Nanodrop 1000 uv vis spectrophotometer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The NanoDrop 1000 UV-Vis spectrophotometer is a compact and easy-to-use instrument designed for the quantification and analysis of small-volume samples. It utilizes a patented sample-retention technology that requires only 1-2 microliters of sample to measure absorbance across the full UV-Vis spectrum.

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63 protocols using nanodrop 1000 uv vis spectrophotometer

DNA and RNA Extraction from Whole Blood Samples

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DNA was obtained from buffy coats collected in EDTA tubes at mean age 8.1 years old. Briefly, DNA was extracted using the Chemagen kit (Perkin Elmer), in batches by cohort. DNA concentration was determined in a NanoDrop 1,000 UV-Vis Spectrophotometer (Thermo Fisher Scientific) and with Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies).
RNA was extracted from whole blood samples collected in Tempus tubes (Applied Biosystems) using the MagMAX for Stabilized Blood Tubes RNA Isolation Kit (Thermo Fisher Scientific), in batches by cohort. The quality of RNA was evaluated with a 2100 Bioanalyzer (Agilent) and the concentration with a NanoDrop 1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific). Samples classified as good RNA quality had an RNA Integrity Number (RIN) > 5, a similar RNA integrity pattern at visual inspection, and a concentration >10 ng/µl. Mean values for the RIN, concentration (ng/ul) and Nanodrop 260/230 ratio were: 7.05, 109.07, and 2.15, respectively.

Ruiz-Arenas C., Hernandez-Ferrer C., Vives-Usano M., Marí S., Quintela I., Mason D., Cadiou S., Casas M., Andrusaityte S., Gutzkow K.B., Vafeiadi M., Wright J., Lepeule J., Grazuleviciene R., Chatzi L., Carracedo Á., Estivill X., Marti E., Escaramís G., Vrijheid M., González J.R, & Bustamante M. (2022). Identification of autosomal cis expression quantitative trait methylation (cis eQTMs) in children’s blood. eLife, 11, e65310.

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Buffy Coat DNA and Whole Blood RNA Extraction

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DNA was obtained from buffy coat collected in EDTA tubes at mean age 8.1 years old.
Briefly, DNA was extracted using the Chemagen kit (Perkin Elmer) in batches of 12 samples.
Samples were extracted by cohort. DNA concentration was determined in a NanoDrop 1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific) and with Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies).
RNA was extracted from whole blood samples collected in Tempus tubes (Applied Biosystems) using the MagMAX for Stabilized Blood Tubes RNA Isolation Kit (Thermo Fisher Scientific). RNA extraction was performed in batches of 12-24 samples and by cohort.
The quality of RNA was evaluated with a 2100 Bioanalyzer (Agilent) and the concentration with a NanoDrop 1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific

Ruiz-Arenas C., Hernández-Ferrer C., Vives-Usano M., Marí S., Quintela I., Mason D., Cadiou S., Casas M., Andrušaitytė S., Gützkow K.B., Vafeiadi M., Wright J., Lepeule J., Gražulevičienė R., Chatzi L., Carracedo Ã., Estivill X., Martı́ E., Escaramís G., Vrijheid M., González J.R., & Bustamante M. (2020). Identification of autosomal cis expression quantitative trait methylation (cis eQTMs) in children’s blood.

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Total RNA Isolation and cDNA Synthesis

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Total RNA was isolated from micromass cultures using the TRI Reagent (Applied Biosystems; Thermo Fisher Scientific) according to the instructions of the manufacturer. After the addition of 20% chloroform, samples were centrifuged at 4°C at 10 000g for 15 min. Samples were incubated in 500 µl of RNase free isopropanol at –20°C for 1 h, then total RNA was harvested in RNase‐free water and stored at –80°C. RNA concentration and purity were determined by a Nanodrop 1000 UV‐Vis spectrophotometer (Thermo Fisher Scientific). For gene expression analyses, 1 μg of RNA was reverse‐transcribed into complementary DNA (cDNA) using the High‐Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), as per the protocol supplied by the manufacturer. The assay mixtures for reverse transcriptase reactions contained 1 µg RNA, 0.112 µM oligo(dT), 0.5 mM dNTP, 200 units of High Capacity RT (Applied Bio‐Systems) in 1× RT buffer. cDNA was stored at –20°C.

Vágó J., Katona É., Takács R., Dócs K., Hajdú T., Kovács P., Zákány R., van der Veen D.R, & Matta C. (2022). Cyclic uniaxial mechanical load enhances chondrogenesis through entraining the molecular circadian clock. Journal of Pineal Research, 73(4), e12827.

