Veriti 96 well thermal cycler

Manufactured by Thermo Fisher Scientific

Sourced in United States, Singapore, Japan, Germany, Australia, Canada, France, China, Switzerland

The Veriti 96-Well Thermal Cycler is a laboratory instrument used for the amplification of DNA samples. It provides precise temperature control and cycling capabilities to facilitate polymerase chain reaction (PCR) processes.

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593 protocols using veriti 96 well thermal cycler

Basal and Induced CDK6 Thermal Stability Assay

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For basal CDK6 thermal stability assay, cells resuspended in 1 ml PBS containing protease inhibitor cocktail (Roche) were aliquoted with 100 μl cell suspension each into nine PCR tubes (~ 3 x 10⁶ cells). Heat the PCR tubes at different temperature endpoints (35-59 °C) for 3 min in the Veriti 96-well thermal cycler (Thermo Fisher). Remove and incubate the tubes at room temperature for 3 min. Snap-freeze the samples immediately in liquid nitrogen. Freeze and thaw the cells three times using liquid nitrogen and a thermal cycler at 25 °C. Cell lysates were centrifuged at 20,000 x g for 15 min at 4 °C. Supernatants were collected for Western blots.
For PB or MS140-ve-induced thermal shift assay, cells were treated with 1 μM PB or 15 μM MS140-Ve for 2 hr. Cells resuspended in PBS containing protease inhibitor cocktail (Roche) were aliquoted with 100 μl cell suspension each into nine PCR tubes. Samples were heated at different temperature endpoints for 3 min using the Veriti 96-well thermal cycler (Thermo Fisher), followed by incubation at room temperature for 3 min and snap-freezing in liquid nitrogen. After three freeze and thaw cycles, samples were centrifuged at 20,000 x g for 15 min at 4 °C. Supernatants were collected for Western blot analysis.

Wu X., Yang X., Xiong Y., Li R., Ito T., Ahmed T.A., Karoulia Z., Adamopoulos C., Wang H., Wang L., Xie L., Liu J., Ueberheide B., Aaronson S.A., Chen X., Buchanan S.G., Sellers W.R., Jin J, & Poulikakos P.I. (2021). Distinct CDK6 complexes determine tumor cell response to CDK4/6 inhibitors and degraders. Nature cancer, 2(4), 429-443.

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Basal and Induced CDK6 Thermal Stability Assay

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16S rRNA Amplicon Library Preparation

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Amplicon libraries were prepared as described by the Illumina protocol [25 ]. Briefly, a PCR of 25 cycles was performed to amplify the V3–V4 region of the 16S rRNA gene prior to the incorporation of the Illumina overhang adapters (Second PCR: 10 cycles) using 2.5 µL of DNA sample (5 ng/µL), 0.5 µL of each primer, and 12.5 µL of 2x KAPA HiFi Hotstart ReadyMix (Kapa Biosystems, Merck, Darmstadt, Germany). Bacterial communities were assessed using universal primers (Bac341F: CCT ACG GGN GGC WGC AG and Bac805R: GAC TAC HVG GGT ATC TAA TCC) [26 (link)] covering the V3–V4 regions of the 16S rRNA gene. The third PCR amplification (8 cycles) was carried out using Nextera XT index primers (Illumina, San Diego, CA, USA). Amplifications were conducted on a Veriti^® 96-Well Thermal Cycler (Applied Biosystems^®, Thermo Scientific, Waltham, MA, USA). The PCR products were purified with AMPure XP beads and Veriti^® 96-Well Thermal Cycler (Applied Biosystems^®, Thermo Scientific, Waltham, MA, USA), as described in the protocol. Amplicon libraries were then sequenced on the Illumina MiSeq (Illumina, San Diego, CA, USA), generating 2 × 300 bp paired-end reads.

Naghizadeh M., Klaver L., Schönherz A.A., Rani S., Dalgaard T.S, & Engberg R.M. (2022). Impact of Dietary Sodium Butyrate and Salinomycin on Performance and Intestinal Microbiota in a Broiler Gut Leakage Model. Animals : an Open Access Journal from MDPI, 12(1), 111.

