All PCR reactions were run on a PTC-240 DNA Engine Tetrad 2 Cycler (MJ Research, Bio-Rad Laboratories, Copenhagen, learn more Denmark) and the products were verified by gel electrophoresis before proceeding to DGGE analysis. Analysis of cecal microbiota by denaturing gradient gel electrophoresis (DGGE) DGGE was carried out as previously described [41]

using a DCodeTM Universal Mutation Detection System instrument and gradient former model 475 according to the manufacturer’s instructions (Bio-Rad Labs, Hercules, California). The denaturing gradient was formed with two 9% acrylamide (acrylamide-bis 37.5:1) stock solutions (Bio-Rad) in 1 × TAE (20 mM Tris, 10 mM acetate, 0.5 M EDTA, pH 7.4). The gels were made with denaturing gradients ranging from 25 to 65% for analysis of the amplified 16S rRNA fragments. The 100% denaturant solution contained 40% formamide and 7 M urea. PCR product (13 μl) were mixed RG7422 supplier with 3 μl loading dye before loading. Gels were run in 1 × TAE at 60°C for 16 hr at 36 V, 28 mA, stained with ethidium bromide for 15 min, destained for 20 min, and viewed by UV-B trans illumination at 302 nm. The BioNumerics software, version 4.60 (Applied Maths, Sint-Martens-Latem, Belgium) was used for identification of bands and normalization of band patterns from DGGE gels.

Pearson correlation and Principal Component Analysis (PCA) based on DGGE pattern profiles were performed using the same software. Subtraction of averages over the characters was included in the PCA analysis. Excision, cloning and sequencing of selected bands from DGGE gels Bands of specific interest were excised from DGGE gels with a sterile razor, placed in 40 μl sterile water, and incubated at 4°C for diffusion of DNA into the water. Methocarbamol 33 μl of the sterile water (containing the DNA) was treated with S1 nuclease [42]. For sequencing of bands retrieved from universal DGGE gels, the S1 nuclease treated DNA was used in a PCR with HDA1/2 primers without

GC-clamp (4 min at 94°C, 20 cycles consisting of 30 s at 94°C, 30 s at 56°C, and 1 min at 68°C, and finally 7 min at 68°C). Subsequently the PCR products were directly cloned into pCR®4-TOPO (Invitrogen, Taastrup, Denmark) according to the manufacturer’s instructions, and electroporated into electrocompetent E. coli TOP10 cells (Invitrogen) with a single pulse (2500 V, 400Ω, 25 μF) by use of a Gene Pulser apparatus (Bio-Rad Laboratories, Richmond, California). Plasmid DNA was isolated from the cells using the Qiagen Mini Spin Prep kit (QIAGEN), and subjected to PCR (HDA1/2-GC) as earlier described. The PCR products were run on a DGGE gel to check the purity and confirm the melting behavior of the excised band. The inserts were sequenced by GATC (Konstanz, Germany) using primers T3 and T7. The obtained sequences were compared to known sequences in the Ribosomal Database (RDP, Michigan State University, Release 9.61), and aligned using BLAST (bl2seq) and the GenBank database.