Nanodrop 3300

Manufactured by Thermo Fisher Scientific

Sourced in United States, Italy, United Kingdom, Germany, Belgium

The NanoDrop 3300 is a UV-Vis spectrophotometer that measures the absorbance of microscale sample volumes. It is designed to quantify and characterize a wide range of biomolecules, including nucleic acids, proteins, and other compounds. The NanoDrop 3300 utilizes a patented microvolume sample retention system that requires only 1-2 microliters of sample for analysis.

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Lab products found in correlation

125 protocols using nanodrop 3300

Frontal Cortex RNA Extraction Protocol

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Frontal cortex samples were removed from − 80 °C and placed in RNAlater (Ambion) which had been chilled to − 20 °C before being placed at 4 °C and allowed to thaw for 24 h under gentle agitation to assist in the perfusion of RNAlater through the tissue during thawing. The tissue was then further dissected to provide a small representative sample of superior frontal gyrus for RNA extraction.
RNA was extracted using the Qiagen RNeasy Midi Kit as per manufacturer's instructions. Samples were quantified using a Nanodrop 3300 (ThermoScientific). cDNA synthesis was carried out on 400 ng of RNA using the Affinity Script multi temperature cDNA synthesis kit (Agilent Technologies Part Number 200436) as per manufacturer's instructions. cDNA samples were also quantified using a Nanodrop 3300 (ThermoScientific) prior to PCR.

Brown S.M., Peters R, & Lawrence A.B. (2017). Up-regulation of IGF-1 in the frontal cortex of piglets exposed to an environmentally enriched arena. Physiology & Behavior, 173, 285-292.

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Quantification of CRH Gene Expression in Hair Follicles

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Total RNA from HFs was extracted using TRIzol, the RNA concentration was quantified (NanoDrop 3300; Fisher Scientific, Wilmington, DE, USA) and 1 μg of total RNA was reverse transcribed with the SuperScript First-Strand Synthesis System (Applied Biosystems, Foster City, CA, USA). Primers for real-time PCR amplification were synthesized by Integrated DNA Technologies Inc. (Coralville, IA, USA) according to earlier published PCR detection of the CRH gene (Table S1; see Supporting Information).^{45 (link)} The reaction was performed in triplicate with the SYBR Green I Master Mix (Roche, Manheim, Germany) on a Light Cycler 480 (Roche). The amount of amplified product for each gene was compared with that of β-actin using a comparative ΔΔCT method ± SD, and the data are presented as the fold change.

Fischer T.W., Bergmann A., Kruse N., Kleszczynski K., Skobowiat C., Slominski A.T, & Paus R. (2020). New effects of caffeine on corticotropin-releasing hormone (CRH)-induced stress along the intrafollicular classical hypothalamic–pituitary–adrenal (HPA) axis (CRH-R1/2, IP3-R, ACTH, MC-R2) and the neurogenic non-HPA axis (substance P, p75NTR and TrkA) in ex vivo human male androgenetic scalp hair follicles. The British journal of dermatology, 184(1), 96-110.

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Quantification of CRH Gene Expression in Hair Follicles

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DNA Methylation Profiling in ESCs

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Genomic DNA (gDNA) was purified using the standard phenol-chloroform method. The extracted gDNA was treated with RNAse (Roche) overnight at 37°C. Following another round of Phenol-Chloroform DNA extraction, gDNA was subjected to FspE1 (NEB, R0662S) digestion overnight at 37°C. The digested gDNA was purified by the Phenol-Chloroform method and quantified using the NanoDrop 3300 fluorospectrometer through PicoGreen dye according to the manufacturer’s protocol (Life Technologies, P11495). Quantitative PCR was performed using an equal amount of DNA for each sample. The change in DNA methylation is represented by relative fold change in the Cq value as follows: 2^(Cq(U)-Cq(I)), where Cq(U) is the Cq for the undifferentiated ESC sample, and Cq(I) represents day 3 or day 7 differentiated ESCs. The primers used for DNA methylation-dependent qPCR analysis have been previously described [30]. Standard deviations represent three technical and two biological replicates.
DNA Methylation-Dependent Restriction digest (MDR): Purified genomic DNA was subjected to methylation-sensitive restriction by Hpa II and methylation-insensitive restriction by Msp I overnight at 37°C. Samples were loaded on 0.8% Agarose gel in TAE buffer and bands visualized by Ethidium bromide staining. A smear in the Hpa II digestion lane indicates global loss of DNA methylation.

Miller W.G., Bates A.H., Horn S.T., Brandl M.T., Wachtel M.R, & Mandrell R.E. (2000). Detection on Surfaces and in Caco-2 Cells of Campylobacter jejuni Cells Transformed with New gfp, yfp, and cfp Marker Plasmids. Applied and Environmental Microbiology, 66(12), 5426-5436.