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Linker DNA-AuNP Conjugation Protocol

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First, an annealing reaction of linker DNA-A (or -B) with the complementary spacer DNA in a phosphate buffer solution containing NaCl was conducted on a Veriti 96-Well Thermal Cycler (Applied Biosystems, Waltham, MA, USA) by incubating the mixture at 95 1C for 5 min and cooling to 25 1C. The linker DNA and DNA-AuNPs were then mixed in a phosphate buffer solution with 0.5 M NaCl (for 13 nm DNA-AuNPs) or with 0.25 M NaCl (for 23 nm DNA-AuNPs). For the 23 nm DNA-AuNPs, 6.5 mM HEPES and 22 mM potassium acetate were also contained in the buffer (final concentration). The amount of linker DNA was 25-100 equivalents per particle (for 13 nm DNA-AuNP) or 200 equivalents per particle (for 23 nm DNA-AuNP). The mixture was slowly cooled over 3 days from 65 1C to 25 1C in an incubator (IS401; Yamato Scientific Co., Ltd, Tokyo, Japan) or thermal cycler (Veriti 96-Well Thermal Cycler (Applied Biosystems) or T100 Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA)).

Yokomori M., Suzuki H., Nakamura A., Sugano S.S, & Tagawa M. (2022). DNA-functionalized colloidal crystals for macromolecular encapsulation. Soft matter, 18(36).

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Gene Isoform Amplification Protocol

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The primers described above were used to amplify alternatively spliced isoforms of ADAM12 and MUC4 using Master Mix DreamTaq^TM Hot Start Green (Cat# K9022, Thermo Fisher Scientific) on a Veriti^TM 96-Well Thermal Cycler (Cat# 4375786; Applied Biosystems). The PCR cycling conditions were 95 °C for 5 min, followed by 40 cycles of 95 °C for 1 min, 60 °C for 30 s and 72 °C for 30 s, followed by one cycle of 72 °C for 7 min and a 4 °C hold. All PCR products were analyzed using 2% horizontal agarose gels; a 50 bp ladder was used to estimate the band sizes.

Althenayyan S., AlMuhanna M.H., AlAbdulrahman A., Alghanem B., Alsagaby S.A., Alfahed A., Alasiri G, & Aziz M.A. (2023). Alternatively Spliced Isoforms of MUC4 and ADAM12 as Biomarkers for Colorectal Cancer Metastasis. Journal of Personalized Medicine, 13(1), 135.

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Mapping Genetic Markers in Common Carp

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A total of 1500 genomic and transcript-associated SSR markers previously published for common carp [73 (link), 74 (link)] were also used for initial segregation screening in the mapping family. Polymorphic loci segregated in either female or male parents were genotyped in the progeny through PCR amplification. PCR was performed on a veritiTM 96 well thermal cycler (Applied Biosystems, USA) with a total volume of 12.5 μl, containing 30 ng template DNA, 1.25 μl 10× PCR buffer (TaKaRa, Japan), 0.25 U Taq DNA polymerase (TaKaRa, Japan), 50 μM each dNTP, 0.2 μM each primer and water to the final volume. The thermal cycling was programmed as follows: 5 min at 94 °C, followed by 37 cycles of 94 °C for 30 s, 35 s at appropriate annealing temperature, and 72 °C for 40 s, and the last extension at 72 °C for 10 min. PCR products were size-fractionated on 8% polyacrylamide gels and visualized by ethidium bromide staining.

Feng X., Yu X., Fu B., Wang X., Liu H., Pang M, & Tong J. (2018). A high-resolution genetic linkage map and QTL fine mapping for growth-related traits and sex in the Yangtze River common carp (Cyprinus carpio haematopterus). BMC Genomics, 19, 230.

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Quantitative Analysis of Gene Expression in Thyroid Tissues

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Total RNA was extracted from thyroid tissues and cultured thyroid cells with Trizol reagent (#9109; TaKaRa, Japan), and RNA concentration was determined on a Nanodrop 2000C spectrophotometer (Nano Drop Technologies, USA). Samples with an OD260/OD280 ratio ranging between 1.8 and 2.0 were used for experiments. Reverse transcription reactions were carried out on a VeritiTM 96-well Thermal Cycler (AB Applied Biosystems, Singapore) with 1,000 ng RNA per sample and a 5 × PrimeScript RT reagent kit (#036A; TaKaRa, Japan). PCR amplifications were performed on a LightCycler 480 Real-Time PCR System (Roche, Switzerland) using a SYBR Premix Ex Taq™ II kit (#820A; TaKaRa, Japan), and GAPDH was used as an internal reference. Primers were synthesized by TaKaRa Biotech (specific sequences are shown in Table 1). Each sample was analyzed in duplicate. A melting curve was generated during amplification to verify the absence of primer dimers or incorrectly paired products. Relative mRNA expression levels of the target genes were calculated using the 2^−ΔCp method after they were corrected based on GAPDH expression.

