9800 fast thermal cycler

Manufactured by Thermo Fisher Scientific

Sourced in United States

The 9800 Fast Thermal Cycler is a laboratory instrument designed for the rapid amplification of DNA samples. It features fast heating and cooling rates, enabling efficient and rapid thermal cycling for various applications such as PCR (Polymerase Chain Reaction).

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Thermal cycler (242 citations)

9 protocols using 9800 fast thermal cycler

Screening Cornus-feeding Phyllocnistis for Wolbachia

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Fourteen barcoded specimens of Cornus-feeding Phyllocnistis spp. were screened for infection by Wolbachia spp. (

Rickettsiaceae

). The two genes, wsp and fbpA, were amplified with the three sets of primers: (1) wspecF (5’-CATACCTAT TCGAAGGGATAG-3’), wspecR (5’-AGCTTCGAGTGAAACCAATTC-3’) and (2) wsp81F (5’-TGGTCCAATAAGTGATGAAGAAAC-3’), wsp691R (5’-AAAATTAAACGCTACTCCA-3’), for the gene wsp (respectively 438 bp and ~600bp) and (3) fbpa-F1 (5’-GCTGCTCCRCTTGGYWTGAT-3’), fbpa-R1(5’-CCRCCAGARAAAAYYACTATTC-3’), for the gene fbpA (429 bp) to test for the presence of other

Rickettsiaceae

following the standard protocols (Baldo et al. 2006 (link), Kodandaramaiah et al. 2013 (link)).
PCR was run on a DNA-cycling machine 9800 Fast Thermal Cycler (Applied Biosystems – Foster City, CA). Gel electrophoresis was applied to visualise the amplified products in 1.5 % agarose gel using the gel electrophoresis apparatus (RunOne, EmbiTec, San Diego, CA). The gel was stained with ethidium bromide (EtBr) at a concentration of 0.5 μg/mL for 30 minutes (Lee et al. 2012 ), visualised in ultraviolet light source (wavelength 254 nm) and subsequently photographed using the image system IP-010.SD (Vilber Lourmat, France). The presence of the amplified transgene element on a gel was interpreted as evidence that the insect was infected with Wolbachia or related species parasite.

Kirichenko N., Triberti P., Kobayashi S., Hirowatari T., Doorenweerd C., Ohshima I., Huang G.H., Wang M., Magnoux E, & Lopez-Vaamonde C. (2018). Systematics of Phyllocnistis leaf-mining moths (Lepidoptera, Gracillariidae) feeding on dogwood (Cornus spp.) in Northeast Asia, with the description of three new species. ZooKeys.

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Genotyping Protocols for Genetic Comparison

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Case-control comparisons for Oulu, Kuopio and Tampere cohorts were performed by High Resolution Melt (HRM) analysis (CFX96, Bio-Rad, Hercules, CA, USA) using Type-It HRM reagents (Qiagen, Hilden, Germany). A positive control was included in all analyses, and samples with differing melting curves were further confirmed by Sanger sequencing (ABI3130xl, Applied Biosystem, Foster City, CA, USA). The Helsinki cohorts were genotyped using Custom TaqMan SNP Genotyping Assays and TaqMan Genotyping MasterMix (ThermoFisher Scientific, Waltham, MA, USA). PCR was performed on 7500 Fast Real-Time PCR System or 9800 Fast Thermal Cycler and genotypes were analyzed on 7500 Fast System SDS software v1.3.1 or 7500 software v2.0.6 (Applied Biosystems, Waltham, MA, USA). Heterozygous carriers were included as positive controls in all analyses.

Mantere T., Tervasmäki A., Nurmi A., Rapakko K., Kauppila S., Tang J., Schleutker J., Kallioniemi A., Hartikainen J.M., Mannermaa A., Nieminen P., Hanhisalo R., Lehto S., Suvanto M., Grip M., Jukkola-Vuorinen A., Tengström M., Auvinen P., Kvist A., Borg Å., Blomqvist C., Aittomäki K., Greenberg R.A., Winqvist R., Nevanlinna H, & Pylkäs K. (2017). Case-control analysis of truncating mutations in DNA damage response genes connects TEX15 and FANCD2 with hereditary breast cancer susceptibility. Scientific Reports, 7, 681.

