Temporal temperature gradient gel electrophoresis ABT-737 in vitro (TTGE) PCR amplification of the V3 region of the 16S rDNA (~200 bp) was performed according to Ogier et al. [12] using a Biometra T-Personal thermocycler (Biometra, Göttingen, Germany) with direct amplification using primers HDA1-GC and HDA2 (Microsynth, Balgach, Switzerland) and ~100 ng of bacterial DNA. Ten μl of PCR products were separated on a 2% (w/v) agarose gel to check successful amplification with a molecular weight

standard of TriDye 100 bp DNA Ladder (BioConcept, Allschwil, Switzerland). TTGE analysis was carried out as described by Ogier et al. [12] with the following modifications. The electrophoresis was run in 1.5 × TAE buffer (1.5 mM EDTA, 60 mM tris(hydroxymethyl)-aminomethane, 60 mM acetic acid) at 65 V for 16 h, with a temperature ramp

of 0.3°C h-1 from 66 to 70°C. The gel concentrations were optimized to enable visualization Wortmannin solubility dmso in separate runs of high-GC bacteria (8 M urea; 8.5% (w/v) acrylamide (37.5:1)) and low-GC bacteria (7 M urea; 8% (w/v) acrylamide (37.5:1)) by empirical approach using a ladder of dairy bacteria harboring a wide range of GC-contents (from 49% for Lactobacillus BV-6 research buy plantarum to 60% for Propionibacterium sp.). Volumes of 20 μl (isolates) or 30 μl (complex consortia) of PCR products were mixed with 20 μl loading dye (0.25% (w/v) Orange G, 50% (w/v) sucrose; Fluka, Buchs, Switzerland) and loaded in each well. The detection limit of the method proved similar to Ogier et al. [12],

with detection of bacterial species accounting for at least 1% of the total DNA amount. Identification of single isolates by partial sequencing of 16S rDNA Groups of isolates with identical TTGE profiles were formed and a representative isolate of each group was selected for further 16S rDNA sequencing analyses. A 1400-bp fragment of the 16S Celecoxib rDNA was amplified with universal primers 16SUNI-L and 16SUNI-R [51]. The 50-μl reaction mixture contained ~20 ng DNA (NanoDrop® ND-100, Witec AG, Littau, Switzerland), 2.5 U of Taq DNA polymerase (Euroclone, Pero, Italy), 0.4 μM of each primer (Microsynth, Balgach, Switzerland), 200 μM of each deoxynucleoside triphosphate (Amersham Biosciences, Otelfingen, Switzerland), and the reaction buffer (Euroclone, Pero, Italy) consisting of 10 mM Tris-HCl, 50 mM KCl, and 1.5 mM MgCl2. The amplification was performed in a Biometra T-Personal thermocycler (Biometra, Göttingen, Germany) with the following temperature profile: 94°C for 3 min, 35 cycles of 94°C for 30 s, 54°C for 30 s, 72°C for 60 s, and a final annealing at 72°C for 7 min. Amplified DNA was purified using the GFX-PCR DNA Purification Kit (GE Healthcare Biosciences, Otelfingen, Switzerland). Partial sequencing was carried out with primer 16SUNI-L and the BigDye® Terminator v1.