Biotin chromogenic detection kit

Aliquots of 200–400 ng extra-chromosomal DNA were separated (50 volts for 60 min) on a 0.7 % agarose, 1X TAE gel. Invitrogen (Eugene, OR USA) SYBR Safe DNA gel stain (# S33102) was added to the gel at a 1∶10,000 dilution. DNA was treated and transferred from gel to Positively Charged Nylon Transfer Membrane (#RPN303B, GE Healthcare, UK) using standard Southern blot procedures [32] . Hybridization probes were prepared from PCR products generated using primers 5′-CCTGTTGCTTGGGTAACTGTATC-3′ and 5′-AATGGCAGAAAGTGGCTGG-3′ for identified bacteriophage in the NRS19 strain and primers 5′TGCCATTGTGATGAGGAGGG-3′ and 5′-GCAACGCAGATTGTTTGAGTG-3′ for the identified bacteriophage of the NRS26 strain. These PCR products were purified with QIAquick PCR Purification Kit # 28106 and labeled for use as a Southern blot probe using Biotin DecaLabel DNA Labeling Kit (#K0652, Thermo Scientific (Waltman, MA USA). DNA transferred to nylon membrane was pre-hybridized and hybridized based on standard Southern blot procedures [32] . Southern blot probe detection was performed using Fermentas (Glen Burnie, MD USA) Biotin Chromogenic Detection Kit (#K0661) based on manufacturer's instructions.

For differential screening of subtracted PCR products from the SSH clone library, a DNA dot blot hybridization was performed. To generate single stranded DNA (ssDNA), all DNA or PCR samples were melted at 95°C and immediately chilled on ice. One microliter of ssPCR product was spotted onto a neutral BioBond nylon membrane (Sigma-Aldrich, St. Louis, MO, USA) and fixed by UV cross-linker (Stratagene, La Jolla, CA, USA) using the auto-crosslinking mode (1200 mJ×100/cm²). Spotted membranes were hybridized with either SSH tester (L. reuteri LR) or driver (L. reuteri 20016^T) DNA. Before hybridization, genomic tester and driver DNA were digested with RsaI and then labelled with biotin using the Biotin Decalable DNA labelling Kit (Fermentas, Burlington, CA, USA). Membranes were pre-hybridized with salmon sperm DNA to block unspecific binding sites. Then the membranes were incubated over night at 60°C in hybridization buffer (5×Denhardt's solution, 5 X SSPE buffer, 1% SDS) with 100 ng/ml labelled ssDNA under moderate shaking. To remove unbound and unspecific tester or driver DNA, membranes were washed with a non-stringent buffer (2×SSC, 0.1% SDS) and a stringent (0.1 X SSC, 0.1% SDS) buffer. DNA dots were visualized using the Biotin Chromogenic Detection Kit (Fermentas, Burlington, CA, USA) following the manufacturer's instructions.

Biotinylated probes for Southern blotting were prepared by the Biotin DecaLabel DNA Labeling Kit (Fermentas). Template for the probes was pAL-ID plasmid (containing regions present in expression plasmids p1.1 including origin of replication, a beta-lactamase gene, EMCV IRES, DHFR ORF [17 (link)]) or a PCR product corresponding to the fragment of FVIII-BDD ORF. Genomic DNA was digested with EcoRI (Fermentas) for 16 h, precipitated by ethanol, separated by 0.8% agarose gel. Gel transfer to an Amersham Hybond-N+ membrane (GE Healthcare, USA) was performed according to the manufacturer’s protocol in 20x SSC buffer (3 M NaCl, 0.3 M Na₃C₆H₅O₇) for 16 h. DNA was fixed by heating the dried membranes at 80 °C for 2 h. Prehybridization and hybridization were conducted according to [22 (link)] in the buffer containing 7% SDS, 0.5 M Na₂PO₄, pH 7.2, 1% BSA for 16 h at 65 °C. Membrane was washed according to the manufacturer’s protocol and stained by Biotin Chromogenic Detection Kit (Fermentas).