Zymobiomics dna mini kit

The samples were taken from randomly chosen parts of sausages using the dynamics described within Section 2.1, by making small cuts in several places with a sterile scalpel. All parts collected from three sausages were pooled and mixed into one sample. The DNA extraction was performed using the Zymo BIOMICS DNA Mini Kit (Zymo Research, Irvine, CA, USA) from a minimum of 200 mg/pooled sample from each time point, according to the manufacturer’s protocol. The concentration of isolated DNA was measured using a Qubit Fluorometric Quantitation device (Qubit 4 Fluorometer, Invitrogen, Carlsbad, CA, USA). Amplicon sequencing was performed using a 2 × 300 bp paired-end sequencer on a MiSeq sequencer according to the manufacturer’s instructions (Illumina, San Diego, CA, USA) at a commercial sequencing service (FISABIO, Valencia, Spain). To identify bacterial communities, the V3-V4 region of the 16S rRNA gene was amplified with defined forward (5′-CCTAGCGGGNGGCWGCAG-3′) and reverse (5′-GACTACHVGGGTATCTAATCC-3′) primers [20 (link)].

The honeybee's gut was dissected under sterile conditions and homogenized in a FastPrep-24 Instrument at 4 m/s for 25 sec. The subsequent procedure was done according to Fatimawali et al. [15 (link)]. The extraction of gut bacterial gDNA (genomic DNA) was performed using ZymoBiomics DNA Mini Kit (Zymo Research) following the manufacturer's protocol. The gDNAs were evaluated by electrophoresis on a 0.8% agarose and analyzed using NanoDrop 1000 (Thermo Scientific, Wilmington, DE, USA). The hypervariable V3-V4 regions of 16S rRNA gene were amplified using MyTaq™ HS Red Mix (Bioline, BIO-25044) in Agilent SureCycler 8800 Thermal Cycler with the following reaction condition: initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 15 sec, annealing at 52°C for 30 sec, and extension at 72°C for 45 sec, and then followed by a final extension at 72°C for 3 min.

The STEC strain obtained from ATCC (ATCC43895) was sequenced. For that, its genomic DNA was extracted using the ZymoBIOMICS DNA mini kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocol. Similarly, sequenced phage infections and phage-free cultures from which prophage induction was assessed were also sequenced. The DNA of these samples was extracted using the Phage DNA Isolation Kit (Norgen Biotek Corp., Thorold, ON, Canada) following the manufacturer’s protocol. Any remaining host DNA was degraded by adding 10 µL (20 U) of DNase I from the RNase-free DNase I kit (Norgen Biotek Corp., Thorold, ON, Canada) prior to proteinase K treatment. Bacterial host DNA concentrations were quantified using the Qubit 3.0 Fluorometer and the Qubit dsDNA High Sensitivity Kit (Thermo Fisher Scientific, Waltham, MA, USA).

For DNA extraction, kitchen sponges were cut into halves and the corners and material from the middle part of each half were sampled with upper and lower parts (~2.5 cm³ in total) using sterile scissors. Cuts of each sponge half were pooled as one sample. Afterwards, DNA was extracted using the ZymoBIOMICS DNA Mini Kit (Zymo Research, Irvine, CA, USA) according to manufacturer´s specifications with some modifications. Briefly, after filtration, the Zymo-spin IV spin filter was repeatedly washed with 100 µL of DNAse free water for 3 or 4 times and the flow-through of the different washing steps was collected in separate tubes. To both the flow-through and the initial washing solution, binding buffer was added, and the solutions were successively applied onto a single Zymo-spin IIIC-Z Colum. Subsequently, the extracted DNA was eluted with 50 µL DNase/RNase free water and again purified using a Zymo-spin II column and 50 µL DNase/RNase-free water. After that, DNA concentration was determined with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using the Qubit dsDNA HS Assay Kit (Thermo Scientific, Waltham, MA, USA). To obtain a higher DNA concentration, all extracts stemming from the same sponge were combined to a single DNA extract of 50 µL by ethanol precipitation [25 ]. For library preparation, DNA concentration was measured again using the Qubit Fluorometer.

Special sterile swabs were taken from all of the sampling points and protected by DNA/RNA shields (Zymo Research, Irvine, CA, USA) during transport. The extraction of ultra-pure DNA was completed using the Zymo BIOMICS DNA Mini Kit (Zymo Research) for several swabs from each sampling point, following the manufacturer’s protocol. The DNA yield was measured using Qubit Fluorometric Quantitation (Qubit 4 Fluorometer, Invitrogen™, Waltham, MA, USA). Library preparation, using Nextera XT Index Kit (FC-131-1096), and amplicon sequencing step was performed using a 2×300 bp paired-end run on a MiSeq Sequencer, according to manufacturer’s instructions (Illumina) in commercially available service (FISABIO, Valencia, Spain). To target the ITS II region, the primers [39 (link)] were as follows: forward primer ITS3_KYO2 (5′- GATGAAGAACGYAGYRAA-3′), and reverse primer ITS4_KYO1 (5′-TCCTCCGCTTWTTGWTWTGC-3′).

Strain GD1 was cultured on NB medium overnight at 30°C. Bacterial cells were harvested and washed three times in 0.3% sterile NaCl. The extraction of ultra-pure DNA was done using the ZymoBIOMICS DNA Mini Kit (Zymo Research, USA), following the manufacturer protocol. The DNA yield was measured using Qubit Fluorometric Quantitation (Qubit 4 Fluorometer, Invitrogen^™, USA). The genome of strain GD1 was sequenced using a 2 × 300 bp paired-end run (MiSeq Reagent kit v3) on a MiSeq platform, according to manufacturer’s instructions (Illumina) in commercial service (FISABIO, Valencia, Spain). Total of 1,220,080 paired reads were generated.

The genomic DNAs (gDNAs) of bacteria were extracted from soil using ZymoBiomics DNA Mini kit (Zymo Research) according to the protocol provided by the manufacturer. Amplification of hypervariable V3-V4 regions of 16S rRNA were performed using MyTaq™ HS Red Mix (Bioline, BIO-25044) in Agilent SureCycler 8800 Thermal Cycler. The reaction conditions were as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 15 sec, annealing at 52°C for 30 sec, extension at 72°C for 45 sec, and then followed by final extension at 72°C for 3 min. Preparation of 16S rRNA libraries and bioinformatic analysis were performed following the previous research [12 (link)].