Gelgreen

EMSA was performed to investigate whether N6amt1–Trm112 possesses DNA-binding ability with several dsDNA and ssDNA oligos of different lengths and sequences. Specifically, 20μl of reaction mixture consisted of 50 pmol DNA in a binding buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM KCl, and 10 mM MgAc₂. Reaction was initiated by adding different amounts of N6amt1–Trm112 (250 or 500 pmol) and then incubated at 25 °C for 2 h. The reaction mixture was loaded on a 2.5% agarose gel and visualized under UV with Gel Green (Biotium) staining.

DGGE was performed with DCode™ Universal Mutation Detection System (Biorad, California, USA). The PCR products were loaded onto 10% polyacrylamide gels in 1 × TAE buffer (20 mM Tris, 10 mM glacial acetic acid, and 0.5 mM EDTA pH 8.0) with a denaturing gradient (urea-formamide) that ranged from 40 to 70%. Electrophoresis was carried out at 60°C and a constant voltage of 70 V was applied during 14 h. After electrophoresis the gel was stained using GelGreen™ for 30 min (Biotium, California, USA) before being visualized on a UV transilluminator (Biorad, California, USA). The dominant bands were excised from the gel, eluted in 10 mM Tris–HCl, 50 mM KCl, 1.5 mM MgCl, 0.1% Triton-x 100, pH 9.0, at 95°C for 20 min and centrifuged (10,000 rpm, 5 min). The DNA was reamplified by PCR with the conditions mentioned in the “PCR amplification” section. The PCR products from reamplification were purified and sent to sequencing at Macrogen Service Center (Maryland, USA). Sequence data were analyzed with BioEdit v 7.1 software (Ibis Bioscience, California, USA) and submitted to the non-redundant nucleotide data base at GenBank^® using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/) and Ribosomal Database Project (http://rdp.cme.msu.edu/index.jsp) for bacterial identification.

Assembly products were analysed on a 1% agarose gel with 1xTAE and run at 80 V. For imaging gels, the DNA was post-stained with GelRed (Biotium) and visualized with a UV illuminator. Three hours run time was sufficient for separating the main band at 10 kb from the side product at 9 kb (Supplementary Figure S2). Extension of our method to longer DNA lengths would benefit from using longer PCR parts to enable straightforward separation of the side products. For extraction, the gel was stained with GelGreen (Biotium) and the bands cut out under blue light to prevent UV induced damage of the DNA. The DNA was then purified with a commercial silica bead kit (Qiaex II, Qiagen). The DNA was quantified with a Qubit fluorometer (ThermoFisher) with typical yields of 0.5–2 ng/μl.

Sequences of the test G4-oligonucleotides and the corresponding partially complementary oligonucleotides used in PCR-stop assays are listed in Supplementary Table S3. The reactions were performed in 1× PCR Combination buffer, containing 20 pmol of each pair of oligonucleotides, 0.2 mM dNTPs, 2.5 U Hot Start Taq polymerase (733–1331, VWR, Radnor, PA, USA), and increasing amounts of GA from 0 µM to 100 µM. PCR products were amplified in a Veriti Thermal Cycler (Applied Biosystems) with the following cycling conditions: 95 °C for 15 min, followed by 30 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. Amplified products were resolved on 3% agarose gel in 1× TBE (100 mM Tris base, 100 mM boric acid, 2 mM EDTA) and stained with GelGreen (41005, Biotium, Fremont, CA, USA). Gel Image was analyzed on ImageQuant LAS 4000. Three independent reactions were conducted per concentration, and representative lanes were displayed.

The consensus sequence of E. canis gltA from selected samples was used to generate LAMP primers using the online LAMP primer designing software, Primer Explorer version 5 (http://primerexplorer.jp/e/) (Table 1).
LAMP reaction mixtures were prepared by mixing 2.5 µL (1×) 10× isothermal amplification buffer, 6 mM MgSO₄, 1.4 mM dNTP mix, a primer mix containing 0.2 µM F3/B3 primers, 1.6 µM FIP/BIP primers, and 0.4 µM LF/LB primers, 8 U Bst 2.0 WarmStart^® DNA Polymerase (New England Biolabs, Inc., Ipswich, MA, USA), 1 µL colori-fluorometric indicator (CFI), 2 µL DNA, and nuclease-free water to achieve a final volume of 25 µL. CFI contains 3 mM hydroxylnaphthol blue (HNB; MP Biomedicals, Aurora, OH, USA) and 0.35% v/v GelGreen (10 000 × Sol, Biotium, Hayward, CA, USA) dissolved in distilled water [17 (link)]. For optimizing LAMP condition, six PCR-positive and two PCR-negative samples were subjected to a LAMP assay for 60 min with varying temperatures between 60 °C to 65 °C. A negative control (nuclease-free water) was included in each run. The reaction was then terminated by heating at 80 °C for two minutes. After determining the optimum temperature, reaction time was varied to 30, 45, and 60 min.

RT-PCR was performed as described previously [30 (link)] for canine GAPDH [93 (link)], NANOG, OCT4, and SOX2 [30 (link)]. Total RNA was isolated from passage 2 cells and cDNA was synthesized. PCR reactions (20 μl) were performed and products were separated via agarose gel electrophoresis for visualization using Gel Green (Biotium, Hayward, CA, USA).