Faecal and salivary microbiota compositions were profiled by sequencing the hypervariable V3–V4 regions of the 16S rRNA gene. Sequencing was performed by LifeSequencing S.L. (Valencia, Spain) on an Illumina MiSeq instrument (San Diego, California, USA). The V3–V4 region was PCR-amplified with universal primers S-D-Bact-0341-b-S-17 primer (forward 5′-CCTACGGGNGGCWGCAG-3′) and S-D-Bact-0785-a-A-21 primer (reverse 5′-GACTACHVGGGTATCTAATCC-3′) [20 (link)] designed for dual indexing following the Illumina 16S Metagenomic Sequencing Library Preparation protocol (Part # 15044223 Rev. B). In brief, PCR amplification was performed in two steps: (1) in a first step, the V3–V4 region was amplified with the addition of universal adaptors to the amplification products. All amplicons were purified (AMPure XP, Beckman, Danvers, MA) to remove short amplification products and quantified using the Quant-iT PicoGreen dsDNA kit (Invitrogen, Carlsbad, California, USA). (2) In the second PCR step, the amplicons from the first step were amplified by targeting the universal adapters and with the addition of sample specific indexes and sequencing adaptors. The final amplicons were purified (AMPure XP) and quantified using the Quant-iT PicoGreen ds DNA kit (Invitrogen). All samples were pooled in equal amounts and sequenced in a 300 bp paired-end mode.
Quant it picogreen dsdna kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Ireland, Germany, China
The Quant-iT PicoGreen dsDNA kit is a fluorescence-based assay used to quantify double-stranded DNA (dsDNA) in solution. It provides a sensitive and accurate way to measure dsDNA concentration.
Automatically generated - may contain errors
Lab products found in correlation
Papain, by Merck Group (6 mentions)
Miseq system, by Illumina (5 mentions)
Infinite 200 microplate reader, by Tecan (5 mentions)
Synergy ht, by Agilent Technologies (5 mentions)
Cell death detection elisaplus kit, by Roche (4 mentions)
Human calprotectin elisa kit, by Hycult Biotech (4 mentions)
Ampure xp, by Beckman Coulter (3 mentions)
Bioruptor, by Diagenode (3 mentions)
Powersoil dna isolation kit, by Qiagen (3 mentions)
Dq 300 fluorometer, by Hoefer (3 mentions)
Uric acid assay kit, by Abcam (3 mentions)
Agencourt ampure xp bead, by Beckman Coulter (3 mentions)
Synergy 2, by Agilent Technologies (3 mentions)
Miseq platform, by Illumina (3 mentions)
Spectramax m5 plate reader, by Molecular Devices (3 mentions)
Nextera dna sample preparation kit, by Illumina (3 mentions)
Hiseq sequencer, by Illumina (3 mentions)
Human mpo elisa kit, by Abnova (3 mentions)
Powermag microbiome rna dna isolation kit, by Qiagen (3 mentions)
Miseq instrument, by Illumina (3 mentions)
207 protocols using quant it picogreen dsdna kit
1
16S rRNA Microbiome Sequencing Protocol
2
Quantifying Cartilage Proteoglycan Content
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteoglycan content in cartilaginous tissue was measured by dimethylmethylene Blue (DMMB) assay, as described before (Melnik et al., 2019 (link)), and the values were normalized to DNA amount in lysed cells measured with Quant-iT PicoGreen dsDNA kit (Invitrogen, Eugene, United States). For this, 20 μl of the digested pellet sample were mixed with 80 μl TE buffer (200 mM Tris HCl, 20 mM EDTA) and PicoGreen solution, and fluorescence in samples was measured at 485/535 nm.
3
Quantifying Cell Proliferation in 3D Scaffolds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell proliferation was determined using Quant-iT™ PicoGreen ® dsDNA Kit (Invitrogen, #P11496) at day 0 (4 h post-seeding) 3, 6 and 9 days (n = 4). Scaffolds were collected in 1.5 mL Eppendorf tubes and frozen at −80 °C immediately after harvesting. To extract DNA from frozen samples, we used a 1 mL of digestion solution composed to each seeded scaffold. Digestion solution: Papain 125 μg/mL (Sigma-Aldrich, #P4762), l -Cysteine 0.242 mg/mL (Sigma-Aldrich, #C1276) and EDTA 0.33 M (Sigma-Aldrich, #E6511) in sterile DPBS (GibcoTM, #14190). Samples were digested in a 1.5 mL eppendorf tube at 65 °C overnight. The following day, samples were completely disaggregated by vortexing, and centrifuged 1 min. Briefly, DNA samples were diluted 1:10 in 1×TE buffer. 100 μL of diluted sample were incubated with 100 μL PicoGreen® reagent (1:1) and incubated 5 min using dark flat-bottomed 96-well plate. The fluorescence was read at excitation 485 nm and emission 525 nm and was compared to a DNA standard curve.
4
DNA Quantification for Cell Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proliferation was assessed by DNA quantification. At each time interval, the constructs were washed in PBS 1x (Gibco™, Waltham, MA, USA) and stored frozen at −80 °C until analysis. For DNA quantification, the constructs were digested in 20 UI/mL Papain solution (Worthington Biochemical Corporation, Lakewood, NJ, USA) at 65 °C for 1 h and total DNA (ng DNA/mg construct) was determined using a Quant-iT Pico Green® dsDNA kit (P7581; InvitrogenTM; Waltham, MA, USA), following the manufacturer’s instructions.
