DNA was extracted from a sputum sample obtained from a patient diagnosed with pneumonia, as previously described20 ,39 (link). As a negative control, Dulbecco’s phosphate-buffered saline (Nacalai Tesque, Kyoto, Japan) was used as the solvent for sample collection and processed according to the same procedure in all subsequent experiments. The samples were subjected to mechanical cell disruption by bead beating using EZ-Beads (Promega, Madison, WI, USA), followed by DNA purification using a Maxwell RSC Blood DNA kit (Promega).
Ez beads
Manufactured by Promega
Sourced in United States, Japan
EZ-Beads is a lab equipment product designed for the rapid purification of biomolecules. It utilizes magnetic beads to enable efficient capture, washing, and elution of target molecules from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s phosphate buffered saline, by Nacalai Tesque (1 mentions)
Maxwell rsc blood dna kit, by Promega (1 mentions)
Brain heart infusion broth, by BD (1 mentions)
Clc genomics workbench software ver 20, by Illumina (1 mentions)
Miseq reagent kit v3 600 cycle kit, by Illumina (1 mentions)
Miseq platform, by Illumina (1 mentions)
3 protocols using ez beads
1
Pneumonia Sputum DNA Extraction
2
Francisella Cultivation and Lysis for Protein Analysis
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The FT strain SCHU P9 (Uda et al, 2014 (link)) and the FN strain U112 were cultured in brain heart infusion broth (Becton, Dickinson and Company) supplemented with cysteine (BHIc) (Mc Gann et al, 2010 (link)). Bacterial strains were diluted to OD595 = 0.001 and cultured with shaking for 12 h. Approximately 2 × 108 bacterial cells were washed with PBS twice and suspended in 500 μl of H2O. FN cells were disrupted by sonication using VP-050 (Taitech) at PWR 80 for 10 s × 10 times on ice. FT cells were disrupted using EZ-Beads (Promega) and beads crusher μT-01 (Taitech) for 10 s × 10 times while being cooled on ice. After centrifugation at 13,000g for 20 min at 4°C, supernatants were filtered through 0.22-μm filter and were stored at −80°C until use.
3
Genomic Analysis of PVL and TSST-1 Strains
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We performed draft whole-genome sequencing of PVL-and TSST-1-positive strains. Genomic DNA was extracted using the bead-beating method with EZ-Beads (Promega K.K., Tokyo, Japan), followed by processing with a combination of magLEAD 6gC and magDEA Dx SV (Precision System Science, Chiba, Japan). DNA library preparation and sequencing were performed using Illumina DNA Prep (Illumina, Inc.., San Diego, CA, USA) and the Illumina MiSeq platform (Illumina, Inc.) for 300 bp paired-end reads using the MiSeq reagent kit v3 600-cycle kit (Illumina, Inc.). Illumina reads were assembled using the CLC Genomics Workbench software ver. 20.0.4 (Qiagen, Hilden, Germany). The assembled contigs were then analysed for multi-locus sequence typing (MLST) using MLST 1.8, SCCmec typing with SCCmec Finder 1.2, and the identification of virulence genes using VirulenceFinder 2.0 (all available on the Centre for Genomic Epidemiology website: http://www. genomi-cepidemiology.org/) [13e15].
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