PCR cycling consisted of an initial denaturation at 94°C for 6 min; followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 45 s, and extension at 72°C for 2 min; and a final extension at 72°C for 3 min. Amplified DNA was verified by electrophoresis on 2% agarose gels. Restriction digest The PCR products from the four replicates were pooled into two samples, purified with QIAquick PCR purification kit (Qiagen, Hilden, selleck inhibitor Germany), and finally eluted in a volume of 30 μl EB buffer (10 mM Tris, pH 8.5). Then 15 μl purified PCR product was digested overnight (or 3 hours) at 37°C with 0.02 U of Hha1 (Boehringer, Mannheim,

Germany) in a 20 μl reaction mixture. Terminal-restriction fragment length polymorphism Each sample was analysed as two replicate fragments (T-RFs) by electrophoresis on an automatic sequence analyzer (ABI-PRISM-373-DNA-Sequencer; PE Biosystems, Foster City, California). Aliquots (2 μl) of T-RFs were mixed with

2 μl of deionized formamide, 0.4 μl of loading buffer (PE Biosystems), and 0.6 μl of DNA fragment length standard (MegaBace ET900, GE Healthcare, Hillerød, DK). The T-RF mixture was denatured at 94°C for 2 min and chilled on ice prior to electrophoresis. Five https://www.selleckchem.com/products/salubrinal.html microliter aliquots of the mixture were loaded on a 36-cm, 6% denaturing polyacrylamide gel. Electrophoresis settings were 2,500 V and 40 mA for 10 h, using the B filter set. Due to sequence species specific variations in the ribosomal gene, a restriction digest will give rise to T-RF

of different size, and when many species are mixed as in the intestinal microbiota this can be visualized as a pattern of peaks in an electropherogram, a fingerprint profile. These profiles were collected by the software and analysed by the use of BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium). The length of each band was determined by comparing it towards the internal standard to ladder. From each sample two replicates were compared, and weak bands that were only represented in one of the two were rejected to exclude false T-RFs from the fingerprint. After normalization of all profiles towards the internal standard, they were compared using BioNumerics. The comparisons between cages were based on calculating the Dice similarity coefficient and the unweighted pair group method using arithmetic averages for clustering. Principal Component Analysis (PCA) was performed to reflect the grouping and relatedness of samples. Pyrosequencing of ribosomal genes Samples (n = 10) from the same cage types (CC, FC, and AV), and sampling date (before inoculation and 4 weeks PI.), were pooled by mixing 250 ng of purified DNA from each sample in one tube, in total making up 6 samples.