Tris acetate edta buffer

Ethidium bromide (EtBr), ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), ethylenediaminetetra acetic acid (EDTA), isopropyl-β-D-thiogalactoside (IPTG), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) coelenterazine, carbenicillin, CaCl₂, Carbonyl cyanide m-chloro phenylhydrazone (CCCP), Calmidazolium (CDZ), Verapamil, coelenterazine, Trifluoperazine (TPZ), Chlorpromazine (CPZ), Phosphate Buffer Solution (PBS), Triton X-100, Tris-Acetate EDTA buffer (TAE), and aequorin oligonucleotides were all purchased from Sigma-Aldrich (St. Louis, MO, United States). Taq polymerase master mix for PCR (Promega, Madison, WI, United States), agarose molecular grade and 100 bp molecular ruler were obtained from Bio-Rad, Hercules, CA, United States). Kanamycin, Gentamicin, Streptomycin, Vancomycin, Ciprofloxacin, and Erythromycin (St. Louis, MO, United States). BamHI and HindIII (Rowley, MA, United States), T4 ligase (Rowley, MA, United States).

Published primers were used to amplify the X region of the spa gene [13 (link)]. Each 25 µl PCR reaction mix included 10.5 µl of sterile nuclease-free water, 12.5 µl of Dream Taq mix (Thermo Fisher Scientific,Waltham, Massachusetts, USA) and 0.5 µl (10 pmol) of both the forward and reverse primers with 1 µl (approximately 50 ng) of template DNA. Cycling was performed in a GeneAmp 9700 PCR System (Applied Biosystems, Foster City, California, USA) with the following conditions: initial denaturation at 95 °C for 5 min, and 35 cycles of denaturation at 95 °C for 45 s, primer annealing at 60 °C for 30 s, extension at 72 °C for 90 s followed by a final extension at 72 °C for 10 min. Amplification products were resolved on a 1.5 % agarose in 1× Tris Acetate EDTA buffer (Sigma-Aldrich, St Louis, Missouri, USA) for 60 min at 95V and visualized with EZ Vision DNA stain (Amresco, Inc., Cleveland, Ohio, USA) using a UV transilluminator (UVP LLC, Upland, California, USA).

The PCR screening of the genetic determinants of the main putative virulence factors involved in the B. cereus gastrointestinal toxi-infections was performed using the GoTaq^® Green Master Mix (Promega, Fitchburg, MA, USA). The different primers used are shown in Table 2. Thermal cycling parameters were 5 min at 94 °C, then 32 cycles of 1 min at 94 °C, 1 min at the annealing temperature (depending on the primer pair, see Table 2) and 1 min at 68 °C, and finally 10 min at 68 °C. The success of amplification was confirmed by gel electrophoresis at 100 V for 28 min on 0.8% agarose gel (Santa Cruz Biotechnology, Dallas, TX, USA) in Tris-Acetate-EDTA buffer (Sigma-Aldrich, St. Louis, MO, USA).
For several B. cereus strains, the presence of Nhe and HBL toxins in the supernatant was also assessed using the immunodetection kit Duopath Cereus Enterotoxins (Merck, Darmstadt, Germany).

Definite identification of MRSA isolates was dependent on the detection of their characteristic mecA gene [15 (link)]. Briefly, genomic DNA was extracted using GeneJET Genomic DNA Purification Kit according to the manufacturer’s instructions (Thermo Fisher Scientific Inc., Vilnius, Lithuania). The mecA gene was amplified, by PCR technique, using a Thermal cycler (Biometra UNO–Thermoblock, Germany) in presence of 1 µL from each particular 10 pmol primer (Table 1), 12.5 µL Cosmo PCR Red Master Mix (Willowfort, Birmingham, UK), 1 µL of 50 ng genomic DNA, and nuclease-free double distilled water to final reaction volume of 25 µL. The mecA gene amplification cycling program includes 1 cycle of initial denaturation at 94 °C for 4 min (min) followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and elongation at 72 °C for 1 min, and lastly 1 cycle of final elongation at 72 °C for 10 min. The obtained PCR products were loaded into 1.5% agarose gel containing 5 µL ethidium bromide (0.05 mg/mL) followed by electrophoresis at 100 V in Tris-acetate EDTA buffer (Sigma Aldrich, Hamburg, Germany). The UV transilluminator (HERMLE Labortechnik GmbH, Wehingen, Germany) was used to observe the mecA gene-specific bands and determine their molecular size with the aid of a 100 bp DNA ladder (Geneaid Biotech Lt., New Taipei City, Taiwan) as a marker [16 (link)].

Larval genomic DNA was prepared using the HotSHOT protocol (60 (link)). Briefly, single injected 1 dpf embryos were dissociated in PCR tubes containing 10 μl of 50 mM NaOH at 95°C for 20 min. After cooling down, 1 μl Tris–HCl, pH 7.5, was added and the preparation was mixed. 1 μl of this mixture was used as a genomic DNA template in PCRs using the primers listed in Table S6. Amplicons were analyzed on 2.5% agarose gels prepared with 1x Tris–acetate–EDTA buffer (Sigma-Aldrich).