Veriti 96 well thermocycler

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Veriti 96-well thermocycler is a laboratory instrument used for DNA amplification through the polymerase chain reaction (PCR) process. It provides precise temperature control and cycling capabilities to facilitate the essential steps of PCR, including denaturation, annealing, and extension.

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Thermocycler (175 citations) Veriti (149 citations) Veriti thermocycler (99 citations) Veriti 96 well (21 citations) 96-well thermocycler (6 citations) Veriti 96-well Fast Thermocycler (3 citations) Veriti 96 thermocycler (2 citations) Veriti® 96-Well Fast Thermal Cycler thermocycler (2 citations)

34 protocols using veriti 96 well thermocycler

Cellulose-Binding Enzyme Kinetics Assay

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Purified N. crassa CBH-1 enzyme at 15 µg/ml and MuLac (4-methylumbelliferyl β-d-lactopyranoside; Sigma) at a final concentration of 1 mM were incubated together in 0.05 M sodium acetate buffer (pH 5) in 50-µl volumes in VWR 12-well 0.2-ml PCR strip tubes. Strip tubes were incubated for 15 min across temperatures using Applied Biosystems Veriti 96-well thermocycler in veriflex mode. After 15 min, the temperature was raised to 95°C for 5 min to deactivate the enzyme. Reactants were transferred to Corning half-area-well 96-well clear bottom plates, and fluorescence was measured at 445 nm after excitation with 365 nm.
To assay TeCBH-1 from culture supernatant, MuLac (final concentration of 1 mM) was incubated with 25 µl of filtered culture supernatant for a final 50-µl volume in 0.05 M sodium acetate buffer (pH 5). Strip tubes were incubated using Applied Biosystems Veriti 96-well thermocycler in veriflex mode for 1 h and raised to 95°C for 5 min to deactivate the enzyme. The 445-nm emission/365-nm excitation was measured after incubation.

Wu V.W., Dana C.M., Iavarone A.T., Clark D.S, & Glass N.L. (2017). Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes. mBio, 8(1), e02231-16.

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DNA Barcoding of Muscle Samples

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Total DNA was extracted from muscle fragments following the protocol of the Canadian Center for DNA-Barcoding (CCDB), available at http://www.ccdb.ca. A segment of the 5' region of the mitochondrial COI gene was amplified using different combinations of primers, including L5698-Asn [65 (link)], FishF1, FishF2, FishR1 and FishR2 [15 (link)], C_FishF1t1–C_FishR1t1 cocktail [66 ], and H7271-COI [67 ]. Polymerase chain reactions (PCR) were run in a 12.5 μl volume containing: 1 μl DNA (concentration 50 ng/μl), 0.25 μl each of the forward and reverse primers (concentration 10 mM), 1.25 μl of reaction buffer, 0.2 μl of 200 mM dNTPs mix, 0.37 μl of MgCl₂ and 0.0625 μl (5 units/μl) of Platinum Taq DNA polymerase (Invitrogen).
The samples were amplified in a Veriti^® 96-well thermocycler (Applied Biosystems), with initial denaturation of 5 minutes at 96°C followed by 35 cycles at 96°C for 45 seconds, 54°C for 45 seconds, 72°C for 1 minute, and final extension at 72°C for 1 minute. The amplified PCR products were cleaned up with ExoSAP-IT (USB Corporation) and sequenced in both directions using the BigDye Terminator v3.1 Cycle Sequencing kit (Life Technologies) in an ABI3130 Genetic Analyzer automated sequencer (Applied Biosystems).

Rossini B.C., Oliveira C.A., de Melo F.A., Bertaco V.D., de Astarloa J.M., Rosso J.J., Foresti F, & Oliveira C. (2016). Highlighting Astyanax Species Diversity through DNA Barcoding. PLoS ONE, 11(12), e0167203.

