arbitrary value of 100 %.
Bile acids were separated by high performance liquid chromatography (HPLC) as described previously (Torchia et al., 2001). Bile acid species were identified based on retention times of certified bile acid standards (Steraloids) and quantified against known amounts of the bile acid standards using area under the curve method.