2), 220 μl SDS (10% w/v) and 150 μl proteinase K (>600 mAU/ml, solution) and incubated for 2 hours in water bath at 60°C. One ml of saturated NaCl solution was added and the suspension was gently inverted twice. Pellets were harvested through centrifugation (5000 × g) at room temperature for 15 minutes. After the transfer of clean supernatants in new tubes, DNA was CDK inhibitor precipitated with 2.5 volumes of cold ethanol (95%) and resuspended in 300 μl of TE buffer [32]. Amplification of gene hsp60 and restriction with HaeIII Universal primers were used to amplify approximately 600 bp of the hsp60 gene in the Bifidobacterium spp. investigated. These

primers H60F (5‘-GG(ATGC)GA(CT)GG(ATGC)AC(ATGC)AC(ATGC)AC(ATGC)GC(ATGC)AC(ATGC)GT-3’) and H60R (5’-TC(ATGC)CC(AG)AA(ATGC)CC(ATGC)GG(ATGC)GC(CT)TT(ATGC)AC(ATGC)GC-3’) were designed by Rusanganwa et al. [30] on the basis of the conserved protein sequences GS-9973 MK0683 chemical structure GDGTTATV and AVKAPGFGD in HSP60. Amplifications were performed in 20 μl volumes with 1.5 μM of each primer (Eurofins MWG Operon, Ebersberg, Germany), 10 μl 2X HotStarTaq Plus Master Mix (Qiagen, Italy) (1,5 mM MgCl2, 1 U Taq, 0.2 mM dNTP, final concentration) and 150 ng/μl DNA. The PCR cycle consisted of an initial denaturation of 5 min at 95°C followed

by 35 cycles of denaturation (30s at 94°C), annealing (30s at 61°C) and extension (45 s at 72°C). The PCR was completed with a final elongation of 10 min at 72°C. The PCR amplification was performed with a PCR Verity 96-well thermal cycler (Applied Biosystems, Milan, Italy). After amplification, the product was visualized via agarose gel (1.3% w/v) in 1X TBE buffer

and visualized with ethidium bromide under UV light. A 100 bp DNA ladder (Sigma-Aldrich) was used as a DNA molecular weight marker. Bands were excised from agarose gel (Additional file 1: Figure cAMP S1) and DNA was eluted with NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel GmbH & Co. KG, Germany) in order to avoid possible non-specific amplifications. 3 μl of the eluted DNA was re-amplified in a 30 μl PCR reaction (see above). BSA was added to the reaction (5% v/v, Fermentas). The PCR products (2 μl) were checked for non-specific amplification on agarose gel. 20 μl (~6 μg) of PCR amplicons were digested with HaeIII enzyme. Restriction digestion was carried out for 2 h at 37°C in 30 μl reaction mixture with 1X SM Restriction Buffer (Sigma-Aldrich), 1.5 μl HaeIII (10 U/μl, Sigma-Aldrich) and water. Digestion products were stained with ethidium bromide and visualized under UV-light (GelDoc™, BioRad), after agarose gel electrophoresis (3.0% agarose (w/v), TBE 1X) at 210 V (3 h). A 20 bp DNA ladder (Sigma-Aldrich) was used. The obtained pictures were elaborated with a free software GNU Image Manipulation Program (Gimp 2.8) only to invert colors and increase contrast.