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Multiplex PCR and CE-SSCA for BRAF and NRAS Mutation Analysis

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BRAF and NRAS mutation analysis was performed on extracted DNA by multiplex PCR and capillary electrophoresis-single strand conformation analysis (CE-SSCA) using an in-house procedure. PCR was done using the 2720 Thermal Cycler (Applied Biosystems^®, Foster City, USA) and the BRAF/NRAS mutational analysis was done using the ABI 3130XL Genetic Analyzer (Applied Biosystems^®, Foster City, USA). For BRAF, a V600E mutation will result in a characteristic pattern in the CE-SSCA at all different temperatures and therefore requires no further confirmation by sequencing. For any unclear results, Direct DNA Sanger sequencing was performed for confirmation. For NRAS, any mobility shift identified by CE-SSCA in codon 61 or codon 12/13 was confirmed by Direct DNA Sanger sequencing. This was done using the 9800 fast thermal cycler (Applied Biosystems^®, Foster City, USA) and the ABI 3500 Genetic analyzer (Applied Biosystems^®, Foster City, USA). Sequencing data was analysed using Mutation Surveyor software.

Abdelraouf F., Sharp A., Maurya M., Mair D., Wotherspoon A., Leary A., Gonzalez de Castro D., Bhosle J., Nassef A., Gaafar T., Popat S., Yap T.A, & O’Brien M. (2015). Focused molecular analysis of small cell lung cancer: feasibility in routine clinical practice. BMC Research Notes, 8, 688.

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16S rDNA PCR Amplification and Purification

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The MicroSeq 500 16S rDNA PCR (Applied Biosystems, United States) was used to amplify the V1-V2 region of the 16S. Sample DNA and the master mix were mixed in a 1:1 ratio and the reaction was carried out in a 9800 Fast Thermal Cycler (Applied Biosystems, USA) with the following conditions: initial denaturation of 95 °C for 10 min and 30 cycles of 95 °C for 30s, 60 °C for 30 min and 72 °C for 45 s, followed by a final extension of 72 °C for 10 min. PCR products were resolved by electrophoresis on a 2% agarose gel (Embi Tech, United States). Samples with a 500–600 bp PCR fragment were purified with the ExoSap-IT kit (Affymetrix, United States).

Stavnsbjerg C., Frimodt-Møller N., Moser C, & Bjarnsholt T. (2017). Comparison of two commercial broad-range PCR and sequencing assays for identification of bacteria in culture-negative clinical samples. BMC Infectious Diseases, 17, 233.

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Mitochondrial DNA Amplification from Environmental Samples

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Genomic DNA was independently extracted from each specimen of assembly 1. Specimens were grinded and processed with the QIAamp DNA Mini Kit (QIAgen, Germany) in accordance with the manufacturer’s instructions. DNA was eluted in 50 μL of PCR-grade water and stored at −20 °C.
The mitochondrial DNA target encompassing the cytb-nd1 region was amplified according to Latrofa et al. (2012 (link)) with minor modifications. Two microliters of DNA was used in a 25-μL final volume PCR reaction, including standard reaction buffer 2 mM MgCl₂, 0.2 mM each dNTP, and 0.7 U of Thermus sp. DNA polymerase (Biotools, B&M Labs, Spain), and 15 pmol of each primer PhleF (5′-AAT AAA TTA GGA GGA GTA ATT GC-3′) and PhleR (5′-GCC TCG AWT TCG WTT ATG ATA AAT T-3′) (Sigma-Genosys).
Amplification was performed on a 9800 Fast Thermal Cycler (Applied Biosystems), standard ramp enabled, with the following conditions: initial denaturation of 5 min at 94 °C, 40 cycles at 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 1 min, and final extension at 72 °C for 10 min. All the amplified products were resolved on 2 % agarose gels stained with Pronasafe Nucleic Acid Staining (Laboratorios CONDA, Spain) and visualized under UV light.

Llanes-Acevedo I.P., Arcones C., Gálvez R., Martin O., Checa R., Montoya A., Chicharro C., Cruz S., Miró G, & Cruz I. (2016). DNA sequence analysis suggests that cytb-nd1 PCR-RFLP may not be applicable to sandfly species identification throughout the Mediterranean region. Parasitology Research, 115, 1287-1295.