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Detecting T. gondii and G. duodenalis DNA

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To detect DNA from T. gondii, a nested PCR was performed as described (Luna et al., 2019 (link); Triviño-Valencia et al., 2016 (link)). The first amplification used oligonucleotides (Toxo N1) 5′-GGAACTGCATCCGTTCATGAG-3′ and (Toxo C1) 5′-TCTTTAAAGCGTTCGTGGTC-3′. The nested PCR used the oligonucleotides (Toxo N2) 5′-TGCATAGGTTGCCAGTCACTG-3′ and (Toxo C2) 5′-GGCGACC AATCTGCGAATACACC-3′.
Conventional PCR was performed to detect the glutamate dehydrogenase (gdh) gene from assemblages A and B of G. duodenalis, with the GoTaq Green Master Mix kit, following manufacturer's recommendations. Oligonucleotide sequences were used, as described (Read et al., 2004 (link)): for Assemblage A, forward 5′-CCGCGAGATCGGGTACCTGTA-3′ and reverse 5′-GCCGGAGAC AGAGACACCG-3′ (637 bp) and for Assemblage B, forward 5′-ATCCTT AAGTTCCTCGGC-3′ and reverse 5′-ATCGGTTATCTGTTTGGAC-3′ (232 bp). The products were visualized in agarose gel at 1.5% by running 30 min at 100 V.
Amplifications were made in Veriti 96-well Thermocycler (Applied Biosystems, Waltham, MASS, USA). During each PCR assay, a negative control of the reaction was available that consisted in adding all the reagents, except for the DNA and a positive control that contained DNA from the parasite.

Pinto-Duarte V.A., Hérnandez-Arango N.M., Marin-Gallego B.J., Toloza-Beltrán P.A., Lora-Suarez F.M, & Gómez-Marín J.E. (2022). Detection of Giardia duodenalis and Toxoplasma gondii in soil and water samples in the Quindío River basin, Colombia. Food and Waterborne Parasitology, 28, e00175.

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Isolation and Conversion of Poly(A) mRNA from SCNT Blastocysts

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Poly(A) mRNA of the SCNT blastocysts was isolated using a Dyna-beads® mRNA
Direct™ kit according to the manufacturer’s protocol. The
cryopreserved embryo samples were melted and mixed with 40 μL of
Dynabeads oligo(dT)₂₅ and shaken for 8 min at room temperature to
induce hybridization of poly(A) mRNA tails with the oligo(dT)₂₅ on
the beads. The beads with attached mRNA were washed twice with each 100
μL of washing buffer A and B. Beads were separated from the supernatant
using a DynaMag™-Spin Magnet (Invitrogen, Carlsbad, CA, USA). Elution of
the poly(A) mRNA from the beads was carried out by incubation with 12.5
μL of 10 mM Tris-HCl (elution buffer) at 75℃ for 5 min. The cDNA
synthesis was carried out using AccuPower®RocketScript™ Cycle RT
Premix (Bioneer, Daejeon, Korea) according to the manufacturer’s
protocol. Each 10 μL of mRNA was used for a template. The reaction was
conducted by Veriti® 96-well Thermo cycler (Applied Biosystems, Foster
City, CA, USA) at 4℃ for 5 min, followed by 5 cycles at 37℃ for 15
sec, 50℃ for 5 min, and 98℃ for 5 min. The cDNA products were
conserved at 4℃ before use.

Kim M.J., Jung B.D., Park C.K, & Cheong H.T. (2021). Development of Porcine Somatic Cell Nuclear Transfer Embryos Following Treatment Time of Endoplasmic Reticulum Stress Inhibitor. Development & Reproduction, 25(1), 43-53.

