Genejet genomic dna purification kit

Amblyomma hebraeum ticks were placed in sterile 2 ml microcentrifuge tubes containing 180 μl of a lysis buffer (GeneJet genomic DNA purification kit, Thermo Fisher Scientific, Landsmeer, the Netherlands) and were frozen at − 20 °C. Adult ticks were tested individually, nymphs in pools of ten ticks. Thereafter, metal beads (5 mm in diameter) were added to the frozen samples, which were disrupted in a TissueLyser (Qiagen Benelux BV, Venlo, the Netherlands) for 3 min at 50 Hz. The DNA was extracted from the triturated samples by using a GeneJet genomic DNA purification kit (Thermo Fisher Scientific, Landsmeer, the Netherlands) according to the instructions of the manufacturer. Extracted DNA was eluted in 150 μl elution buffer and used directly or stored at − 20 °C. After extraction, DNA was PCR amplified and tested by reverse line blot hybridisation (RLB).

From the total of 133 bacterial isolates tested with Bioscreen, we chose 17 isolates (based on maximizing diversity in CRISPR spacers, morphology and phage resistance) for full genome sequencing. We also sequenced population-level phage DNA from week 16 samples and clonal bacterial isolates from different time points. For bacterial genomic DNA extraction, isolates were taken from the freezer and grown overnight. DNA of turbid cultures was extracted using the GeneJet Genomic DNA Purification Kit (Thermo Fisher). For phage DNA extraction, lysates from week 16 were used to infect B245. Confluent soft-agar bacterial lawns were collected, mixed with 4 ml of Shieh media, centrifuged (10.000 rpm, 10 minutes, Sorvall RC34) and filtered. Phage precipitation was made with ZnCl2 followed by removal of host DNA with nucleases^{49 (link)}. After Protease K treatment, the material was mixed with Guanidine:Ethanol (1 part 6 M guanidine and 2 parts 99% ethanol, v.v.) and the extraction finished using the GeneJet Genomic DNA Purification Kit (Thermo Fisher). All samples were sequenced using Illumina 150PE BGISEQ platform at BGI Group. We were unable to obtain phage sequence data from the replicate b of the lake water with mucin condition due to technical problems.

For the evaluation of the three biofilm-encoding genes (algD, pelf, and pslD) in P. aeruginosa, the polymerase chain reaction (PCR) method was employed. DNA extraction from the samples was conducted using the GeneJET Genomic DNA Purification Kit from Thermo Fisher Scientific, USA, following the manufacturer’s protocol. Specific primers for the amplification of the target genes were designed using Primer3 software, based on the nucleotide sequences obtained from GenBank (Table S4). The PCR reaction was carried out in a total volume of 25 µL, consisting of 12.5 µL of Taq Master Mix, 0.3 µM of each forward and reverse primer, 100 ng of template DNA, and nuclease-free water to complete the volume.
The PCR amplification conditions included an initial denaturation step at 95 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 40 s, and extension at 72 °C for 40 s. A final elongation step was performed at 72 °C for 5 min. After the PCR amplification, the products were detected using gel electrophoresis on a 1.5% agarose gel at 80 volts for 45 min. The gel was then stained with Midori green and visualized, imaged, and analyzed using a gel documentation system (Analytic Jena, Biometra model, Biodoc analyzer, Jena, Germany).

The GA1 genome sequence was reconstructed using a combined approach of two sequencing technologies which generated short paired-end reads and long reads. The resulted sequences were then used for hybrid assembly. More precisely, genomic DNA was extracted and purified from B. velezensis GA1 using the GeneJET genomic DNA purification kit (ThermoFisher Scientific). The first half of extracted DNA was sent to the GIGA sequencing facility (Liège, Belgium) and used as the DNA template for Illumina MiSeq sequencing after being prepared using the Nextera library kit (Illumina). The sequencing run generated 150-bp paired-end reads, which were trimmed and corrected using an in-house python script and SPAdes 3.14 (68 (link)) before assembly. The second half of the extracted DNA was used to generate long reads with a MinION Oxford Nanopore platform. A DNA library was constructed using the rapid sequencing kit (SQK-RAD0004; Oxford Nanopore). Adapters were trimmed from generated reads with Porechop software (https://github.com/rrwick/Porechop). Trimmed reads were then filtered by size (>500) and Q-score (>10) using NanoFilt implemented in NanoPack (69 (link)). Finally, the hybrid assembly was performed using the hybridSPAdes algorithm implemented in SPAdes 3.14 (70 (link)).

The DNA isolation from sarcocysts was conducted using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) according to the manufacturer’s recommendations. The partial 28S rRNA was amplified using KL-P1F/KL-P1R primer pairs (Table 1) [35 (link)]. Each PCR reaction was carried out in a 25 µL mixture containing 12.5 µL of DreamTaq PCR Master Mix (Thermo Fisher Scientific Baltics, Vilnius, Lithuania), 4 µL of DNA template, 0.5 µM of both forward and reverse primers and nuclease-free water. The PCR was initiated with the initial hot start at 95 °C for 5 min followed by 35 cycles of 94 °C for 45 s, annealing at 52 °C for 60 s and 72 °C for 80 s, and a final extension at 72 °C for 7 min. The visualisation, purification, and sequencing of PCR products followed the previously described protocol [36 (link)]. In order to detect essentially similar DNA sequences, and evaluate the interspecific and intraspecific genetic variability of detected Sarcocystis parasites, the 28S rRNA sequences generated in this study were compared with those of various Sarcocystis spp. using the nucleotide BLAST program (http://blast.ncbi.nlm.nih.gov/, accessed on 10 March 2024).