Generuler dna ladder

DNA from the fecal samples was extracted using the Stool DNA Isolation Kit (Norgen Biotek, Ontario, Canada). The bacterial DNA of D. desulfuricans MB (DSM 6949) and D. vulgaris DSM 644, for use as positive controls, were isolated using the MagAttract HMW DNA Kit (Qiagen GmbH, Hilden, Germany). PCR products were purified using the SanPrep Column PCR Product Purification kit (BBI Life Sciences, Shanghai, China). Gel electrophoresis was performed in 0.9% or 1.5% (w/v) agarose gel containing 0.1 μg/ml ethidium bromide, followed by visualization under UV light. The size markers used were 100 bp GeneRuler DNA ladder or 1 kb GeneRuler DNA ladder (Thermo Fisher Scientific, Waltham, MA, USA).

DNA was quantified with the Qubit fluorometer (ThermoFisher, USA), following the manufacturer's instructions. The integrity of the DNA (10 µL), with 2 µL of the 6X loading buffer (6X MassRuler, Loading Dye Solution, Fermentas) was evaluated after carrying out an agarose gel electrophoresis (SeaKem LE, Cambrex, USA) at 1%, dissolved in Tris-Acetate-EDTA Buffer (TAE), stained with GelRed (Gel Stain, Biotium, Cat: 41003). Electrophoresis was performed at 100 volts (FB1000 Power Source, Fisher Scientific, USA). To visualize the PCR amplifications and the RFLPs, electrophoresis was carried out on a 2% agarose gel, following the same methodology. The size of the amplified PCR, and of its fragments obtained after digestion with the enzymes (RFLP), was compared with a marker of 50 bp (DNA ladder GeneRuler, #SM0371, ThermoFisher, USA) or 100 bp (DNA ladder GeneRuler, #SM0241, ThermoFisher, USA). Image analysis was performed with a UV transilluminator (Slimline Series; Spectroline), the image was captured with an image digitizer (Enduro™ GDS, Labnet International, Inc., USA). Both the confirmation of the size of the amplified by PCR and the analysis of the RFLP were carried out with the TotalLab 1D software, version 14.0.

Reagents were purchased from Sigma-Aldrich unless noted otherwise. Restriction endonucleases, T4 ligase and calf intestinal alkaline phosphatase were from New England Biolabs. RNA was isolated using the Pure Link RNA Mini kit from Ambion and transcribed into first-strand cDNA using SuperScript III first-strand cDNA synthesis kit (Invitrogen). Phusion high-fidelity polymerase, dNTPs, DreamTaq DNA polymerase, GeneRuler DNA ladders, PageRuler plus prestained protein ladder, HisPur cobalt resin and the enhanced chemiluminescence (ECL) system for western blot were from Thermo Scientific. All oligonucleotides used in this study are listed in Supplementary Table 1 and were synthetized by IDT or LifeTechnologies (Invitrogen). Reagents for cell culture were from Biowest. The pG140 and pG152 vectors were provided by Markus Meissner (University of Glasgow, UK). The following primary antibodies were used in western blot or immunofluorescence assays: mouse or rabbit anti c-myc (monoclonal antibodies, M4439 and C3956, Sigma), anti-PentaHis antibody (Qiagen), rabbit anti-TgHsp90 (Echeverria et al., 2010 (link)) kindly provided by Sergio Angel (National University of San Martín, Argentina) and anti-TgDHO (Robles Lopez et al., 2006 (link)).