Magna pure lc dna isolation kit 2

DNA extraction was performed using the MagNa Pure LC DNA Isolation kit II (Roche®) and a MagNa Pure Instrument. Protocol was used as indicated by the company with some modifications following Dzidic et al. 2018 [15 (link)]. In summary, samples were lysed using 3 × 10 seconds cycles of ultrasounds, enzymatic digestion with an enzyme cocktail of lysozyme (100 mg/ml), lysostaphin (5 kU/ml) and mutanolysin (2.5 kU/ml), followed by protein degradation with Proteinase K.
After cleaning and measuring the DNA, the V3-V4 hypervariable region of the 16S rRNA gene was amplified using universal primers optimized for Illumina sequencing, following Dzidic et al. 2018 [15 (link)]. Library was constructed following the 16S rRNA gene Metagenomic Sequencing Library Preparation Illumina protocol (Part #15,044,223 Rev. A) and sequenced at the sequencing service at the FISABIO Institute (Valencia) using the 2 × 300 bp paired-end Illumina protocol.

Samples were extracted using the MagNa Pure LC DNA Isolation kit II and a MagNa Pure Instrument (Roche Molecular Systems Inc., Pleasanton, CA, USA). The protocol was used as indicated by the manufacturer with some modifications following Dzidic et al [31 (link)]. In brief, samples were lysed using 3 × 10-second cycles of ultrasound and enzymatic digestion with an enzyme cocktail of lysozyme (100 mg/ml), lysostaphin (5 kU/ml), and mutanolysin (2.5 kU/ml). Finally, proteins were degraded using Proteinase K. Then, the DNA was resuspended in 100 μl of ultrapure DNAse-free water.
After measuring the DNA by fluorimetry, the V3-V4 hypervariable region of the 16S rRNA gene was amplified using universal primers optimised for Illumina sequencing, following Dzidic et al [31 (link)]. The library was constructed using the Metagenomic Sequencing Library Preparation Illumina protocol (Part #15044223, Rev. A) and sequenced with the standard procedure recommended by the manufacturer at the sequencing service in FISABIO (Valencia, Spain) using 2 × 300 bp paired-end sequencing with an Illumina MiSeq instrument. Data has been deposited in the SRA database (Bioproject: PRJNA598825).

Abscess drainage fluid was analyzed using a qPCR-based assay targeting insertion sequence IS711, which is unique to the genus Brucella (Hinić et al., 2008 (link)). DNA was extracted using a MagNA Pure LC instrument and a MagNA Pure LC DNA Isolation Kit II (Roche Molecular Biochemicals) according to the manufacturer’s instructions. TaqMan Universal PCR Master Mix with AmpErase UNG (Applied Biosystems) was used along with 0.5 μL of input DNA and nuclease-free water (Promega). Each PCR assay was performed in triplicate.

Samples were extracted using the MagNa Pure LC DNA Isolation kit II (Roche®) and a MagNa Pure Instrument. Protocol was used as indicated by the company with some modifications following Dzidic et al. 2018 [20 (link)]. In summary, samples were lysed using 3 × 10 s cycles of ultrasounds, enzymatic digestion with an enzyme cocktail of lysozyme (100 mg/ml), lysostaphin (5 kU/ml) and mutanolysin (2.5 kU/ml). Finally, proteins were degraded using Proteinase K. DNA was resuspended in 100 ul of ultrapure DNAse-free water.
After measuring the DNA by fluorimetry, the V3-V4 hypervariable region of the 16S rRNA gene was amplified using universal primers optimized for Illumina sequencing, following Dzidic et al. 2018 [20 (link)]. Library was constructed using the Metagenomic Sequencing Library Preparation Illumina protocol (Part #15044223 Rev. A) and sequenced with the standard procedure recommended by the manufacturer, at the sequencing service in FISABIO (Valencia, Spain) using 2 × 300 bp paired-end sequencing with an Illumina MiSeq instrument. Data have been deposited in the SRA database (Bioproject PRJNA542627: Microbiology of Molar–Incisor Hypomineralization lesions, SRR9098974-SRR9099019).

DNA was isolated from a placenta biopsy using the MagnaPureLC DNA isolation kit II (Roche). For QF-PCR aneuploidy screening the QST*Rplus V2 kit (Elucigene Diagnostics, AN0PLB2) was used according to the manufacturer’s instructions. The amplified sample was analyzed with the ABI 3500 (Applied Biosystems, Foster City, California, USA). Interpretation of results was performed using guidelines from the manual, the 2012 ACC/CMGS ‘QF-PCR for the diagnosis of aneuploidy best practice guidelines’ V3.01, 22 and the CCMG ‘Practice Guidelines for Prenatal QF-PCR’.