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Sanger Sequencing of pUC19 Plasmid

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The cycle sequencing reactions (10 µl) were performed on a 9800 Fast Thermal Cycler (Applied Biosystems) with the Cycle Sequencing Mix (0.5 µl) of the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) containing 1 ng template and 6 pmol of sequencing primer pUC19-seq-rev under the following thermal cycling conditions: initial denaturation (98 °C, 1 min); and 25 cycles of denaturation (96 °C, 10 s), annealing (60 °C, 15 s), and extension (68 °C, 2.5 min). Upon completion, the residual dye terminators were removed from the reaction with Agencourt CleanSEQ (Beckman-Coulter, Danvers, MA). Products were eluted off the beads with deionized water and sequenced directly on a 3730 DNA Analyzer (Applied Biosystems). Sequencing traces were collected using Applied Biosystems Data Collection software v3.0 and analyzed with the Applied Biosystems Sequencing Analysis v5.2 software.

Malyshev D.A., Dhami K., Lavergne T., Chen T., Dai N., Foster J.M., Corrêa IR J.r, & Romesberg F.E. (2014). A Semi-Synthetic Organism with an Expanded Genetic Alphabet. Nature, 509(7500), 385-388.

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Gene Expression Analysis of ADMSCs

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Gene expression profiles of ADMSCs after 14 days of culture in formulation S‐50 were examined using quantitative real‐time polymerase chain reaction (qRT‐PCR). Seeded cells were pelleted from PNIPAAm‐g‐CS + MPs gels by dissolution in sodium citrate‐EDTA buffer and subsequent centrifugation. Total RNA was extracted using the Pure Link RNA Extraction Mini Kit (Ambion, Life Technologies), quantified in terms of concentration and purity with a nanodrop (Applied Biosystems), and reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Target genes (Table S1) were amplified in 20 μL reactions using 20 ng of cDNA, Fast SYBR Green Master Mix (Fisher Scientific), 500 nM primer concentrations, and an Applied Biosystems 9800 Fast Thermal Cycler. Relative gene expression was calculated using the delta‐delta Ct method (2^−ΔΔCt) and normalized to ADMSCs on day 0 after encapsulation and the housekeeping gene GAPDH.

Christiani T., Mys K., Dyer K., Kadlowec J., Iftode C, & Vernengo A.J. (2021). Using embedded alginate microparticles to tune the properties of in situ forming poly(N‐isopropylacrylamide)‐graft‐chondroitin sulfate bioadhesive hydrogels for replacement and repair of the nucleus pulposus of the intervertebral disc. JOR Spine, 4(3), e1161.

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Sanger Sequencing of pUC19 Plasmid

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Malyshev D.A., Dhami K., Lavergne T., Chen T., Dai N., Foster J.M., Corrêa IR J.r, & Romesberg F.E. (2014). A Semi-Synthetic Organism with an Expanded Genetic Alphabet. Nature, 509(7500), 385-388.

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Microbial 16S rDNA Amplification by PCR

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Polymerase chain reaction (PCR) was done on the genomic DNA after dilution and standardizing concentration to 50 ng/ul. The PCR machine used was 9800 Fast Thermal Cycler from Applied Biosystems. PCR targeting the 16S rDNA was done using universal primers P3 (forward, >5 ′ -ATTAGATACCCTGGTAGTCC-3 ′ ) and P5 (reverse, >3 ′ -GGTTACCTTGTTACGACTTC-5 ′ ). The PCR reaction mix contained 12.5 µl one Taq 2X mastermix with standard buffer from Biolabs, 1.25 µl of 10 µM primer P3F, 1.25 µl of 10 µM primer P5R, 9.0 µl sterile nuclease free PCR water, and 1.0 µl of sample DNA. After optimization, the PCR conditions were as follows: an initial DNA denaturation at 94 • C for 3 min then denaturation at 94 • C for 45 s, annealing at 53 • C for 45 s, and extension at 72 • C for 2 min (35 cycles). The final extension was carried out at 72 • C for 5 min. Amplified DNA was held at 4 • C.

Gitonga N.M., Njeru E.M., Cheruiyot R., & Maingi J.M. (2021). Genetic and Morphological Diversity of Indigenous Bradyrhizobium Nodulating Soybean in Organic and Conventional Family Farming Systems. Frontiers in Sustainable Food Systems, 4.

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