Nanodrop nd 1000 spectrometer

Manufactured by Avantor

Sourced in Germany

The NanoDrop ND-1000 is a spectrophotometer designed for the measurement of nucleic acid and protein concentrations. It utilizes a patented sample retention system that requires only 1-2 microliters of sample. The device provides accurate quantification of samples at concentrations from 2-3700 ng/μL for dsDNA.

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15 protocols using nanodrop nd 1000 spectrometer

Protein Purification and Characterization

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RfaH used in crystallization experiments and in vitro transcription assays was produced as described (Vassylyeva et al., 2006 (link)), as was RfaH used in NMR experiments (Burmann et al., 2012 (link)), and RNAP for in vitro transcription assays (Svetlov and Artsimovitch, 2015 (link)). All expression plasmids are listed in Table 2.
The purity was checked by SDS-PAGE, the absence of nucleic acids was checked by recording UV/Vis spectra on a Nanodrop ND-1000 spectrometer (PEQLAB, Erlangen, Germany). Concentrations were determined by measuring the absorbance at 280 nm (A₂₈₀) in a 10 mm quartz cuvette (Hellma, Müllheim, Germany) on a Biospectrometer basic (Eppendorf, Hamburg, Germany).

Zuber P.K., Artsimovitch I., NandyMazumdar M., Liu Z., Nedialkov Y., Schweimer K., Rösch P, & Knauer S.H. (2018). The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand. eLife, 7, e36349.

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DNA Isolation from Frozen Tissues

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Pulmonary DNA was isolated using a Qiagen kit for the preparation of genomic DNA with slight modifications of the manufacturer’s instructions (Qiagen, Venlo, Netherlands). Briefly, 100 mg samples of frozen tissues were homogenized in 9.5 mL of the lysis buffer G2. The homogenate was digested with proteinase K (500 μL, 10 mg/mL) and DNAse-free RNAse A (20 μL, 10 mg/mL in water) for 2 h at 37°C. The mixture was cooled to 4°C and centrifuged at 10,000g for 10 min. The supernatant was loaded onto a Qiagen Genomic-tip 100/G previously equilibrated with 4 mL QBT-buffer. The column was washed twice with 7.5 mL QC solution. The DNA was eluted with 5 mL QF solution that was heated to 70°C, precipitated by adding 2.5 mL 2-propanol and separated by centrifugation at 15,000g for 15 min. The pellet was washed with 1 mL cold 70 % ethanol, centrifuged again and dissolved in 300 μL water. The DNA concentration was determined spectrophotometrically with a Nanodrop ND-1000 spectrometer (peqlab Biotechnologie, Erlangen, Germany).

Monien B.H., Schumacher F., Herrmann K., Glatt H., Turesky R.J, & Chesné C. (2014). Simultaneous Detection of Multiple DNA Adducts in Human Lung Samples by Isotope-Dilution UPLC-MS/MS. Analytical Chemistry, 87(1), 641-648.

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RNA Isolation Using TRIzol Reagent

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After withdrawal of the apical and basolateral medium, cells were covered with TRIzol Reagent (Invitrogen, Germany) as described by the manufacturer’s protocol and scraped off the membrane. After adding chloroform to the cell lysate, the supernatant was extracted and RNA was precipitated using isopropanol alcohol. Using 75% ethanol, the RNA was purified and stored in RNA-free water peqGOLD (PEQLAB, Germany) at –80 °C until further processing. Five independent experiments were performed. The RNA was isolated using TRIzol Reagent as per the manufacturer’s directions (Sigma-Aldrich, Taufkirchen, Germany). RNA integrity was checked using the 2100 Bioanalyzer platform (Agilent). RNA concentrations were measured on a NanoDrop ND-1000 spectrometer (PEQLAB, Erlangen, Germany). The absence of genomic DNA was checked by PCR amplification of the porcine GAPDH gene. All RNA samples were stored at –80 °C until downstream analyses were performed.

Nossol C., Landgraf P., Kahlert S., Oster M., Isermann B., Dieterich D.C., Wimmers K., Dänicke S, & Rothkötter H.J. (2018). Deoxynivalenol Affects Cell Metabolism and Increases Protein Biosynthesis in Intestinal Porcine Epithelial Cells (IPEC-J2): DON Increases Protein Biosynthesis. Toxins, 10(11), 464.

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Beetle RNA Extraction Protocol

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Two legs from the same individual beetle were frozen in liquid nitrogen and ground to a fine powder using mortar and pestle. The powder was directly transferred into Tri Reagent^® (Sigma-Aldrich, Munich, Germany) and RNA was extracted with the Direct-zol™ RNA MiniPrep (Zymo, Freiburg, Germany) following the manufacturer’s protocol. RNA quality was determined with a NanoDrop ND 1000 spectrometer (PeqLab, Erlangen, Germany) and RNA integrity was checked with the Agilent RNA Nano Chip Assay (Agilent, Santa Clara). Only RNA samples of high quality (OD 260/280 > 2 and OD 260/230 > 1.8) and high integrity were used for library preparation.