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Genomic DNA Isolation and Duplex-PCR for nifH and nodC

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The bacterial isolates were grown in liquid YM medium for three days for the fast-growing isolates and six days for the slow-growing. An aliquot of 1 mL of each broth was used for the DNA extraction using the Wizard^® Genomic DNA Purification System (Promega, USA) according to the manufacturer's instructions. nifH and nodC genes were co-amplified in a duplex-PCR reaction as described by Fernandes Júnior et al.²⁴ For the nifH, the primers PolF (TGCGAYCCSAARGCBGACTC) and PolR (ATSGCCATCATYTCRCCGGA)²⁵ (link) were used. For nodC, the primers NodCF (AYGTHGTYGAYGACGGTTC) and NodCR(I) (CGYGACAGCCANTCKCTATTG)²⁶ (link) were applied. For a single isolate that showed positive amplification of the nifH amplicon, a complementary uniplex-PCR was performed with the primers nodCForB (CTCAATGTACACARNGCRTA) and nodCRevB (GAYATGGARTAYTGGYT)⁵⁷ (link) targeting the nodC amplification of β-rhizobia.
The reactions were performed in a Veriti 96-well thermocycler (Applied Biosystems, USA) and the PCR products were submitted to horizontal electrophoresis in agarose gel (0.8%, w/v). The gel was stained with Gel Red (Biotium) and visualized in a UV chamber. The bacterial isolates positive for both amplicons were selected for the next steps.

Rodrigues D.R., Silva A.F., Cavalcanti M.I., Escobar I.E., Fraiz A.C., Ribeiro P.R., Ferreira Neto R.A., Freitas A.D, & Fernandes-Júnior P.I. (2018). Phenotypic, genetic and symbiotic characterization of Erythrina velutina rhizobia from Caatinga dry forest. Brazilian Journal of Microbiology, 49(3), 503-512.

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Quantitative RNA Expression Analysis

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RNA isolations were performed using Qiagen Mini RNA isolation kit (Qiagen, #74136) for >500k cells or Qiagen micro RNA isolation kit (Qiagen, #74034) for <= 500k cells and RNA concentration was measured via NanoDrop (Thermo Scientific, Waltham, MA, USA).
cDNA synthesis was done using high capacity cDNA reverse Transcription kit (Applied Biosystems cat #4368813) and AppliedBiosystems Veriti 96-well thermocycler.
Q-PCR analysis was performed using TaqMan probes (ThermoFisher Scientific: EWS-FLI1 Hs03024497_ft, GAPDH Hs02786624_g1, RPLP0 Hs00420895_gH, ICAM-1 Hs00164932_m1, PD-L1 Hs00204257_m1, PD-L2 Hs00228839), Taqman Universal PCR Master Mix (Applied Biosystems, cat #4304437) and StepOnePlus Real-Time PCR system. RT-PCR experiments were conducted at a minimum in biological triplicates, with technical triplicates performed for each sample in an experiment.

Bailey K.M., Julian C.M., Klinghoffer A.N., Bernard H., Lucas P.C, & McAllister-Lucas L.M. (2019). EWS-FLI1 low Ewing sarcoma cells demonstrate decreased susceptibility to T-cell-mediated tumor cell apoptosis. Oncotarget, 10(36), 3385-3399.

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ISSR Genetic Diversity Analysis Protocol

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ISSR primers were synthesized by Macrogen (Seoul, South Korea) and Sigma (UK). A set of 43 ISSR primers were screened for reproducibility and suitability for genetic diversity analysis after optimization of primer annealing temperatures according to their Tm values. DNA was diluted to a working concentration of 20 ng/μl. Amplifications were performed in 25-μl volumes using PCR beads (GE Healthcare, Spain) [26 ] on a Veriti 96-well thermocycler (Applied Biosystems), with the tubes vortexed and briefly spun (≈10 s) after addition of all components. PCR products were separated on a 1.3% agarose gel in 1× Tris/Borate/EDTA buffer TBE containing 0.5 μg/ml ethidium bromide and electrophoresed at 5 V/cm. Gels were photographed using a Syngene bio-imaging gel documentation system (Ingenius).

Gaafar A.R., Al-Qurainy F, & Khan S. (2014). Assessment of genetic diversity in the endangered populations of Breonadia salicina (Rubiaceae) growing in The Kingdom of Saudi Arabia using inter-simple sequence repeat markers. BMC Genetics, 15, 109.

