superscript iii rt pcr platinum taq high fidelity kit  (Thermo Fisher)


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    Structured Review

    Thermo Fisher superscript iii rt pcr platinum taq high fidelity kit
    ZIKV insertional mutant library screening protocol and deep sequencing results. (A) Representative images of individual mutants from the library of ZIKV cDNA clones (blue), each bearing a single 15-nucleotide insertion, represented by a colored line (not drawn to scale). (B) 293T cells were transfected with the mutant plasmid library to select for mutant genomes that could initiate RNA replication and virus release. (C) Forty-eight hours after transfection, the supernatant was collected and passaged on Vero cells to select mutants that retained the ability to spread. (D) At 48 h postinfection, the supernatant was passaged once more on Vero cells to select for the fittest mutants. At the time of supernatant collection, RNAs from all <t>three</t> of the cell populations were subjected to <t>RT-PCR</t> to amplify the viral genomes. The subsequent RT-PCR products were then deep sequenced. (E) Raw number of deep sequence reads with inserts at each location for the input plasmid library. The ZIKV genome schematic and bars in the graph show structural genes in red, nonstructural genes in blue, UTRs in black, and the intron used to stabilize the plasmid in bacteria in yellow. This intron was not present in the rescued viruses. Alternating gray and white shading within each graph differentiates the genomic regions. (F to H) Frequencies of insertions at each location following normalization to the representation in the input library after plasmid DNA transfection (F), infected cell passage 1 (G), and infected cell passage 2 (H), with a schematic of the genome shown below. Numbering along the x axis indicates the nucleotide position in the ZIKV genome, while the y axis gives the log raw number of reads (E) or the log fold change over input (F to H), with horizontal dotted lines representing the cutoff of the mutants enriched two standard deviations above the average. Values are averages for three independent screens (see Materials and Methods for details).
    Superscript Iii Rt Pcr Platinum Taq High Fidelity Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 1 article reviews
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    superscript iii rt pcr platinum taq high fidelity kit - by Bioz Stars, 2020-02
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    Images

    1) Product Images from "Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion"

    Article Title: Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion

    Journal: Journal of Virology

    doi: 10.1128/JVI.00698-17

    ZIKV insertional mutant library screening protocol and deep sequencing results. (A) Representative images of individual mutants from the library of ZIKV cDNA clones (blue), each bearing a single 15-nucleotide insertion, represented by a colored line (not drawn to scale). (B) 293T cells were transfected with the mutant plasmid library to select for mutant genomes that could initiate RNA replication and virus release. (C) Forty-eight hours after transfection, the supernatant was collected and passaged on Vero cells to select mutants that retained the ability to spread. (D) At 48 h postinfection, the supernatant was passaged once more on Vero cells to select for the fittest mutants. At the time of supernatant collection, RNAs from all three of the cell populations were subjected to RT-PCR to amplify the viral genomes. The subsequent RT-PCR products were then deep sequenced. (E) Raw number of deep sequence reads with inserts at each location for the input plasmid library. The ZIKV genome schematic and bars in the graph show structural genes in red, nonstructural genes in blue, UTRs in black, and the intron used to stabilize the plasmid in bacteria in yellow. This intron was not present in the rescued viruses. Alternating gray and white shading within each graph differentiates the genomic regions. (F to H) Frequencies of insertions at each location following normalization to the representation in the input library after plasmid DNA transfection (F), infected cell passage 1 (G), and infected cell passage 2 (H), with a schematic of the genome shown below. Numbering along the x axis indicates the nucleotide position in the ZIKV genome, while the y axis gives the log raw number of reads (E) or the log fold change over input (F to H), with horizontal dotted lines representing the cutoff of the mutants enriched two standard deviations above the average. Values are averages for three independent screens (see Materials and Methods for details).
    Figure Legend Snippet: ZIKV insertional mutant library screening protocol and deep sequencing results. (A) Representative images of individual mutants from the library of ZIKV cDNA clones (blue), each bearing a single 15-nucleotide insertion, represented by a colored line (not drawn to scale). (B) 293T cells were transfected with the mutant plasmid library to select for mutant genomes that could initiate RNA replication and virus release. (C) Forty-eight hours after transfection, the supernatant was collected and passaged on Vero cells to select mutants that retained the ability to spread. (D) At 48 h postinfection, the supernatant was passaged once more on Vero cells to select for the fittest mutants. At the time of supernatant collection, RNAs from all three of the cell populations were subjected to RT-PCR to amplify the viral genomes. The subsequent RT-PCR products were then deep sequenced. (E) Raw number of deep sequence reads with inserts at each location for the input plasmid library. The ZIKV genome schematic and bars in the graph show structural genes in red, nonstructural genes in blue, UTRs in black, and the intron used to stabilize the plasmid in bacteria in yellow. This intron was not present in the rescued viruses. Alternating gray and white shading within each graph differentiates the genomic regions. (F to H) Frequencies of insertions at each location following normalization to the representation in the input library after plasmid DNA transfection (F), infected cell passage 1 (G), and infected cell passage 2 (H), with a schematic of the genome shown below. Numbering along the x axis indicates the nucleotide position in the ZIKV genome, while the y axis gives the log raw number of reads (E) or the log fold change over input (F to H), with horizontal dotted lines representing the cutoff of the mutants enriched two standard deviations above the average. Values are averages for three independent screens (see Materials and Methods for details).

