Nanodrop uv spectrometer

Detailed data on demographic characteristics, medical history, family history of cancer, lifestyle factors, and anthropometric measures were collected in-person by trained interviewers. Blood samples, as a source of DNA, were initially collected from approximately 850 participants until we transitioned to collection of saliva samples using Oragene™ kits (DNA Genotek Inc., Kanaya, Ontario, Canada) as a source of DNA. Pathology data including ER status, grade, and stage were collected and abstracted from patient records by trained staff.
Genomic DNA was extracted from blood samples using FlexiGene™ DNA isolation kits (Qiagen Inc., Valencia, CA) and from saliva samples using Oragene™ kits. DNA was then evaluated and quantified by Nanodrop UV-spectrometer (Thermo Fisher Scientific Inc., Wilmington, DE) and PicoGreen-based fluorometric assay (Molecular Probes, Invitrogen Inc., Carlsbad, CA). Samples were stored at −80°C until analysis.

Approximately 200 μg FVIII was subjected to the filter-aided sample preparation (FASP) procedure as reported [27 (link)]. In brief, 200 μg FVIII were mixed with 50 μL of lysis buffer (20 mM Tris-HCl, 4% (v/v) SDS, 100 mM DTT, pH 7.6), and incubated for 5 mins at 95°C. After mixing with 200 μL of UA solution (8 M urea in 0.1 M Tris/HCl, pH 8.5), the sample was loaded into a 30 kDa Microcon filtration device and centrifuged at 13,000 g until the volume was reduced to less than 10 μL. The concentrate was washed twice in the device with 200 μL of UA solution. After centrifugation, the concentrate was mixed with 100 μL of 50 mM IAA in the UA solution and incubated in the dark at room temperature (RT) for 30 mins, followed by brief centrifugation for 20 mins. Then, the protein concentrate was washed two more times against the UA solution. The so yielded sample was diluted twice with 100 μL of 40 mM NH₄HCO₃ for digestion. Sequencing grade trypsin was mixed with the sample at an enzyme to protein ratio of 1:50 for overnight digestion at 37°C. Finally, digested peptides were transferred into a filtration device and subjected to centrifugation, and washed with 50 μL of 0.5 M NaCl for 20 mins for 6 times. The concentration of peptides was determined by a Thermo Nanodrop UV-spectrometer, applying an extinction coefficient of 1.1 for 0.1% (g/L) solution at 214 nm.

Bacterial genomic DNA for strain As-13-3 was extracted using Axygen® AxyPrep™ Bacterial Genomic DNA Miniprep Kit (Axygen American).
A 2 mL sample was collected from each culture in the exponential phases using the RNA Bacteria Protect Reagent (Qiagen, Valencia, CA, USA). Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol and subsequently treated with DNase I (Invitrogen, Carlsbad, CA, USA). The RNA yield was estimated using a Nanodrop UV Spectrometer (Thermo Scientific, Wilmington, DE, USA).