Nanodrop

Manufactured by Agilent Technologies

Sourced in United States, Germany, Italy

The Nanodrop is a compact and efficient spectrophotometer designed for the measurement of small volume samples. It allows for the quantification and analysis of DNA, RNA, and protein concentrations using only 1-2 microliters of sample.

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Nanodrop 2100 spectrophotometer Nanodrop 2000c spectrophotometer Nanodrop N-1000 NanoDrop 1000 spectrophotometer NanoDrop 8000 NanoDrop® ND-1000 UV spectrophotometer Epoch 2 nanodrop reader NanoDrop 8000 Spectrophotometer NanoDrop® ND-100 spectrophotometer Nanodrop-Synergy H1 Multi-Mode Reader

270 protocols using nanodrop

Pancreatic Tumor Cell Line and Tissue Analysis

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The MIA PaCa2, HPAC, and PANC-1 cell lines were obtained as frozen aliquots from ATCC (http://www.atcc.org/). A total of six de-identified pancreatic tumor frozen specimens were available for this study through an IRB approved tissue banking protocol. Genomic DNA and total RNA were isolated using Omega BioTek (http://www.omegabiotek.com/) chemistries according to the manufacturer's protocols. DNA was quantitated using NanoDrop and Qubit, and RNA was quantitated using NanoDrop and Agilent BioAnalyzer.

Goecks J., El-Rayes B.F., Maithel S.K., Khoury H.J., Taylor J, & Rossi M.R. (2015). Open pipelines for integrated tumor genome profiles reveal differences between pancreatic cancer tumors and cell lines. Cancer Medicine, 4(3), 392-403.

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Genomic DNA and RNA Isolation

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Genomic DNA and total RNA were isolated using Omega BioTek chemistries according to the manufacturer’s protocol from 5 μM sections for each isolate. DNA was quantitated using NanoDrop and Qubit, and RNA was quantitated using NanoDrop and Agilent BioAnalyzer.

Saba N.F., Dinasarapu A.R., Magliocca K.R., Dwivedi B., Seby S., Qin Z.S., Patel M., Griffith C.C., Wang X., El-Deiry M., Steuer C.E., Kowalski J., Shin D.M., Zwick M.E, & Chen Z.G. (2020). Signatures of somatic mutations and gene expression from p16INK4A positive head and neck squamous cell carcinomas (HNSCC). PLoS ONE, 15(9), e0238497.

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RNA Isolation, cDNA Synthesis, and qRT-PCR Analysis

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Total RNA was isolated from cells using trizol reagent (Invitrogen, Carlsbad, CA, United States) and RNA purity was determined by NanoDrop spectrophotometer (BioTek, Winooski, VT, United States). Further, 2 μg RNA was used for cDNA synthesis using maxima first cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s protocol. Briefly, RNA was treated with Maxima Reverse Transcriptase enzyme and reaction buffer. Thereafter, RT-PCR and analysis were done on ABI 7500 Fast real-time PCR system (Waltham, MA, United States) at cycling conditions as: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 30 s at 60°C, and 60 s at 72°C. The analysis was done using comparative Ct method. β-actin was used as an internal control (Khan et al., 2016 (link)). Results are normalized to β-actin and depicted as relative expression (fold change). Primers used in the qRT-PCR are as follows:

Negi S., Pahari S., Das D.K., Khan N, & Agrewala J.N. (2019). Curdlan Limits Mycobacterium tuberculosis Survival Through STAT-1 Regulated Nitric Oxide Production. Frontiers in Microbiology, 10, 1173.

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DNA Extraction and Purity Assessment from Vegetable Oils

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Before conducting the PCR reaction on the extracted DNA from crude and refined soybean, corn, and canola oils, the quality of the extracted DNA was assessed using agarose gel electrophoresis. The results are presented in Fig. 1.Fig. 1

Agarose gel electrophoresis of genomic DNA extracted from crude Soybean Oil. Well 1: DNA extraction using the CTAB method, wells 2 and 3: DNA extracted using the MBST kit, well 4: DNA extracted using the manual hexane-based method, M: Molecular marker.

To quantify the amount of extracted DNA from oil, a NanoDrop device (BioTek Epoch, USA) was utilized. Using this instrument, DNA absorbance at 260 nm (nucleic acid absorption wavelength) and 280 nm (protein absorption wavelength) was measured, and ultimately, the A₂₆₀/A₂₈₀ absorption ratio, which is an indicator of DNA purity, was obtained.

Vahdani M., Sahari M.A, & Tanavar M. (2024). Quantitative and qualitative analysis of three DNA extraction methods from soybean, maize, and canola oils and investigation of the presence of genetically modified organisms (GMOs). Food Chemistry: Molecular Sciences, 8, 100201.

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Quantitative gene expression analysis

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RNA was extracted using ISOLATE-II RNA MiniKits (Bioline, London, UK) with on-column DNase treatment. RNA was quantified using a Nanodrop (BioTek, Winooski, VT), and reverse-transcribed using a SensiFAST cDNA synthesis kit (Bioline). cDNA was amplified in triplicate with gene-specific primers (Invitrogen) using a CFX384 real-time PCR machine (BioRad). Thermal cycling conditions were 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Data were analysed using the ΔΔCt method with expression normalized to the housekeeping gene and sham-operated animals used as the referent controls.

El-Rashid M., Nguyen-Ngo D., Minhas N., Meijles D.N., Li J., Ghimire K., Julovi S, & Rogers N.M. (2020). Repurposing of metformin and colchicine reveals differential modulation of acute and chronic kidney injury. Scientific Reports, 10, 21968.

