Qiasymphony dsp mini kit

Strains were pre-lysed in a lysis buffer composed of 2 mg lysozyme (Sigma-Aldrich, Dorset, UK), 120 U mutanolysin (Sigma-Aldrich), 400 μg RNase A (Qiagen, Manchester, UK), 20 μL proteinase K (>600 mAU/mL) and 15 μL of overnight culture. Cell suspensions were incubated for 1 h at 37 °C, 2 h at 56 °C followed by 1 h at 80 °C. DNA was extracted using the QIAsymphony SP system and the QIAsymphony DSP mini kit (Qiagen) according to the manufacturers’ recommendations. DNA concentrations were measured using the Quant-iT dsDNA Broad-Range Assay Kit (Life Technologies, Paisley, UK) and GloMaxR 96 Microplate Luminometer (Promega, Southampton, UK). For sequencing preparation, a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) was used followed by sequencing using a HiSeq 2500 System (Illumina) and the 2 × 100-bp paired-end mode.

DNA isolation was performed by use of either the QiaSymphony DSP mini kit (Qiagen, Hilden, Germany) or the Maxwell 16 Blood DNA Purification Kit (Promega, Nacka, Sweden) according to the manufacturer's instructions.
Protein coding exons and flanking intronic regions of the
PROS1gene were amplified using previously reported primers.
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The primers were modified with M13 linkers to facilitate sequencing. The polymerase chain reaction (PCR) products were purified by use of exonuclease and shrimp alkaline phosphatase digestion (ExoSAP-IT) as recommended by the manufacturer (Life Technologies Europe BV, Roskilde, Denmark). The purified fragments were bidirectionally sequenced using M13 sequencing primers (M13F: 5′ - GTAAAACGACGGCCAG – 3′ and M13R: 5′ – CAGGAAACAGCTATGAC – 3′) and BigDye terminator version 1.1 (Life Technologies). The sequencing reactions were ethanol-precipitated and separated on an Applied Biosystems 3500 or 3500xl Genetic Analyzer (Life Technologies). Sequence traces were aligned to NM_000313 (
PROS1) by use of SeqScape software (version 2.7, Life Technologies).
Nomenclature of variants follows current guidelines.
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