Guo Q., Wu Y., Hou Y., Liu Y., Liu T., Zhang H., Fan C., Guan H., Li Y., Shan Z, & Teng W. (2018). Cytokine Secretion and Pyroptosis of Thyroid Follicular Cells Mediated by Enhanced NLRP3, NLRP1, NLRC4, and AIM2 Inflammasomes Are Associated With Autoimmune Thyroiditis. Frontiers in Immunology, 9, 1197.

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Genomic DNA Extraction and UBE3 Gene Analysis

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Total genomic DNA was extracted using the Plant Genomic DNA Kit (DP305) from Tiangen Biotech (Beijing) Co., Ltd. China. The nuclear DNA UBE3 gene locus was amplified using the primer pair H_UBE3_23f (5′-TCGCCTCCAAGTTCAGTG-3′) and H_UBE3_838r (5′-CTCCCATAGGTGTAGTTCCA-3′). Taq DNA polymerase and PCR buffer (TaKaRa Code: DR100B) were from TaKaRa Biotechnology Co., Ltd. (Dalian, China). The PCR protocol were as follows: preheating at 94 °C for 4 min, 34 cycles at 94 °C for 45 s, annealing at 52 °C for 45 s and elongation at 72 °C for 1.2 min, followed by a final extension at 72 °C for 10 min. PCR amplification of the regions of interest was performed in an Applied Biosystems VeritiTM 96-Well Thermal Cycler (Model#: 9902, made in Singapore). The amplicons were resolved simultaneously on 2% agarose gels (Promega, the USA) run in 1 × TAE buffer at 3 V cm⁻¹ for 2.5 h and were stained with ethidium bromide. The fragments (PCR products) were directly sequenced with the same primer pair mentioned above using a 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA).

Suo Z., Chen L., Pei D., Jin X, & Zhang H. (2014). A new nuclear DNA marker from ubiquitin ligase gene region for genetic diversity detection of walnut germplasm resources. Biotechnology Reports, 5, 40-45.

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Expression analysis of PAC1, VPAC1, and VPAC2 receptors

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Total RNAs from cultured cells were isolated using the SV Total RNA isolation system (Promega, Madison, WI, United States) according to the manufacturer’s instructions. The total RNAs were reverse transcribed with Superscript III (Thermo Fisher Scientific, Waltham, MA, United States). RT-PCR was performed with GoTaq^® Green Master Mix (Promega) using Applied Biosystems^TM Veriti^TM 96-Well Thermal Cycler. The following primers were used: 5′-ATGAGTCTTCCCCAGGTTG-3′ (forward) and 5′-ACCGACAGGTAGTAATAATCC-3′ (reverse) for the PAC1 receptor; 5′-AGTGAAGACCGGCTACACCA-3′ (forward) and 5′-TCGACCAGCAGCCAGAAGAA-3′ (reverse) for the VPAC1 receptor; 5′-ATGGACAGCAACTCGCCTC TCTTTAG-3′ (forward) and 5′-GGAAGGAACCAACACATAA CTCAAACAG-3′ (reverse) for the VPAC2 receptor; 5′-AC CACAGTCCATGCCATCAC-3′ (forward) and 5′-TCCA CCACCCTGTTGCTGTA-3′ (reverse) for GAPDH. PCR was performed for 40 cycles at 95°C for 30 s; 55°C for 30 s; and 72°C for 30 s.

Takeuchi S., Kawanai T., Yamauchi R., Chen L., Miyaoka T., Yamada M., Asano S., Hayata-Takano A., Nakazawa T., Yano K., Horiguchi N., Nakagawa S., Takuma K., Waschek J.A., Hashimoto H, & Ago Y. (2020). Activation of the VPAC2 Receptor Impairs Axon Outgrowth and Decreases Dendritic Arborization in Mouse Cortical Neurons by a PKA-Dependent Mechanism. Frontiers in Neuroscience, 14, 521.

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cDNA Synthesis from Total RNA

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The total RNA collected was transformed into cDNA by reverse transcriptase in the presence of oligo-dT priming using the MiScript II RT Kit (Qiagen, Catalog No. 218161). The RT reaction took place in a Veriti^TM 96-Well Thermal Cycler (Applied Biosystems, USA) at 37 °C for 1 h before being briefly incubated at 95 °C to inactivate the reaction.

Mostafa S.A., Mohammad M.H., Negm W.A., Batiha G.E., Alotaibi S.S., Albogami S.M., Waard M.D., Tawfik N.Z, & Abdallah H.Y. (2022). Circulating microRNA203 and its target genes' role in psoriasis pathogenesis. Frontiers in Medicine, 9, 988962.

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