    Techniques Used: Mutagenesis, Library Screening, Sequencing, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Infection

    Related Articles

    Clone Assay:

    Article Title: Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection
    Article Snippet: .. SUN1, SUN2, and LMNA cDNAs were derived from THP-1 total RNA using the SuperScript III One-Step RT-PCR system with Platinum Taq DNA polymerase (ThermoFisher Scientific) cloned into TopoTA pCR2.1 (ThermoFisher Scientific) and subcloned into pCSxW ( ). .. The SUN1 isoform cloned and used in the assays encoded 888 amino acids and represents a novel isoform with closest similarity to isoform ENST00000405266 but containing an additional exon (ENSE00003501736), which has been described in other alternatively spliced SUN1 isoforms (e.g., ENST00000429178.5) and with a described common single nucleotide polymorphism (SNP; rs6461378), leading to the H118Y amino acid change.

    Amplification:

    Article Title: HIV Nef-M1 Effects on Colorectal Cancer Growth in Tumor-induced Spleens and Hepatic Metastasis
    Article Snippet: .. Using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase Kit (Invitrogen, Cat # 1257-026), 5 μg of total RNA was primed reversed transcribed into cDNA in 50 μl reaction mixtures at 55° C for 30 min and was followed immediately by pre-denaturation consisting of one cycle at 94° C for 2 min. PCR amplification using the One-Step for 35 cycles of denaturing at 94° C for 15 sec, annealing at 60° C for 30 sec, and extension at 68° C for 2 min, followed by the final extension at 68° C for 5 minutes. ..

    Article Title: Direct Acting Antiviral Agents in Korean Patients with Chronic Hepatitis C and Hemophilia Who Are Treatment-Naïve or Treatment-Experienced
    Article Snippet: .. The extracted RNA was reverse transcribed and amplified by the PCR method using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, San Diego, CA, USA) with the pairs of primers as follows: sense (5872–5891) 5′AAGAGGCTCCACCAGTGGAT-3′ and antisense (6730–6749) 5′-CGCCGGAGCGTACCTGTGCA-3′. .. The targeted HCV genome was amplified by nested PCR using PrimeSTAR Max DNA Polymerase (TaKaRa), with the pairs of primers as follows: sense (5893–5912) 5′-AATGAGGACT GCTCCACGCC-3′ and antisense (6690–6709) 5′-GTG AAGAATTCGGGGGCCGG-3′.

