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.