Meyer J.M., Markov G.V., Baskaran P., Herrmann M., Sommer R.J, & Rödelsperger C. (2016). Draft Genome of the Scarab Beetle Oryctes borbonicus on La Réunion Island. Genome Biology and Evolution, 8(7), 2093-2105.

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Porcine PBMC Transcriptome Profiling

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In total, 16 samples (balanced for mixing group) were selected for subsequent analyses. Using 4 ml of blood, peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on a Histopaque (Sigma-Aldrich, Taufkirchen, Germany) density gradient. Total RNA was isolated from individual PBMC-samples using the column-based NucleoSpin RNA II Kit (MACHEREY-NAGEL, Düren, Germany). RNA integrity was checked by a 2100 Bioanalyser (Agilent) and agarose gels containing ethidium bromide. RNA concentration was measured by a NanoDrop ND-1000 spectrometer (PEQLAB, Erlangen, Germany). The absence of genomic DNA was checked by a PCR amplification of the porcine GAPDH gene (forward primer 5’-AAGCAGGGATGATGTTCTGG-3’; reverse primer 5’-ATGCCTCCTGTACCACCAAC-3’). All samples were stored at -80°C until downstream analyses was performed. For the microarray experiments individual biotin-labelled cRNA was synthesized by the GeneChip 3’ Express Kit (Affymetrix, Santa Clara, CA, USA). According to the manufacturer’s protocol the cRNA was fragmented and hybridized on Affymetrix GeneChip porcine 24 k Arrays. After a washing and staining procedure the arrays were scanned (Affymetrix, Santa Clara, CA, USA).

Oster M., Muráni E., Ponsuksili S., D’Eath R.B., Turner S.P., Evans G., Thölking L., Kurt E., Klont R., Foury A., Mormède P, & Wimmers K. (2014). Transcriptional responses of PBMC in psychosocially stressed animals indicate an alerting of the immune system in female but not in castrated male pigs. BMC Genomics, 15(1), 967.

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Genome-wide Transcriptome Analysis in Pigs

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Total RNA was isolated using TRI Reagent per manufacturer’s directions (Sigma-Aldrich, Taufkirchen, Germany), then treated with DNase and purified with the column-based NucleoSpin RNA II-Kit (Macherey-Nagel, Düren, Germany). RNA integrity was determined by visualization on a 1% agarose gel containing ethidium bromide and the concentration was measured using the NanoDrop ND-1000 spectrometer (PEQLAB, Erlangen, Germany). DNA contamination was assessed by PCR amplification of the porcine RPL32 gene (forward primer: 5’-AGCCCAAGATCGTCAAAAAG-3′; reverse primer: 5’-TGTTGCTCCCATAACCAATG-3′). All RNA samples were stored at − 80 °C.
Each RNA sample was transcribed to DNA using the Ambion WT Expression Kit (Ambion, Austin, TX, USA). The DNA preparations were fragmented and labelled with the WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA, USA). DNA preparations were hybridized on genome-wide snowball arrays (Affymetrix), which were invented for genome-wide analysis of the pig transcriptome [22 (link)]. Raw data was generated with Affymetrix GCOS 1.1.1 software and deposited in a MIAME-compliant database [23 (link)], the National Center for Biotechnology Information Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo; accession number: GSE94448).

Just F., Oster M., Büsing K., Borgelt L., Murani E., Ponsuksili S., Wolf P, & Wimmers K. (2018). Lowered dietary phosphorus affects intestinal and renal gene expression to maintain mineral homeostasis with immunomodulatory implications in weaned piglets. BMC Genomics, 19, 207.

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RNA Extraction and cDNA Synthesis

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Total RNA was isolated using TRI Reagent per manufacturer’s directions (Sigma-Aldrich, Taufkirchen, Germany), then treated with DNase and purified with the column-based NucleoSpin RNA II-Kit (Macherey–Nagel, Düren, Germany). RNA integrity was determined by visualisation on a 1% agarose gel containing ethidium bromide and the concentration was measured using the NanoDrop ND-1000 spectrometer (PEQLAB, Erlangen, Germany). Absence of DNA contamination was verified by PCR amplification of the porcine RPL32 gene (forward primer: 5′-AGCCCAAGATCGTCAAAAAG-3′; reverse primer: 5′-TGTTGCTCCCATAACCAATG-3′). All RNA samples were stored at −80 °C. First-strand cDNA was synthesised from 2 μg of total RNA using random primers (Promega, Fitchburg, WI, USA) and oligo d(T) 13VN in the presence of Superscript III reverse transcriptase (Invitrogen, Karlsruhe, Germany). The final cDNA was diluted with Aqua dest to a total volume of 100 µl.