    Article Title: Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth
    Article Snippet: RT-PCR was performed using 1 μg of RNA and SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instruction. .. The thermal cycling process consisted of reverse transcription for 30 minutes at 55°C and 2 minutes at 94°C, followed by 35 cycles of PCR amplification.

    Article Title: Prevalence of resistance-associated substitutions to direct-acting antiviral agents in hemodialysis and renal transplant patients infected with hepatitis C virus
    Article Snippet: .. NS3, NS5A and NS5B amplification Viral RNA was extracted from 200 µL of each plasma sample using the QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany) according to manufacturer recommendations. cDNA synthesis and the first round of PCR of the NS3, NS5A and NS5B genes were performed using the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA). .. Next, a nested PCR was performed using Platinum Taq DNA Polymerase (Thermo Fisher Scientific).

    Article Title: Mechanisms Associated with HIV-1 Resistance to Acyclovir by the V75I Mutation in Reverse Transcriptase *
    Article Snippet: .. HIV-1 RNA was reverse-transcribed into cDNA, and a 2878-bp nucleotide fragment encompassing protease and reverse transcriptase was amplified in an outer PCR using SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Invitrogen) and outer primers AV190-1 and CR1 ( ). .. A 2853-bp nucleotide fragment was amplified using Expand High Fidelity PCR System (Roche Diagnostics) and the inner primers AV190-2 and CR2.

    Article Title: Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection
    Article Snippet: SUN1, SUN2, and LMNA cDNAs were derived from THP-1 total RNA using the SuperScript III One-Step RT-PCR system with Platinum Taq DNA polymerase (ThermoFisher Scientific) cloned into TopoTA pCR2.1 (ThermoFisher Scientific) and subcloned into pCSxW ( ). .. The LULL1, LBR, NET26, NET39, and LUMA cDNAs were a gift from Rose Goodchild (KU Leuven) and Barbara Klupp ( ) and were cloned into pCSxW following PCR amplification.

    Article Title: Oxidized galectin-1 reduces lipopolysaccharide-induced increase of proinflammatory cytokine mRNA in cultured macrophages
    Article Snippet: .. RT-PCR and real-time RT-PCR Total RNA was isolated from cultured peritoneal macrophages in each dish using an RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. cDNA was reverse transcribed and amplified using the SuperScript III One-Step RT-PCR system with platinum Taq DNA polymerase (Invitrogen). .. The primer pairs were synthesized by Invitrogen, and their sequences, PCR product sizes, and GenBank acc are shown below: Galectin-1: 5′-ATGGCCTGTGGTCTGGTCGCC-3′ and 5′-TCACTCAAAGGCCACACACTT-3′ (408 bp, NM_019904), GAPDH: 5′-TTCAACGGCACAGT CAAGG-3′ and 5′-CATGGACTGTGGTCATGAG-3′ (373 bp, NM_009084), IL-1β: 5′-GAAGCTGTGG CAGCTACCTATGTCT-3′ and 5′-CTCTGCTTGA GAGGTGCTGATGTAC-3′ (520 bp, NM_031512), IL-6: 5′-GACTGATGTTGTTGACAGCCACTGC-3′ and 5′-TAGCCACTCCTTCTGTGACTCTAACT-3′ (509 bp, NM_012589), and iNOS: 5′-GGAGAGATTTTTCACGA CACCC-3′ and 5′-CCATGCATAATTTGGACTTGCA-3′ (493 bp, NM_012611).

    Article Title: Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239?nef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients
    Article Snippet: .. Next, four overlapping PCR amplicons which span the entire SIVmac239 coding sequence were amplified by using the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity (Invitrogen, Carlsbad, CA). .. These products were gel purified and fragmented with a Nextera DNA sample prep kit (Epicentre, Madison, WI).