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Quantification of Osteogenic Gene Expression

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A quantitative real time polymerase chain reaction (qRT-PCR) was utilized to quantify the presence of specific gene markers indicative of osteogenesis. The genes of interest and their primer sequences are outlined in Table 3. At 5 weeks of culture, samples (n = 3) were removed from media and frozen at −80°C. Total RNA extraction was completed using a TRIzol-based assay. Each scaffold was dissolved in 300 μl TRIzol and centrifuged at 13,000 rpm for 30 seconds. TRIzol solutions were then transferred to new RNA-free microcentrifuge tubes and mixed with a half volume of isopropanol through manual pipette agitation before proceeding with the Zymo Research Microprep Kit instructions (Zymo Research, Direct-zol RNA Microprep Kit, R2062). Isolated RNA concentrations were then quantified (NanoDrop 3300) and reverse-transcribed into cDNA using Superscript IV VILO Mastermix Reverse Transcriptase (Life Technologies, 11756500).
qRT-PCR was performed using a final concentration of 0.4 ng/μl cDNA, 1 μM forward and reverse primers, and 1X PowerTrack SYBR Green PCR Master Mix (Life Technologies, A46109). Melt curves were assessed on four samples to verify the suitability of each primer set. Relative gene expression was determined using the ΔΔCT method with GAPDH serving as the housekeeping gene and PCL scaffolds incubated in basal media as the reference control.

Fainor M., Mahindroo S., Betz K.R., Augustin J., Smith H.E., Mauck R.L, & Gullbrand S.E. (2023). A tunable calcium phosphate coating to drive in vivo osseointegration of composite engineered tissues. Cells, tissues, organs, 212(5), 383-398.

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RNA Extraction and qRT-PCR for SDH Genes

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Extraction of total RNA from 100mg of powder was performed using the RNeasy Plant Mini kit (Qiagen, Hilden, Germany). The concentration of total RNA isolated from berries was quantified with Nanodrop 3300 (ThermoScientific, Wilmington, USA). One µg of isolated RNA was used for reverse transcription using ImProm-II^TM Reverse Transcription System (Promega) in a total volume of 20 µl. One µl of synthesized cDNA was amplified in triplicate by quantitative real time PCR using specific primers of SDH genes (Supplementary Table S1) and the Power SYBER-Green PCR Master-kit (Applied Biosystems, Applera France, Courtaboeuf, France). PCR was carried out with the 7 300 Real-Time PCR System (Applied Biosystems) and analysed with 7 300 System SDS Software v 1.3.1.
To normalize VvSDH expression, Elongation factor 1α (EF1α) was used as the reference gene (Monteiro et al., 2013 (link)). Relative SDH expression was calculated from the cycle threshold (Ct) according to formula 2 exp-(Ct_SDH–Ct_EF1α).

Bontpart T., Marlin T., Vialet S., Guiraud J.L., Pinasseau L., Meudec E., Sommerer N., Cheynier V, & Terrier N. (2016). Two shikimate dehydrogenases, VvSDH3 and VvSDH4, are involved in gallic acid biosynthesis in grapevine. Journal of Experimental Botany, 67(11), 3537-3550.

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Microbial Community Composition in Anaerobic Digestate

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As already described in Gazzola et al. (2022) (link), anaerobic digestate samples were collected at different sampling times to perform the analysis of microbial community composition over the reactor operation. A small aliquot (2 mL) was centrifuged at 15,000 rpm for 2 min, the resulting pellet was immediately stored at −20°C and used for DNA extraction with DNeasy PowerSoil Pro Kit (QIAGEN - Germantown, MD) following the manufacturer’s instructions. After checked the quality (1.6 < A260/280 < 1.8 and A260/230 > 2) with a Nanodrop 3,300 (Thermo Scientific, Italy), the genomic DNA was used for the high-throughput sequencing of 16S rRNA gene. The V1-V3 region of bacterial 16S rRNA gene (27F 5′- AGAGTTTGATCCTGGCTCAG-3; 534R 5-ATTACCGCGGCTGCTGG-3) was sequenced following library preparation and protocol described in Crognale et al. (2019) (link). The samples were paired end sequenced (2 × 301bp) on a MiSeq platform (Illumina) using a MiSeq Reagent kit v3, 600 cycles (Illumina, United States) following the standard guidelines for preparing and loading samples, with 20% of Phix control library. Bioinformatic analysis were performed using QIIME2 v. 2018.2 (Bolyen et al., 2019 (link)) as described in Crognale et al. (2021) (link). The Dataset is available through the Sequence Read Archive (SRA) under accession PRJNA1052454.

