Tris borate edta buffer tbe

For the experiments at different ionic strengths, 5 µM λ-DNA (48502 bp, New England Biolabs, Ipswich, MA, USA) was mixed with 2 µM YOYO (Invitrogen, Waltham, MA, USA) and 520 µM netropsin (Sigma-Aldrich, St. Louis, MO, USA) in Tris-Borate-EDTA buffer (TBE, Sigma-Aldrich, St. Louis, MO, USA); the samples were incubated at 50 °C for 30 min and diluted in MilliQ water to obtain the different ionic strengths. 3% (v/v) β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) was added to suppress photonicking and photobleaching.

After thermal cycling each PCR reaction underwent restriction endonuclease digestion. Briefly, 10 μl PCR product was combined with 5 U restriction enzyme (Table 1), 3 μl 10× Cut Smart buffer (New England Biolabs) and molecular biology grade water to a final volume of 30 μl, prior to incubation for 60 min at the temperature required for each enzyme (as shown in Table 1). Restriction endonucleases were purchased from New England Biolabs, using high fidelity (-HF) versions where necessary to permit use of the same Cut Smart^® buffer in all reactions. Subsequently, each digested PCR reaction was resolved by agarose gel electrophoresis using a 2% (w/v) UltraPure agarose gel in 1× Tris-borate-EDTA buffer (TBE; all Sigma-Aldrich), including 0.01% (v/v) SafeView nucleic acid stain (NBS Biologicals). The results of electrophoresis were visualised using a U:Genius Gel Documentation System (Syngene).

OV50 was identified using Maldi-TOF [18 (link)] and by 16S rDNA gene sequencing. For this purpose, we recovered total genomic DNA using the NucleoSpin Microbial DNA Mini Kit (Macherey-Nagel, Düren, Germany) and amplified the 16S rDNA gene using universal primers S1 (5′ AGAGTTTGATC(A,C)TGGCTCAG-3′) and S2 (5′GG(A,C)TACCTTGTTACGA(T,C)TTC-3′) and the PCR program previously reported [19 ]. The PCR products were electrophoresed on 2% (w/v) agarose gel for 2 h at a constant voltage of 75 V in Tris-Borate-EDTA buffer (TBE) (Sigma-Aldrich, Schnelldorf, Germany). Gels were stained using GelRed (Biotium, Fremont, CA, USA) and visualized using the GelDoc device (Bio-Rad, Hercules, CA, USA). The obtained sequences were analyzed using BLAST (Basic Local Alignment Search Tool) on the NCBI website (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov accessed on 13 May 2022).