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Fecal DNA Extraction Protocol

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After microbiological analysis, the fecal samples were stored at −80°C until DNA extraction. Samples collected from each subject on different days (n = 9; T0–T8) were thawed on ice and mixed vigorously for 2–3 min with a sterile spatula; then, 250 mg of each sample was weighed and processed with a DNeasy® PowerLyzer® PowerSoil® Kit (Qiagen, Hilden, Germany) with the following modifications: tubes containing samples were incubated at 65°C for 10 min after addition of solution C1. Before extraction, mechanical lysis of the cells was carried out using a Precellys 24 bead homogenizer (Bertin Technologies, Montigny le Bretonneux, France). Then, the extraction was conducted according to the manufacturer’s specifications. The DNA extracted from fecal samples was quantified by using a NanoDrop (BioTek Instruments, Inc., CA, United States). Finally, the DNA was stored at −80°C until molecular analysis.

Arioli S., Koirala R., Taverniti V., Fiore W, & Guglielmetti S. (2018). Quantitative Recovery of Viable Lactobacillus paracasei CNCM I-1572 (L. casei DG®) After Gastrointestinal Passage in Healthy Adults. Frontiers in Microbiology, 9, 1720.

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Fecal DNA Extraction and Real-Time PCR

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Stool samples were collected from eligible participants and stored at −80 °C in an ultra-freezer. For molecular tests, DNA was extracted from faecal by Sambio™ Stool DNA Extraction Kit (Azma gene company, Iran) according to the manufacturer's recommendation. The purity of DNA was determined by UV methods (NanoDrop, Epoch, BioTek, USA). The DNA concentration was normalized for all samples to 30 ng/μL and stored at −20 °C until to do the Real-Time PCR assay.

Nabizadeh E., Sadeghi J., Rezaee M.A., Hamishehkar H., Hasani A., Kafil H.S., Sharifi Y., Asnaashari S., Kadkhoda H, & Ghotaslou R. (2023). The profile of key gut microbiota members and short-chain fatty acids in patients with sepsis. Heliyon, 9(7), e17880.

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Colorectal Cancer Tumor Profiling

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Patient samples were scrutinised following the set criteria. A total of 15 CRC tumors with paired normal colorectal mucosa samples were collected from RGCIRC (Rajiv Gandhi Cancer Institute and Research Centre), India, from 2016 to 2019. It was ensured that the tumor was sporadic and the patient had not received chemotherapy or radiotherapy before surgery. The pathologist ensured the total oncogenic area of cancerous cells was not less than 80%. Informed consent was obtained from each patient. The ethical approval of the study was approved by the Institute Ethics Committee, Motilal Nehru National Institute of Technology Allahabad (Ref. No. IEC17-18/027). Total RNA was extracted from tumor tissues using RNeasy Mini Kit (Qiagen, cat no.74104), and RNA concentration and quality were estimated using nanodrop (BioTek Take3). The cDNA preparation and RT-PCR analysis were done as described above.

Saini K.K., Chaturvedi P., Sinha A., Singh M.P., Khan M.A., Verma A., Nengroo M.A., Satrusal S.R., Meena S., Singh A., Srivastava S., Sarkar J, & Datta D. (2023). Loss of PERK function promotes ferroptosis by downregulating SLC7A11 (System Xc⁻) in colorectal cancer. Redox Biology, 65, 102833.

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16S Metagenomic Sequencing Workflow

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Each sequenced sample was prepared according to the Illumina 16S Metagenomic Sequencing Library protocol to amplify the V3 and V4 regions (519F-806R). DNA quality was measured using PicoGreen reagent and Nanodrop (BioTek, Winooski, VT, USA). Input gDNA (10 ng) was used for PCR amplification. The barcoded fusion primer sequences used for PCR amplifications were as follows: 519F, 5′-CCTACGGGNGGCWGCAG-3′; 806R, 5′-GACTACHVGGGTATCTAATCC-3′. Then the final purified product was quantified by quantitative PCR using a KAPA Library Quantification kit for Illumina Sequencing platforms (Roche, Mannheim, Germany) according to the manufacturer’s protocol, and the quality was assessed using a LabChip GX HT DNA High Sensitivity Kit (PerkinElmer, Waltham, MA, USA). Next, paired-end (2 × 300 bp) sequencing was performed using the MiSeq platform (Illumina, San Diego, CA, USA).

Oh S., Lee H.J, & Park K.U. (2023). Metagenomic characterization of the microbiomes in five different body habitats of otherwise healthy individuals with periodontal disease. Frontiers in Cellular and Infection Microbiology, 13, 1257816.

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ODE Sample DNA Removal Protocol

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In order to eliminate any interference by the prokaryotic or eukaryotic DNA that may be present in 0.2 μm filtered ODE samples, we performed the DNase treatment. Total RNA in an ODE sample was measured using NanoDrop (Biotek synergy 2). Then RNA concentration was used as a criterion for the amount of DNase (Turbo DNase, Thermo Fisher Scientific catalog # AM2239) to be added in 50 μL volume of 100% ODE to remove any traces of DNA effectively.

Massey N., Shrestha D., Bhat S.M., Kondru N., Charli A., Karriker L.A., Kanthasamy A.G, & Charavaryamath C. (2021). Organic dust induced mitochondrial dysfunction could be targeted via cGAS-STING or cytoplasmic NOX-2 inhibition using microglial cells and brain slice culture models. Cell and tissue research, 384(2), 465-486.

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