Nanodrop one onec microvolume uv vis spectrophotometer

Genomic DNA was isolated using DNeasy Blood & Tissue (Qiagen, Hilden, Germany) and DNA concentrations were measured using a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Primers were designed to anneal around the Cas9 cutting site within the BRCA1 gene. To detect small indel variations, a short sequence around the Cas9 cutting site was amplified, while to detect larger variations a longer fragment was amplified (Table 3)
For PCR, GoTaq^® Flexi Polymerase (Promega, Walldorf, Germany) with the Green GoTaq^® Flexi Reaction Buffer supplemented with 5 mM MgCl₂, 200 µM dNTPs, 100 pmol/µL forward and reverse primer, and 100 ng genomic DNA as template was used. PCR was performed using a Primus 25 advanced^® Thermocycler with the following conditions: Initial denaturation (95 °C for 2 min) followed by 35 cycles of denaturation (95 °C for 30 s), annealing (54 °C for 30 s) and extension (72 °C for 50 s), ending with a final extension (72 °C for 10 min). Fragments were then loaded on to a 2% agarose gel, run for 30 min at 120 V, and stained with ethidium bromide, or analyzed by BioAnalyzer 2100 Expert (B.02.08.SI648) using DNA 7500 chips (Agilent Technologies, Santa Clara, CA, USA).

To determine the expression levels of osteoblastogenesis and osteoclastogenesis, the total RNA was isolated using the TRIzol reagent following the manufacturer’s instructions. The purity and concentrations of RNA were measured by a NanoDrop One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at an absorbance of 260/280 nm. Complementary DNA was prepared by 1 µm of total RNA using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas, Hanover, NH, USA). qRT-PCR was processed by a SYBR Green I qPCR Kit (TaKaRa, Shiga, Japan) and the fluorescence was measured by a CFX Connect™ Real-Time System (Bio-Rad). The specific primers for osteoblast and osteoclast differentiation are listed in Supplementary Table S1. The relative gene expressions were normalized by mouse Gapdh for osteoblasts and mouse Hprt for osteoclasts, and the fold change was determined using the 2^−ΔΔCt method.

Peripheral blood (PB) leukocytes were isolated by Buffy Coat procedure from 37 CML patients, according to LabNet recommendations, and 5 healthy donors. Total RNA was extracted using Maxwell 16 LEV simplyRNA Blood kit (Promega, Madison, WI, USA), following the manufacturer’s instructions. RNA samples were then quantified by NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and aliquoted for RT-qPCR and ddPCR experiments. CML samples were divided into 5 groups based on the MR levels [3 (link),6 (link)] obtained in routine RT-qPCR analysis as follows: group 1, MR < 3, n = 10; group 2, MR 3, n = 9; group 3, MR 4, n = 8; group 4, MR 4.5 and 5, n = 10, and group 5, healthy donors, n = 5.

RNA was isolated from spinal cords using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Spinal cords were weighed and suspended in 10 μl/mg of RLT buffer. Three hundred microliters of each sample was diluted 1:2 with RLT for a 30 mg/600 μl mixture. Ten microliters per milliliter of BME was added to the samples. Spinal cords were then homogenized by gentle pipetting. Spinal cord lysates were transferred to QIAshredder tubes and centrifuged at 13,000 rpm for 15 s. Six hundred microliters of 70% alcohol was added to each QIAshredder, spun at 13,000 rpm for 15 s, and transferred to separate RNAeasy columns. The RNAeasy columns were centrifuged twice for 15 s at 13,000 rpm, discarding the flow-through after each step. Seven hundred microliters of Buffer wash RW1 were added to each column. Samples were spun 15 s at 13,000 rpm, with the flow-through discarded. Two successive washes of 500 μl of Buffer RPE were added to the samples and spun at 13,000 rpm for 2 min. The columns were placed in collection tubes and then washed with 50 μl of RNase-free water to elute the RNA. RNA quantity (nanograms/microliter) and quality (A260/280) were measured using a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Scientific, Waltham, MA, USA).

DNA extraction was performed using the Precellys Evolution homogenizer (Bertin Technologies SAS, Montigny-le-Bretonneux, France). Each bacterial colony was homogenized using acid-washed 425–600 μm glass beads obtained from Sigma-Aldrich, St. Louis, MO, USA. The extract was then boiled for 5 minutes at 100 °C and centrifuged for 5 minutes, and the supernatant was gently transferred. DNA concentration and purity were measured using Qubit fluorometer with dsDNA high-sensitivity assay kit (Invitrogen, Waltham, Ma, USA) and NanoDrop™ One/OneC Microvolume UV–Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, Ma, USA), using the ratio of absorbance at 260 nm vs. 280 nm. The genomic libraries were prepared using the Nextera Flex kit according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was performed using iSeq 100, generating 151-bp paired-end reads (Illumina, San Diego, CA, USA).

To evaluate the nucleic acid occurrence in the three fractions after ultrafiltration, the quantity of DNA molecules has been assessed by the NanoDrop™ One/One^C Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific Inc., London, UK); absorbances at 260 nm and 280 nm were measured and concentrations were calculated as: $$\mathrm{DNA} \mathrm{concentration} \left(\mathrm{ng}/\mathsf{\mu }\mathrm{L}\right)={ \mathrm{A}}_{260\mathrm{nm}}·\frac{50 \mathrm{ng}/\mathsf{\mu }\mathrm{L}}{1\mathrm{a}.\mathrm{u}.}$$
The quality of the DNA has been evaluated by the direct measurements of the ratios A₂₆₀/A₂₈₀.
For the electrophoresis analysis, equal volumes of feed, permeate and retentate from the same UF assay were solubilized in an equal volume of loading buffer under stirring overnight at RT. Samples were separated in a 0.8% agarose gel using 0.5X TBE as the running buffer; bromophenol blue was added to the samples as a tracking dye. The 10 kbp DNA ladder was used (Thermo Fisher Scientific Inc.). After 30 min of running at 70 mA, 120 V, the gel image was digitalized using a UV Transilluminator (Consort bvba, Turnhout, Belgium) equipped with a photocamera.
All analytical measurements were performed in triplicate. Results were expressed as mean value ± SD.