Oster M., Gerlinger C., Heide K., Just F., Borgelt L., Wolf P., Polley C., Vollmar B., Muráni E., Ponsuksili S, & Wimmers K. (2017). Lower dietary phosphorus supply in pigs match both animal welfare aspects and resource efficiency. Ambio, 47(Suppl 1), 20-29.

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Extracting High-Quality RNA from Dinospore Cells

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TRI Reagent–fixed and frozen dinospore cells were lysed using a Bio 101 FastPrep instrument (Thermo Savant, Illkirch, France) at maximum speed (6.5 m/s) for 2 × 45 s. Lysed cells were cooled on ice, and 200 μl of chloroform was added and vortexed for 20 s. The samples were transferred to a phase lock tube after 5 min of incubation at room temperature (Eppendorf, Hamburg, Germany) and incubated for another 5 min followed by centrifugation for 15 min at 13,000g and 4°C. The upper aqueous phase was transferred to a new tube and mixed with the same volume isopropanol, ¹/₁₀ volume of 3 M Na-acetate (pH 5.5; Ambion by Life Technologies, Carlsbad, CA, USA), and 2 μl of linear polyacrylamide (Ambion). Total RNA was precipitated for 90 min at −20°C and collected by centrifugation for 20 min at 13,000g and 4°C. The obtained pellet was washed twice, first with 1 ml of 70% ethanol (EtOH) followed by 1 ml of absolute EtOH; the RNA pellet was dried for 1 min at 37°C and resolved in 30 μl of RNase-free water (Qiagen, Hilden). RNA quality check was performed using a NanoDrop ND-1000 spectrometer (PEQLAB, Erlangen, Germany) for purity and RNA Nano Chip Assay on 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to examine the integrity of the extracted RNA.

John U., Lu Y., Wohlrab S., Groth M., Janouškovec J., Kohli G.S., Mark F.C., Bickmeyer U., Farhat S., Felder M., Frickenhaus S., Guillou L., Keeling P.J., Moustafa A., Porcel B.M., Valentin K, & Glöckner G. (2019). An aerobic eukaryotic parasite with functional mitochondria that likely lacks a mitochondrial genome. Science Advances, 5(4), eaav1110.

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Quantitative PCR Analysis of Zebrafish Transcripts

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Total RNA of zebrafish tissues was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The concentrations of RNA samples were assessed with a NanoDrop ND-1000 spectrometer (PeqLab). The quality of the RNA was determined after electrophoresis on an agarose gel to visualize the integrity of the ribosomal 28S, 18S, and 5S RNA bands. The first strand cDNA synthesis of total RNA was performed according to the manufacturer’s protocol (Promega). Quantitative PCR was performed using the Step One Plus Real-Time PCR System (Applied Biosystems) and SYBR Green (Promega) master mix according to the manufacturers’ recommendations. Primer sequences are shown in Supplementary Table 1. The relative expression levels for each gene were calculated by the 2^–ΔΔCT method and normalized using the relative expression of β-actin.

Ruggiero G., Ben-Moshe Livne Z., Wexler Y., Geyer N., Vallone D., Gothilf Y, & Foulkes N.S. (2021). Period 2: A Regulator of Multiple Tissue-Specific Circadian Functions. Frontiers in Molecular Neuroscience, 14, 718387.

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Nematode RNA Extraction and Sequencing

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Nematodes were separated by centrifugation from the culture supernatant, washed once with S-medium and immediately transferred into Tri Reagent^® (Sigma-Aldrich, Munich, Germany). After three rounds of freezing in liquid nitrogen and thawing at 37 °C to break open the cells RNA was extracted with the Direct-zol™ RNA MiniPrep (Zymo, Freiburg, Germany) following the manufacturer’s protocol. RNA quality was determined with a NanoDrop ND 1000 spectrometer (PeqLab, Erlangen, Germany) and RNA integrity was checked with the Agilent RNA Nano Chip Assay (Agilent, Santa Clara, USA). Only RNA samples of high quality (OD 260/280 > 2 and OD 260/230 > 1.8) and high integrity were used for the RACE PCR experiment. Further, the high quality RNA was used to prepare two libraries following Illumina’s truseq RNA-sequencing protocol (Illumina, Eindhoven, Netherlands). Both libraries were sequenced on one 101 bp pair-end flow cell lane of an Illumina HiSeq 2000 Sequencer.

Falcke J.M., Bose N., Artyukhin A.B., Rödelsperger C., Markov G.V., Yim J.J., Grimm D., Claassen M.H., Panda O., Baccile J.A., Zhang Y.K., Le H.H., Jolic D., Schroeder F.C, & Sommer R.J. (2018). Linking Genomic and Metabolomic Natural Variation Uncovers Nematode Pheromone Biosynthesis. Cell chemical biology, 25(6), 787-796.e12.

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