Blood dna purification kit

Genomic DNA (gDNA) from 25 randomly selected CM samples was extracted by an automated Maxwell 16 DNA extraction platform using blood DNA purification kits (Promega, United Kingdom) following previously described protocols (Hoque et al., 2019 (link)). DNA quantity and purity were determined with NanoDrop (ThermoFisher, United States) by measuring 260/280 absorbance ratios. Sequencing libraries were prepared with the Nextera XT DNA Library Preparation Kit (Head et al., 2014 (link)) and paired-end (2 × 150 bp) sequencing was performed on a NextSeq 500 machine (Illumina Inc., United States) at the George Washington University Genomics Core facility. Our metagenomic DNA yielded a total of 596.74 million reads with an average of 23.87 million (maximum = 39.75 million, minimum = 8.89 million) reads per sample (Supplementary Material) before cleaning.

The leptospiral burden in urine, blood and organs was determined by quantitative real-time PCR (q-PCR), as described [15] (link). The Maxwell 16 automat was used to extract total DNA from a drop of urine (5 to 100 µl) or from 50 µl of blood, using the cell LEV DNA, or blood DNA purification kits (Promega), respectively. Total DNA from organs was extracted with the DNAeasy tissue Qiagen kit after mechanical disruption with metallic beads. Primers and probe designed in the lpxA gene of L. interrogans strain Fiocruz [23] (link) were used to specifically detect pathogenic Leptospira spp[15] (link). L. biflexa was detected using the classical RNA 16S gene q-PCR, as described [8] (link). q-PCR reactions were run on a Step one Plus real-time PCR apparatus using the absolute quantification program (Applied Biosystems), with the following conditions for FAM TAMRA probes: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles with denaturation at 95°C for 15 s and annealing temperature 60°C for 1 min, according to the manufacturer's instructions. Results were expressed as the number of leptospires per 100 µl of urine, 50 µl of blood, or per 200 ng of total DNA extracted from organs.

DNA was extracted from the blood buffy coat by automated Blood DNA purification kit on the Maxwell 16 instrument (Promega), according to the manufacturer’s instructions. DNA preparates were stored at -80°C until the analyses were performed.
DNA samples of six SSc patients, which resulted positive to the hemolysis test, were sent to Secugen Diagnostic (Madrid, Spain) for the genetic analyses. Exons and promoter regions of genes for FH, FI and MCP were sequenced and compared to the published sequences in Ensemble, NCBI, and aHUS databases. Genotypes and haplotypes for common polymorphisms (SNPs) in these genes were also analyzed. The two polymorphic variants of MCP promoter region (-366A>G, rs2796268 and -652A>G, rs2796267), of those identified thereby, were assessed with TaqMan allelic discrimination assays designed on demand (Applied Biosystems, Foster City, CA, USA) on a 7500 Real Time PCR instrument (Applied Biosystems).