Magattract dna mini m48 kit

Nasal, buccal and fecal swabs were taken, placed in vials containing serum-free cell-culture medium, and stored at −80°C until further analysis. Viral RNA was extracted from nasal and buccal swabs taken at 0, 2, 4, 6 dpi and from fecal swabs at 3 dpi using the MagAttract® DNA Mini M48 Kit (Qiagen, Hilden, Germany) on the KingFisher Flex Magnetic Particle Processors (Thermo Fisher Scientific, Waltham, USA). Swabs were eluted in 1 mL serum-free cell-culture media of which 100 µl were used for RNA extraction into 100 µl AVE buffer. Real-time reverse transcriptase RT-qPCR for quantification of SIV copy numbers was performed using a pan-Influenza A-M1.2 assay [24] (link) and an in vitro-transcribed RNA standard.

Strains were grown overnight on MacConkey agar. Genomic DNA was isolated for each strain using the Qiagen MagAttract DNA Mini M48 Kit and the Qiagen BioRobot M48 (Qiagen, Hilden Germany) as described by the manufacturer.

Six sections of 10 μm thickness were cut from FFPE tissue blocks. After deparaffinisation, tumour areas were macrodissected from unstained slides. The tumour area was marked on a haematoxylin-eosin (H&E) stained slide by a senior pathologist (E. W., H.-U. S.). DNA was extracted with the MagAttract® DNA Mini M48 Kit (Qiagen, Hilden, Germany) on the BioRobot® M48 (Qiagen). Samples collected before the year 2010 were extracted manually with the QIAamp® DNA Mini Kit (Qiagen). DNA extraction of the subregions were performed with the Maxwell® 16 FFPE Plus Tissue LEV DNA Purification Kit (Promega, Mannheim, Germany) on the Maxwell® 16 (Promega). Fresh-frozen tissues were extracted with the DNeasy® Blood & Tissue Kit (Qiagen) (Figure 1). All extraction procedures were performed following the manufacturers’ instructions.Figure 1

Visual depiction of the different experiments and workflows performed on the GS Junior (Roche) (A) and the MiSeq™ (Illumina) (B) with FFPE and fresh-frozen material.

An SAF-fixated faecal solution (1000 μL) was centrifuged at 10,000 × g for 1 min. Pellets were then washed with phosphate-buffered saline (PBS) pH 7.4 for 1 min, followed by another centrifugation step. Pellets were suspended in a mixture of 280 μl AL lysis buffer and 20 μL proteinase K (Qiagen, Hilden, Germany), and incubated at 56 °C for 1 h with gentle agitation. Suspensions were then frozen at −196 °C for 30 min and, finally, heated at 98 °C for 15 min. DNA extraction was done with a MagAttract DNA Mini M48 kit (Qiagen) in an M48 instrument (Qiagen). Real-time PCR for parasite detection was done with the LightCycler 480 II instrument (Roche Diagnostics GmbH, Mannheim, Germany), according to Verweij et al. [7 (link), 15 (link)], modified by the inclusion of Entamoeba dispar [16 (link)].

DNA was extracted from sections of the most recent FFPE tumor specimens available from biopsies or surgical resections. Optimal tumor regions were identified by clinical breast pathologists (AMM and HKB). Tumors containing a minimum acceptable tumor cellularity of 10% were processed with tumor regions isolated by 1–2 × 1 mm punch from FFPE blocks or manual macrodissection of unstained material from 15 to 20 slides. FFPE samples were deparaffinized and treated with proteinase K, followed by DNA extraction using the QIAmp DNA FFPE Tissue Kit (Qiagen, Germantown, MD) and quantification using the Qubit dsDNA Assay kit on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA). DNA was extracted from peripheral blood samples for germline testing using either standard manual phenol/chloroform extraction methods or automated extraction (MagAttract DNA Mini M48 kit; Qiagen).

Representative fresh-frozen tissues stored at −80°C in a biobank were used for molecular cytogenetic analysis. DNA from 16 tumor samples was isolated using the MagAttract DNA Mini M48 kit (Qiagen, Valencia, CA, USA). HR-CGH was performed according to our modifications of the standard procedure (20 (link)–22 (link)). Chromosomes were karyotyped based on their inverted DAPI appearance and the relative hybridization signal intensity was determined along each chromosome. On average, 10–15 metaphases were analyzed. The description of the CGH copy number changes was based on the recommendations of the ISCN (2009).

Tumor tissue was deparaffinized and macrodissected from unstained slides. The tumor tissue was then lysed with proteinase K overnight. DNA extraction from FFPE-embedded tissue was performed with the BioRobot M48 Robotic workstation and the corresponding MagAttract DNA Mini M48 Kit (Qiagen, Germany). Determination of HPV subtypes was performed applying the HPV Type 3.5 LCD-Array Kit (Chipron, Germany) according to the manufacturer’s instructions as described previously [23 (link)]. With this assay the detection of 32 different HPV subtypes is possible (HPV types 06,11,16,18,31,33,35,39, 42,44,45,51,52,53,54,56, 58,59,61,62,66,67,68,70, 72,73,81,82,83,84,90 and 91).