5
Comprehensive Characterization of MSC-Laden Hydrogels
6
Chickpea Genetic Diversity Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A population of 129 F10 recombinant inbred lines (RILs) from a cross between C. arietinum var. ICC4958 (desi, Fusarium wilt resistant and drought tolerant) and C. reticulatum var. PI489777 (wild, Fusarium wilt susceptible) was utilized. This is an internationally accepted, reference mapping population, developed at the Washington State University, USDA, USA. Genomic DNA was isolated from fresh young leaf tissues of the two mapping parents and the RILs using the GenEluteTM plant genomic DNA Miniprep kit (Sigma). DNA quality was checked by electrophoresis on 0.8% agarose-gels. For high-throughput SNP genotyping, the DNA was quantified using Quant-iT™ Pico Green® dsDNA Kit (Invitrogen) and the fluorescence was measured with Microtiter plate reader (Varioscan from Thermo Scientific).
7
DNA Quantification and Integrity Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The amount of DNA recovered from each sample processed via the four methods described above (Fig 1.3, 1.8, 1.13 and 1.17 ) was quantified with the Quant-iT PicoGreen dsDNA Kit (Invitrogen). DNA integrity and fragment size in samples was determined by gel electrophoresis in a 1% (w/v) agarose (Promega) gel stained with 0.4 μg/mL ethidium bromide. A 100 bp DNA ladder (Promega) was run alongside both samples as a molecular weight marker. DNA was visualised under UV illumination using a Bio-Rad Gel Doc 1000 (Bio-Rad) and photographed using a Kodak DC90 Zoom Digital Camera (Kodak) in conjunction with the Kodak 1D v3.6 image analysis software.
8
ChIP-seq Protocol for Transcription Factor Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For ChIP-seq 108 asynchronously growing cells were crosslinked with 1 % formaldehyde for 10 min at room temperature, followed by quenching with 125 mM glycine for 10 min, washed twice with 1× phosphate buffered saline (PBS), and resuspended in chromatin immunoprecipitation (ChIP) lysis buffer (150 mM NaCl, 1 % Triton X‐100, 0.1 % SDS, 20 mM Tris–HCl pH8.0, 2 mM EDTA). Chromatin was sheared to an average length of 200–500 bp using a Bioruptor sonicator. After overnight incubation with DiaMag magnetic beads (Diagenode, Inc.) and CTCF or BORIS monoclonal or polyclonal antibodies (characterized and described by us [29 (link), 32 (link), 39 (link)]), precipitated chromatin was then washed, de-crosslinked, and digested with proteinase K. The resulting DNA was purified using phenol/chloroform/isoamyl alcohol. DNA concentration was assessed with a Quant‐it PicoGreen dsDNA kit (Invitrogen) and 5–10 ng was used to generate sequencing libraries. ChIP DNA was amplified using a TruSeq ChIP Sample Preparation Kit (Illumina, Inc., USA). Briefly, the immunoprecipitated material was end-repaired, A-tailed, ligated to the sequencing adapters, amplified by 15 cycles of PCR, and size selected (200–400 bp) followed by single end sequencing on an Illumina Genome Analyzer according to the manufacturer’s recommendations.
9
Mitochondrial Respiration Analysis in Adipocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SGBS cells or primary human preadipocytes were differentiated in XF96-PS plates (Seahorse Bioscience, North Billerica MA, USA). After stimulation for 48 h as described in Section 2.4 , cells were washed with XF assay medium containing 5 mM glucose (pH adjusted to 7.5), incubated for 1 h in 37 °C air incubator in fresh XF assay medium containing 5 mM glucose and subjected to respiratory and extracellular acidification analysis as previously described [42] (link). The following injections and final concentrations were used: oligomycin (1 μg/ml), the chemical uncoupler FCCP (0.5 μM), rotenone (4 μM), antimycin A (2 μM) and 2-deoxy-glucose (100 mM). Seahorse data were normalized to DAPI or Quant-iT™ PicoGreen® dsDNA Kit (Invitrogen) as a surrogate for cell number per well.
10
Mapping Transgene Insertion Sites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed whole-genome sequencing to identify the insertion-sites of transgene in the mouse genome. Paired-end 150 bp-long sequencing reads were aligned to a modified version of the reference genome (GRCm38/mm10) using the bowtie aligner (bowtie-bio.sourceforge.net). The reference was modified to include a copy of the original cloning vector. Sequencing reads that uniquely mapped both to the mouse genome and to the cloning vector sequence were used to localize the insertion sites. Raw-sequencing reads in the form of.fastq files were deposited in the European Nucleotide Archive with the accession (E-MTAB-5502). To generate the library, DNA was extracted from liver using the DNA Mini kit (Qiagen) and fragmented using Bioruptor (Diagenode). Briefly, 380 ng DNA in 100 microL in 500 microL tubes were subjected to sonication with the following parameters: power setting low, cycle 30 sec on/90 sec off, time 8 minutes in ice-cold, degassed water, resulting in average 550 bp size fragments. The library was prepared using the TruSeq Nano DNA kit (Illumina). DNA and library quantifications were performed with the Quant-iT PicoGreen dsDNA Kit (Invitrogen). Library quality was assessed by using the High sensitivity Bioanalyzer kit (Agilent).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!