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Quant it picogreen assay kit

Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany

The Quant-iT PicoGreen Assay Kit is a fluorescence-based method for quantifying double-stranded DNA (dsDNA) in solution. The kit contains a proprietary PicoGreen dsDNA-binding dye that produces a fluorescent signal when bound to dsDNA. The intensity of the fluorescent signal is proportional to the dsDNA concentration, allowing for the quantification of dsDNA samples.

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42 protocols using quant it picogreen assay kit

1

Quantifying Cellular Proliferation with EdU and PicoGreen

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The Click-iT EdU Alexa Fluor 488 Imaging Kit (ThermoFisher Scientific) was used with a 17 h pulse of 5-ethynyl-2′-deoxyuridine (EdU) at 10 μM. Samples were then immunostained for DAPI as previously described. The Quant-iT PicoGreen Assay kit (ThermoFisher) was used to quantify the dsDNA content according to the manufacturer's instructions for the Quant-iT PicoGreen Assay kit (ThermoFisher).
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2

16S rRNA Gene Amplicon Sequencing

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Libraries were generated using barcoded PCR primers 27F 5′-AGAGTTTGATCCTGGCTCAG-3′ and 338R 5′-TGCTGCCTCCCGTAGGAGT-3′ annealing to the V1–V2 region of the 16S rRNA gene36 (link). PCR reactions were carried out in quadruplicate using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA). After amplification, quadruplicate PCR reactions were pooled and then purified using a 1:1 volume of SPRI beads (GE HealthCare, Chicago, IL). DNA in each sample was then quantified using Quant-iT PicoGreen Assay Kit (Thermo Fisher) and pooled in equal molar amounts. The resulting library was sequenced on the MiSeq instrument (Illumina, San Diego, CA) using 2 × 250 bp chemistry. Extraction blanks and DNA-free water were subjected to the same amplification and purification procedure to allow for empirical assessment of environmental and reagent contamination. Positive controls, consisting of eight artificial 16S gene fragments synthesized in gene blocks and combined in known abundances, were also included37 (link).
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3

Extracellular Matrix Extraction from S. davawensis

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Extraction method for extracellular matrix of S. davawensis was based on a previous study.47 The colonies were grown on a cellophane membrane placed onto MS medium. At each of the designated incubation time, the cellophane was peeled off and then soaked in 3 mL of 1.5 M NaCl solution for 5 min. The solution was transferred to a tube for centrifugation at 3000 × g for 3 min to remove cells. The resultant supernatant was used for further analyses. The treated cellophane was not used for any experiment. DNA concentration in the extract was quantified using Quant-iT PicoGreen assay kit (Thermo Fisher, MA, USA). For agarose gel electrophoresis, 400 μL of the extract was subjected to ethanol precipitation and then dissolved in 40 μL of 10 mM HEPES-NaOH (pH7.5). Saccharide content was quantified by phenol-sulphate method. One aliquot of 5% phenol was added to the extract, and then 5 aliquots of sulphate was added. After cooling, absorbance at 490 nm was measured. Standard curve of glucose was used for calculation of the saccharide content in the extract. Protein concentration was quantified using Pierce BCA protein assay kit (Thermo Fischer). Standard curve of bovine serum albumin was used for calculation of the protein content in the extract.
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4

Extracting DNA from FFPE Lung Tissue

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DNA was isolated from FFPE lung tissue using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Eight to ten 10 µm sections were pre-treated with xylene to remove the paraffin and with sodium thiocyanate to permeabilize the tissue. Incubation with (ATL) lysis buffer and proteinase K was extended to 36 h to allow complete lysis. DNA concentrations were measured with the Quant-iT Picogreen assay kit (ThermoFisher Scientific) according the manufacturer’s instructions.
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5

Quantifying DNA Content in Scaffolds

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Scaffolds were washed 3 times in PBS and freeze dried for 24 h. Each scaffold was incubated within an ultrasonic bath (6 litre Cavitek Digital, The Allendale Group, UK) in 300 µl of a 2.5 units/ml papain digest solution, 5 mM cysteine HCL and 5 mM EDTA in PBS (all reagents from Sigma Aldrich, UK) at 60 °C for 24 h with periodic sonication. Total DNA content of the samples was calculated using a Quant-iT™ PicoGreen® assay kit (ThermoFisher, UK) as per the manufacturers’ instructions. Fluorescence was read using a microplate reader at Ex 490 nm Em 510–570 nm, N = 4 independent replicates.
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6