Gallipoli A., Angelini F., Angelini S., Braguglia C.M., Montecchio D., Tonanzi B, & Gianico A. (2024). Thermally enhanced solid–liquid separation process in food waste biorefinery: modelling the anaerobic digestion of solid residues. Frontiers in Bioengineering and Biotechnology, 12, 1343396.

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Constructing Bacterial Mock Communities

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The mock communities used in this study were reported previously (71 (link)). Briefly, 16S rRNA gene sequences from 33 bacterial strains, belonging to 27 different phyla, were used to construct the mock communities (Table S11; File S1) (71 (link)). Clones of near full-length 16S rRNA gene fragments were generated using PGEM-T Easy Vector II system (Promega, Inc., Madison, WI). The plasmid concentrations were quantified in triplicate using Quant-iT dsDNA assay kit (Invitrogen, Carlsbad, CA) on a Nanodrop 3300 (Thermo Scientific, Wilmington, DE). The sequences were divided into three clusters mostly (with a few exceptions) based on GC content within the V3–V5 region of the 16S rRNA genes and, considering the strain diversity at the phylum level, the GC content of V4 region: low (51.16% ± 2.1%), moderate (55.1% ± 1.8%), and high (59.3% ± 3.3%), with 11 in each cluster to assess whether overall community GC content of the target sequences causes biases. The sequences of the three clusters were then distributed into three mock communities in equal (3.03%) abundance in bacterial mock community 1 (Bm1) and in a combination of low (0.01%), moderate (0.67%), and high (8.41%) abundance in two different allotments (Bm2 and Bm3) (Table S11). All mock communities had a 16S rRNA gene concentration of 10⁹ copies/µL.

Qin Y., Wu L., Zhang Q., Wen C., Van Nostrand J.D., Ning D., Raskin L., Pinto A, & Zhou J. (2023). Effects of error, chimera, bias, and GC content on the accuracy of amplicon sequencing. mSystems, 8(6), e01025-23.

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Rat Wound Granulation Tissue RNA Isolation and Analysis

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Total RNA content was isolated from fresh wound granulation tissue of rats, homogenized using the TRIzol reagent (10296010, Thermo Fisher Scientific, Rockford, IL, USA) (1 mL per 100 mg tissue), and then mixed with chloroform (200 μL) and centrifuged at 4°C and 12000 g for 10 min. Next, the upper aqueous phase was collected, and mixed with isopropanol (500 μL) to precipitate RNA. Subsequently, the RNA was dissolved in RNA enzyme-free water (10-30 μL) and quantified by Nanodrop (Nanodrop 3300, Thermo Fisher Scientific, Rockford, IL, USA). One μg of total RNA was reverse-transcribed with the TaqMan reverse transcription reagent (N8080234, Thermo Fisher Scientific, Rockford, IL, USA). PCR analysis was performed using the PowerUp SYBR Green premix kit (A25741, Thermo Fisher Scientific, Rockford, IL, USA). Primer sequences are illustrated in Supplementary Table 1.

Zhao Q., Xu J., Han X., Zhang Z., Qu J, & Cheng Z. (2023). Growth differentiation factor 10 induces angiogenesis to promote wound healing in rats with diabetic foot ulcers by activating TGF-β1/Smad3 signaling pathway. Frontiers in Endocrinology, 13, 1013018.

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Quantification of Gene Expression

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After treatment, total RNA was extracted by EZ-press RNA purification kit (Roseville, MN, USA) and RNA concentration was determined with NanoDrop 3300 (Thermo Fisher Scientific). cDNA was synthesized from 1 μg of total RNA using HiScript III 1st strand cDNA synthesis kit (Vazyme, Nanjing, China). The qPCR analysis was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme). The contents of different mRNA targets in different groups were calculated by ^ΔΔCt method. Primers were synthesized by Sangon Biotech (Sangon Biotech, Shanghai, China). Primers used in the experiments were as follows: human-p16-F (5′-GGGGGCACCAGAGGCAGT-3′), human-p16-R (5′-GGTTGTGGCGGGGGCAGTT-3′), human-p21-F (5′-TCCTCATCCCGTGTTCTCCT-3′), human-p21-R (5′-CACCCTGCCCAACCTTAGAG-3′), human-GAPDH-F (5′-GGACCTGACCTGCCGTCTAGAA-3′), human-GAPDH-R (5′-GGTGTCGCTGTTGAAGTCAGAG-3′).

Ren C., Hu C., Wu Y., Li T., Zou A., Yu D., Shen T., Cai W, & Yu J. (2022). Nicotinamide Mononucleotide Ameliorates Cellular Senescence and Inflammation Caused by Sodium Iodate in RPE. Oxidative Medicine and Cellular Longevity, 2022, 5961123.

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