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Quantitative Analysis of IL-6 Expression

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TRIzol was purchased from Life Sciences (Grand Island, NY) and used to extract total RNA. Nucleic acid purity was measured using a Nanodrop 1000 UV-Vis Spectrophotometer (Thermo Scientific, Waltham, MA). cDNA was synthesized with the qScript cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD) following the manufacturer’s protocol. To determine IL-6 and GAPDH mRNA expression, real time quantitative PCR (RT-qPCR) was performed with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) and PerfeCTa SYBR Green SuperMix (Quanta Biosciences). Primers for IL-6 (forward: 5’-TCCAGTTGCCTTCTTGGGAC-3’, reverse: 5’-GTGTAATTAAGCTCCGACTTG-3’) and GAPDH (GAPDH forward, 5’-GATGACATCAAGAAGGTGGTG-3’, reverse, 5’-GCTGTAGCCAAATTCGTTGTC-3’) were purchased from Eurofins MWG Operon (Huntsville, AL). Amplification conditions were as follows: 95°C (2 min) followed by 40 cycles of 95°C (15 s), 55°C (30 s), and 60°C (1 min). A melting curve analysis was performed between 50°C and 95°C. Results were normalized to GAPDH and the H₂O control by using the Relative Livak Method (ΔΔCt).

Abebayehu D., Spence A.J., Caslin H., Taruselli M., Haque T.T., Kiwanuka K.N., Kolawole E.M., Chumanevich A.P., Sell S.A., Oskeritzian C.A, & Ryan J. (2019). Lactic acid suppresses IgE-mediated mast cell function in vitro and in vivo. Cellular immunology, 341, 103918.

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Saliva-Based DNA Isolation and Genotyping

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Genomic DNA was isolated from volunteer saliva sample (400 µL) using High Pure PCR template Preparation Kit (Roche, Basel, Switzerland). The quality and the concentration of the isolated DNA were assessed using Nanodrop 1000 UV-Vis Spectrophotometer (Thermo-Fisher Scientific, MA, USA). A final concentration between 20–100 ng/µL of the isolated DNA was considered great. Then, aliquots were stored at −20 °C until the analysis of polymorphisms. Genotyping was performed by qPCR (LightCycler^®480 FastStart DNA or Genotyping Master mix, Roche, Basel, Switzerland) using the differential melting temperature to determine the alleles of each single nucleotide polymorphism (SNP). The qPCRs were done following these conditions: 95 °C for 5 min, 45 cycles of denaturation at 92 °C for 15 s and annealing 60 °C for 1 min. The melting temperatures were recorded and analysed to determine the presence of specific alleles. The genotyping of all SNP was done using expressly designed probes (TIB Molbiol, Berlin, Germany).

Galmés S., Cifre M., Palou A., Oliver P, & Serra F. (2019). A Genetic Score of Predisposition to Low-Grade Inflammation Associated with Obesity May Contribute to Discern Population at Risk for Metabolic Syndrome. Nutrients, 11(2), 298.

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RNA Isolation from Blood Samples

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Blood samples (2.5 ml) were drawn using PaxGene Blood RNA tubes (PreAnalytix GmbH, Hombrechtikon, Switzerland) and total RNA was then isolated as described in a previous publication [11 (link)]. The integrity of the purified RNA was accessed by 2100 Bioanalyzer RNA 6000 Nano Chips (Agilent Technologies, Inc., Santa Clara, CA, USA) and the quantity of RNA was assessed by NanoDrop 1000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Inc. Waltham, MA, USA). All RNA samples were assessed by RNA integrity number ≥7·0 and 28S:18S rRNA≥1.0.

Hou H., Lyu Y., Jiang J., Wang M., Zhang R., Liew C.C., Wang B, & Cheng C. (2020). Peripheral blood transcriptome identifies high-risk benign and malignant breast lesions. PLoS ONE, 15(6), e0233713.