Metagenomic Analysis of Infant Gut Microbiome

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The Qiagen DNeasy PowerSoil kit was used for DNA extraction from infant fecal samples. Extracted DNA was quantified with the Quant-iT PicoGreen Assay Kit (Thermo Fisher). The NexteraXT kit was used to generate DNA libraries for shotgun metagenomic sequencing. Extraction blanks and DNA-free water samples were prepared for sequencing as negative controls. A laboratory-generated DNA mock community of Vibrio campbellii and Lambda phage was included as a positive control. Sequencing was performed on the Illumina HiSeq 2500 instrument, generating paired-end 125 bp reads.
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7

Comprehensive Nucleic Acid Extraction and Analysis

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Cell line-derived DNA was extracted from freshly isolated cell pellets using the DNeasy Blood and Tissue kit (Qiagen) and eluted in TE buffer. DNA concentration was measured using a Quant-iT PicoGreen assay kit (ThermoFisher) and quality assessed using an Agilent Bioanalyzer 2100 and DNA 1000 kit (Agilent). A total of 250 ng DNA was submitted for DNA sequencing. A total of 1 μg DNA was submitted for DNA methylation analysis.
Total RNA including miR were extracted using the mirVana microRNA Isolation Kit (ThermoFisher) and eluted in nuclease-free water. RNA was quantitated using a Qubit fluorimeter and quality was assessed using a Fragment Analyzer (Advanced Analytical). Only analytes with an RNA Quality Number (RQN) ≥ 7.0 proceeded to RNA sequencing. For miR (small RNA) sequencing, total RNA quality was assessed using the Bioanalyzer 2100 and RNA6000 Nano assay (Agilent); all analytes had RIN ≥ 8.0, except for three cell lines; 2350 (RIN 7.9), 2417 (RIN 6.9) and 2734 (RIN 5.2). All samples proceeded to miR sequencing given that RIN values may be poorly reflective of sample miR quality76 (link).
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8

Quantifying miR-150 Nanoparticle Encapsulation

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The amount of miR-150-loaded in nanoparticles was determined as described previously.22 (link) In brief, the amount of un-entrapped miR-150 mimics in the nanoparticle wash solutions obtained during the formulation step was quantified using the Quant-iT™ PicoGreen Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence resulting from the binding of PicoGreen reagent to miR-150 was measured in a black-bottomed 96-well plate using FLx-800 microplate reader (BIO-TEK Instruments, Winooski, VT, USA). miR-150 loading in nanoparticles was determined by subtracting the total amount of miR-150 recovered in the wash solutions (miR-150w) from the initial amount of miR-150 (miR-150i) added. Encapsulation efficiency was calculated as:
(miR-150imiR-150w/miR-150i)×100
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9

LM-Seq Library Preparation and Sequencing

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One hundred nanograms of total RNA was used to prepare sequencing libraries following the Ligation Mediated Sequencing (LM-Seq) protocol41 (link) and quantified with a Qubit fluorometer (Life Technologies). Final cDNA libraries were quantitated with the Quant-iT PicoGreen Assay Kit (ThermoFisher Scientific), multiplexed, loaded at a final concentration of 1 nM or 2.5 nM, and sequenced as single reads on the NextSeq 2000 (Illumina), respectively.
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10

Long-range Whole Genome Sequencing

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Prior to 10X Genomics lrWGS library construction, genomic DNA samples were checked for fragment size distribution and were quantified. Genomic DNA fragment size distributions were determined with a Caliper Lab Chip GX (Perkin Elmer) to quantify DNA above 40 kb in length. Size selection was performed on 1.2 ug of genomic DNA with an 0.75% Agarose cassette on the Blue Pippin platform (Sage Science) with target specifications set to start at 40 kb and end at 80 kb. Samples were quantified using the Quant-it Picogreen assay kit (Thermo Fisher) on a Qubit 2.0 Fluorometer (Thermo Fisher) and normalized to a starting concentration of 1 ng/uL with TE (0.1 mM EDTA). Starting concentrations of 1 ng/uL were confirmed by picogreen and libraries were subsequently created in accordance with the 10X WGX protocol (10X Genomics). Library size was determined using the DNA 1000 Kit and 2100 BioAnalyzer (Agilent Technologies) and quantified using quantitative PCR (qPCR) (KAPA Library Quantification Kit, Kapa Biosystems). The finished WGX libraries were run on an Illumina HiSeqX platform at paired 151 bp reads with an eight-base single index read at the Broad Institute. Upon completion of sequencing, the resulting BCL files were processed by the Long Ranger Pipeline (10X Genomics) for alignment, variant discovery, and phasing.
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