Genelute bacterial genomic dna kit

Upon completion of the 7- and 13-day experiments, the three discs for each incubation environment were vortexed for at least 1 minute in sterile PBS and pooled prior to bacterial DNA extraction. The stored aliquots of participant and pooled saliva inoculum were allowed to thaw slowly at 4°C overnight. All samples were centrifuged at 16,000 RPM for 10 minutes. The supernatant was discarded and the pellet was treated with lysozyme (Merck) for 30 minutes at 37°C, to enhance extraction of Gram-positive bacteria DNA, followed by extraction using the GenElute Bacterial Genomic DNA Kit (Merck) according to the manufacturer guidelines.
Extracted DNA was quantified using the Qubit fluorometry platform (ThermoFisher Scientific) according to the manufacturer handbook, and analysed using the Qubit fluorometer (Version 4.0). Results were reported in nanograms per microliter (ng/μl).
Variable regions 1–2 of the 16S rRNA gene were amplified by PCR using the 27F (YM modifications) and 338R primers [as previously described by 19], with unique barcodes and Illumina MiSeq (Illumina, USA) adapters. PCR amplicons were purified using the Applied Biosystems SequalPrep Normalization kit (Thermofisher), pooled to approximately 0.5 ng/μl, and sent to the Genome Centre, King’s College London, UK where they were sequenced using the Illumina MiSeq v3 2 × 250 bp flow cell for paired-end sequencing.

Phage DNA extraction based on phenol-chloroform extraction. For detailed information see Supplemental Materials.
Isolation of bacterial DNA: DNA from E. coli was isolated using the GenElute™ Bacterial Genomic DNA Kit (Merck, Darmstadt, Germany) following the manufacturer’s instructions.

For the preparation of the chromosomal bacterial DNA from isolates (2.3.5), GenElute bacterial genomic DNA Kit (Merck KGaA, Darmstadt, Germany) was used following the protocol for gram-positive bacteria, which is more efficient in cell disruption and DNA release. Single colonies of each isolate grown in all TSA plates (2.3.5) were pooled together into 500 μLl of TSB, obtaining a polymicrobial culture. After 1.5 h of incubation at 37 °C, total DNA was extracted. At the last step of the procedure of purification the final elution volume was 200 μL.

The genes encoding for nudA, nudB, nudC [3 (link)], nudD, nudE, nudF, nudI, nudJ and nudL were PCR-amplified from genomic DNA of E. coli K-12 (isolated via GenElute Bacterial Genomic DNA Kit, Merck KGaA, Darmstadt, Germany). The DNA sequence encoding for the hNUDT5 gene was ordered from IDT and also amplified by PCR as well. pET28a-hDcp2 was ordered from Addgene (72214) (Addgene Europe, Teddington, UK). Restriction sites were introduced during amplification and resulting sequences are listed in Supplementary Table S3. The resulting PCR products were digested with XbaI/XhoI and NcoI, and cloned into pET-28c vector (Merck Millipore). After Sanger sequencing, the confirmed plasmids were transformed into E. coli One Shot BL21 (DE3) (Thermo Fisher Scientific, Waltham, MA, USA).

Individual colonies of strains QI0054 and QI0055 (n = 1) were used to inoculate 1 mL BHI and incubated as described above. Genomic DNA was extracted from the resulting cultures using GenElute™ Bacterial Genomic DNA Kit (Merck, UK). DNA was used for library preparation using Illumina Nextera low input tagmentation (Illumina, UK) and whole-genome sequencing using a NextSeq500 instrument (Quadram Institute Bioscience, UK). To close the genomes, DNA was sent to Novogene (China) for long-read sequencing with PacBio (Novogene, China). Short reads were quality trimmed with a minimum quality of 2 and adapters removed using bbduk (v.37.02) (trimq = 2 ftl = 10 qtrim = rl). The quality of the cleaned reads was inspected using FastQC. Hybrid assemblies for strains QI055 and QI0055 were reconstructed using Unicycler with standard settings (v.0.4.7)^{32 (link)}, which resulted in one single scaffold for each isolate. Unicycler used the read error correction module from SPAdes (v.3.12.0) before the initial round of assembly and polished the assembly with Racon (v.1.3.1) and Pilon (v1.22)^{33 (link),34 (link)}. The assembly graphs were inspected with Bandage which revealed that the end of the scaffolds were connected to themselves suggesting a closed circular genome. Genome completeness was assessed with CheckM (v.1.1.3) using 325 marker genes (Bacilli UID285)^{35 (link)}.

Each Streptomyces strain was inoculated into 20 mL tryptic soy broth medium (Sigma‐Aldrich) and incubated at 28°C under shaking (180 rpm) for 3 days. DNA was extracted using the GenElute™ Bacterial Genomic DNA Kit (Merck), according to the manufacturer's instruction with slight modifications: the cells were incubated with the lysozyme solution for 90 min instead of 30 min, they were then incubated with proteinase K and the lysis solution C for 15 min instead of 10 min. The quality and quantity of the extracted DNA samples were evaluated using a 1% agarose gel electrophoresis and a Nanodrop, respectively.