Chargeswitch gdna mini bacteria kit

Vaginal swab samples were collected from patients and archived in −80 °C before use. The swab was expressed by gently dabbing the cotton tip against a 600 μL microcentrifuge tube containing ~200 μL lysis reagent composed of 50:20:1 lysis buffer, resuspension buffer and proteinase K solution by volume (ChargeSwitch gDNA mini bacteria kit, Life Technologies). Afterwards, the tube was inserted into an on-board thermal lysis chamber and incubated for 30 minutes at 65 °C followed by 15 minutes at 95 °C for proteinase inactivation. Swab samples tested at the emergency room were similarly expressed in lysis buffer without proteinase K and lysed using a portable microbead-beating unit (OmniLyse Kit, Claremont BioSciences). 70 μL of the lysate was transferred to the sample inlet of the droplet cartridge.

The strain D. cejpii FS110 (Accession No.KF706672) was isolated from a deep-sea sedimental sample in the South China Sea, and single colonies were incubated on a potato dextrose agar medium for five days at 30 °C [27 (link)]. The total genome DNA of D. cejpii FS110 was extracted using a Charge Switch gDNA Mini Bacteria Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Genome DNA was checked using TBS-380 (Turner Biosystem Inc, Sunnyvale, CA, USA) for the production of high-quality dsDNA (OD260/280 = 1.8–2.0, >10 μg) and subsequent sequence. For the construction of a genome library, the purified genome DNA was broken into short fragments, which were used for Illumina sequencing (Illumina HiSeq Xten, San Diego, CA, USA) and Pacific Biosciences sequencing (Pacific Biosciences RS, San Diego, CA, USA), according to the manufacturer’s instructions. The fragments, including the Illumina and PacBio reads, were assembled by SOAP denovo v2.04 into many long scaffolds and finally integrated into a complete genome sequence [28 (link)]. This whole genome shotgun project of D. cejpii FS110 was deposited at DDBJ/ENA/GenBank under the accession SMSW00000000.

The 24 invasive N. meningitidis strains used in this study (Table 1) were recovered from several meningitis cases treated in hospitals in four Brazilian regions: Bahia (2009), Pernambuco (2010–2012), Minas Gerais (2011), and Rio de Janeiro (2012). The isolates were grown on 5% sheep blood agar. Each subcultivation was started from a single colony, and several colonies were used for genomic DNA extraction with the ChargeSwitch gDNA Mini Bacteria Kit (Invitrogen), according manufacturer’s instructions. Approvals from the institutional review board of the hospitals and the Research Ethics committee of the Meningitis Advisory Committee of the State Department of Health from Rio de Janeiro, Brazilian Ministry of Health in Bahia, and Ezequiel Dias Foundation in Minas Gerais were obtained to conducting the study. All methods were performed in accordance with the required guidelines and regulations.

After growing the selected bacteria in the respective medium (MRS or M17), genomic DNAs from eight dairy starters and L. brevis 145 were isolated and purified by using ChargeSwitch^® gDNA Mini Bacteria Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. One pair of degenerate primers DP1 and DP2, PGDG-2F and PGDG-4R (Table 1) was applied for amplification of partial GAD gene using AmpliTaq^® Gold 360 master mix (Applied Biosystems, Foster, CA, USA). Based on the manufacturer’s instruction, the PCR reaction volume (25 μL) included 12.5 μL of master mix, 0.5 μL of each primer (10 μM), 2 μL (~1 ng) of DNA template and 8.5 μL of DNase-free water. The amplification was carried out in a GeneAmp^® PCR system 2700 (Applied Biosystems) with 35 cycles (94 °C for 30 s, 60 °C for 30 s, and 72 °C for 90 s) for partial GAD gene. Agarose gel (1%; w/v) electrophoresis was carried out for all PCR products. The size of the PCR products was ~1014 bp.