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Influenza H1N1 Detection Protocol

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The target RNA sequence was designed according to the specific sequence fragment of influenza H1N1. Mature LwaCas13a crRNA was designed. It contained a 36-nucleotide (nt) direct repeat sequence and a 25-nt spacer that was completely complementary to the target RNA. The crRNA and target RNA were synthesized by in vitro transcription using T7 RNA polymerase and the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs) to transcribe and Monarch RNA Cleanup Kit to purify RNA. Two microliters of 100 μM T7 promoter, 2 μL of 100 μM ssDNA templates containing T7 promoter sequence, and 14 μL RNase-free water were incubated at 95 °C for 5 min and then gradient cooled to 25 °C. The transcription reaction was established with the above solution, 2 μL T7 RNA polymerase (50 U/μL), and 10 μL NTP Buffer mix (20 mM) at 37 °C for 16 h. Then, 2 μL DNaseI (2 U/μL) was added and mixed to degrade the DNA in the solution. The concentrations of crRNA and target RNA were determined using a Nanodrop 1000 UV–vis spectrophotometer (Thermo Fisher Scientific) and stored at – 80 °C.

Zhou H., Bu S., Xu Y., Xue L., Li Z., Hao Z., Wan J, & Tang F. (2022). CRISPR/Cas13a combined with hybridization chain reaction for visual detection of influenza A (H1N1) virus. Analytical and Bioanalytical Chemistry, 414(29-30), 8437-8445.

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Tris-Neutralization DNA Extraction Protocol

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The total genomic DNA was extracted from a 30 mg sample of dry roots using a modified Tris-neutralization method [29 ], in which an extraction buffer containing 0.5 mol/L NaOH, 1% polyvinyl polypyrrolidone (PVP) and 1% Triton × 100 was used. Extraction of the genomic DNA was performed through the following procedures: The extraction buffer (20 μL) was added in a 1.5 mL EP tube containing the sample powder. This tube was vortexed for 10–15 s followed by boiling for 10–15 s; 80 μL Tris-HCl (0.1 mol/L, pH 8.0) was then added into the mixture, vortexed gently and centrifuged for 2 min at 12,000 r/min; the supernatant was collected for later use. The quality and quantity of the exacted DNA samples were assessed using a NanoDrop 1000 UV/Vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and 1.2% agarose gel electrophoresis.

Tian E., Liu Q., Ye H., Li F, & Chao Z. (2017). A DNA Barcode-Based RPA Assay (BAR-RPA) for Rapid Identification of the Dry Root of Ficus hirta (Wuzhimaotao). Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(12), 2261.

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Circular Dichroism Spectroscopy of Proteins

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Circular dichroism (CD) spectrum of JS7 was collected on a Jasco J-815 spectrometer (DIY = 8 s, scan speed 20 nM/min, 10 iterations 178–260 nM) and interpreted with the CDSSTR program [53 (link),54 (link)], accessed via the Dichroweb server [55 (link)].
Protein/peptide concentrations were determined by direct measurement on a Nanodrop 1000 UV–vis spectrophotometer (Thermo Scientific). JS10 and JS7 (dissolved in water or PBS) were quantified based on calculated extinction coefficients at 280 nm. Peptides 1–3 were dissolved in water to approximately 10 mg/mL and then further dissolved to 5 mg/mL stock solution based on their absorbance at 230 nm.

Schein C.H., Levine C.B., McLellan S.L., Negi S.S., Braun W., Dreskin S.C., Anaya E.S, & Schmidt J. (2021). Synthetic proteins for COVID-19 diagnostics. Peptides, 143, 170583.

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RNA Extraction and Analysis for Bacterial Growth

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Total RNA was extracted from 10 mL of mid- to late exponential phase cultures of ATCC 29149 and ATCC 35913 in YCFA supplemented with one carbon source (Glc or pPGM). Four biological replicates were performed for each carbon source. The RNA was stabilized prior to extraction by adding 1/5 vol of phenol (pH 4.3) : ethanol (1:9) mixture to 1 vol of culture then incubating 30 min on ice and finally pelleting the cells for 5 min at 10,000 g at 4°C. Cell pellets were stored at −80°C before extraction. Extraction was performed using a traditional method using phenol and chloroform. Genomic DNA contamination was removed by DNAse treatment using TURBO DNA-free kit (Life Technologies Ltd, Paisley, UK) according to supplier's recommendations. The purity, quantity and integrity of the extracted RNA were assessed before and after DNase treatment, with NanoDrop 1000 UV-Vis Spectrophotometer (Thermo Fischer Scientific, Wilmington, DE) and with Agilent RNA 600 Nano kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Stockport, UK).

Crost E.H., Tailford L.E., Monestier M., Swarbreck D., Henrissat B., Crossman L.C, & Juge N. (2016). The mucin-degradation strategy of Ruminococcus gnavus: The importance of intramolecular trans-sialidases. Gut Microbes, 7(4), 302-312.

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