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4C-DpnII Library Preparation and Sequencing

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Each 3C library DNA sample was digested with Dpn II (NEB, R0543S). The 4C ligation was in 10 ml with 50 μl T4 DNA ligase (400 U/μl, NEB) at room temperature overnight. DNA was then ethanol precipitated and purified with the QIAquick PCR purification kit (Qiagen, 28106). The 4C-DpnII library was then amplified on the Veriti 96-Well Thermo Cycler (Applied Biosystems). Limited PCR (round-1) was conducted with the MYC-promoter region as the bait. The PCR products were purified using PCR purification kit and re-amplified by limited PCR (round-2) with primers containing Illumina sequencing adapters (Read 1 and Read 2 adapters, respectively). Primer sequences are in Supplementary Table S1. 4C library purification was performed using Ampure beads (Fisher, NC9959336) and then analyzed by deep sequencing (85 M PE150 reads per sample) using the Illumina platform (Genewiz Inc.).

Guo H., Wu Y., Nouri M., Spisak S., Russo J.W., Sowalsky A.G., Pomerantz M.M., Wei Z., Korthauer K., Seo J.H., Wang L., Arai S., Freedman M.L., He H.H., Chen S, & Balk S.P. (2021). Androgen receptor and MYC equilibration centralizes on developmental super-enhancer. Nature Communications, 12, 7308.

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Standard Molecular Cloning Techniques

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The cloning techniques used in this study were carried out using standard protocols (Ausubel et al., 1994 ). Plasmid DNA was isolated using the Plasmid Miniprep kit (Qiagen) and AscI and NdeI restriction enzymes were purchased from New England BioLabs. PCR reactions were performed in Veriti 96 Well Thermocycler (Applied Biosystems). Agarose gels were prepared using the multiSUB Midi electrophoresis unit, 10 cm × 10 cm UV Tray, 2 × 16 sample combs, loading guides, and dams. 1 or 2% agarose gels were run at 150 V for 25 min on the Fisherbrand multiSUB Midi Horizontal Gel System then stained for 30 min in 0.5 μg/ml ethidium bromide in ultrapure water. Bands were visualized using G:Box machine by SynGene and associated software. Lastly, samples were sent to Genewiz for Sanger Sequencing².

Blackburn S.A., Shepherd M, & Robinson G.K. (2021). Reciprocal Packaging of the Main Structural Proteins of Type 1 Fimbriae and Flagella in the Outer Membrane Vesicles of “Wild Type” Escherichia coli Strains. Frontiers in Microbiology, 12, 557455.

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RNA Isolation and Quantification from Mouse Muscle

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RNA was isolated from ∼50 mg of mouse quadriceps tissue in 1 mL of TRIzol (Invitrogen) using a TissueLyser (Qiagen) according to the manufacturer’s instructions. The RNA was resuspended into 50 µL DEPC treated water (Bioline). RNA was purified and DNAseI (Qiagen) treated using RNeasy Mini protocol (Qiagen). Quality and quantity was assessed using a 2100 Bioanalyser (Agilent Technologies) 6000 RNA kit (Agilent Technologies) according to the manufacturer’s instructions. One microgram (1 µg) of RNA was reverse transcribed using Super-Script™ III Reverse Transcriptase (Invitrogen), random pdN6 primers (Roche), 0.1 mM DTT (Invitrogen) and 10 mM dNTP (Invitrogen) in a 20 µL volume for 90 minutes at 50°C on a Veriti 96-well thermocycler (Applied Biosystems). All cDNA was diluted 1∶5 in DEPC treated water (Bioline) prior to RT-qPCR. Except for the Rn18S and Gapdh, the cDNA were diluted to 1∶100 due to the high abundance of these transcripts. Diluted cDNA was aliquoted into working volumes of 10 µl and stored at -80 °C until use. A maximum of three freeze-thaw cycles were performed for each aliquot.

Thomas K.C., Zheng X.F., Garces Suarez F., Raftery J.M., Quinlan K.G., Yang N., North K.N, & Houweling P.J. (2014). Evidence Based Selection of Commonly Used RT-qPCR Reference Genes for the Analysis of Mouse Skeletal Muscle. PLoS ONE, 9(2), e88653.

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