    Article Title: Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica
    Article Snippet: .. PCR amplification was carried out using the SuperScript III One-Step RT-PCR System, Platinum Taq DNA Polymerase (Invitrogen) and either the primers ZNC1iD_3 and ZNC1iR_4 for the amplification of ZNC1 cDNA or HIS1F and HIS1R for the amplification of HIS1 cDNA. .. The cDNA was amplified using the following parameters: 1 cycle of 30 min at 60°C, 2 min at 94°C; 30 cycles of 1 min at 96°C, 1 min at 55°C, 1 min at 72°C; and 1 cycle of 7 min at 72°C.

    Article Title: Prevalence of hepatitis C virus (HCV) variants resistant to NS5A inhibitors in naïve patients infected with HCV genotype 1 in Tunisia
    Article Snippet: Paragraph title: NS5A amplification ... After reverse transcription (RT) using the SuperScript® III One-Step RT-PCR system with Platinum® Taq High Fidelity DNA Polymerase (Invitrogen, Cergy-Pontoise, France), a nested-polymerase chain reaction (nested-PCR) was performed with AmpliTaq Gold DNA Polymerase (Applied Biosystem, Courtaboeuf, France).

    Positive Control:

    Article Title: Detection of Japanese Encephalitis Virus RNA in Human Throat Samples in Laos – A Pilot study
    Article Snippet: NS3 RT-qPCR was performed with SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase kit (Thermo Fisher, Waltham, USA): 50 μL reaction volume; 600 nM forward (5′-GCAATGTGYCTCCAAAGAGC-3′) and reverse (5′-GTCGATGACCCTGCTCGC-3′) primers; 300 nM probe (5′FAM-TCCTATGAYACAGAATAYCCAAA-MGB NFQ 3′); 15 μL RNA. .. RNA positive control (UVE/JEV/UNK/TW/RP9-190 strain, GenBank KF907505, EVA 001V-02344) and no template control were also included to each RT-qPCR run.

    Synthesized:

    Article Title: Oxidized galectin-1 reduces lipopolysaccharide-induced increase of proinflammatory cytokine mRNA in cultured macrophages
    Article Snippet: RT-PCR and real-time RT-PCR Total RNA was isolated from cultured peritoneal macrophages in each dish using an RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. cDNA was reverse transcribed and amplified using the SuperScript III One-Step RT-PCR system with platinum Taq DNA polymerase (Invitrogen). .. The primer pairs were synthesized by Invitrogen, and their sequences, PCR product sizes, and GenBank acc are shown below: Galectin-1: 5′-ATGGCCTGTGGTCTGGTCGCC-3′ and 5′-TCACTCAAAGGCCACACACTT-3′ (408 bp, NM_019904), GAPDH: 5′-TTCAACGGCACAGT CAAGG-3′ and 5′-CATGGACTGTGGTCATGAG-3′ (373 bp, NM_009084), IL-1β: 5′-GAAGCTGTGG CAGCTACCTATGTCT-3′ and 5′-CTCTGCTTGA GAGGTGCTGATGTAC-3′ (520 bp, NM_031512), IL-6: 5′-GACTGATGTTGTTGACAGCCACTGC-3′ and 5′-TAGCCACTCCTTCTGTGACTCTAACT-3′ (509 bp, NM_012589), and iNOS: 5′-GGAGAGATTTTTCACGA CACCC-3′ and 5′-CCATGCATAATTTGGACTTGCA-3′ (493 bp, NM_012611).

    Quantitative RT-PCR:

    Article Title: Oxidized galectin-1 reduces lipopolysaccharide-induced increase of proinflammatory cytokine mRNA in cultured macrophages
    Article Snippet: .. RT-PCR and real-time RT-PCR Total RNA was isolated from cultured peritoneal macrophages in each dish using an RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. cDNA was reverse transcribed and amplified using the SuperScript III One-Step RT-PCR system with platinum Taq DNA polymerase (Invitrogen). .. The primer pairs were synthesized by Invitrogen, and their sequences, PCR product sizes, and GenBank acc are shown below: Galectin-1: 5′-ATGGCCTGTGGTCTGGTCGCC-3′ and 5′-TCACTCAAAGGCCACACACTT-3′ (408 bp, NM_019904), GAPDH: 5′-TTCAACGGCACAGT CAAGG-3′ and 5′-CATGGACTGTGGTCATGAG-3′ (373 bp, NM_009084), IL-1β: 5′-GAAGCTGTGG CAGCTACCTATGTCT-3′ and 5′-CTCTGCTTGA GAGGTGCTGATGTAC-3′ (520 bp, NM_031512), IL-6: 5′-GACTGATGTTGTTGACAGCCACTGC-3′ and 5′-TAGCCACTCCTTCTGTGACTCTAACT-3′ (509 bp, NM_012589), and iNOS: 5′-GGAGAGATTTTTCACGA CACCC-3′ and 5′-CCATGCATAATTTGGACTTGCA-3′ (493 bp, NM_012611).

    Article Title: Detection of Japanese Encephalitis Virus RNA in Human Throat Samples in Laos – A Pilot study
    Article Snippet: .. NS3 RT-qPCR was performed with SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase kit (Thermo Fisher, Waltham, USA): 50 μL reaction volume; 600 nM forward (5′-GCAATGTGYCTCCAAAGAGC-3′) and reverse (5′-GTCGATGACCCTGCTCGC-3′) primers; 300 nM probe (5′FAM-TCCTATGAYACAGAATAYCCAAA-MGB NFQ 3′); 15 μL RNA. .. RNA positive control (UVE/JEV/UNK/TW/RP9-190 strain, GenBank KF907505, EVA 001V-02344) and no template control were also included to each RT-qPCR run.

    Electrophoresis:

    Article Title: Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica
    Article Snippet: PCR amplification was carried out using the SuperScript III One-Step RT-PCR System, Platinum Taq DNA Polymerase (Invitrogen) and either the primers ZNC1iD_3 and ZNC1iR_4 for the amplification of ZNC1 cDNA or HIS1F and HIS1R for the amplification of HIS1 cDNA. .. Aliquots from each reaction were analyzed by electrophoresis on 2% agarose gels and staining with ethidium bromide.

    In Silico:

    Article Title: Detection of Japanese Encephalitis Virus RNA in Human Throat Samples in Laos – A Pilot study
    Article Snippet: Primers and probes were designed to identify the best in-silico matching from a multiple genome alignment of all JEV sequences (303) above 9,000 base pairs available on GenBank. .. NS3 RT-qPCR was performed with SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase kit (Thermo Fisher, Waltham, USA): 50 μL reaction volume; 600 nM forward (5′-GCAATGTGYCTCCAAAGAGC-3′) and reverse (5′-GTCGATGACCCTGCTCGC-3′) primers; 300 nM probe (5′FAM-TCCTATGAYACAGAATAYCCAAA-MGB NFQ 3′); 15 μL RNA.

    Expressing:

    Article Title: Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth
    Article Snippet: RT-PCR was performed using 1 μg of RNA and SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instruction. .. SHED differentiation was monitored by detection of dentin matrix protein-1 (DMP-1) gene expression (sense primer 5’-CAGGAGCACAGGAAAAGGAG-3’ and antisense primer 5’-CTGGTGGTATCTTGGGCACT-3’, expected product size: 213 bp).

    Article Title: Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica
    Article Snippet: The HIS1 mRNA was used as an internal control due to the constitutive expression of the HIS1 gene. .. PCR amplification was carried out using the SuperScript III One-Step RT-PCR System, Platinum Taq DNA Polymerase (Invitrogen) and either the primers ZNC1iD_3 and ZNC1iR_4 for the amplification of ZNC1 cDNA or HIS1F and HIS1R for the amplification of HIS1 cDNA.

    Derivative Assay:

    Article Title: HIV Nef-M1 Effects on Colorectal Cancer Growth in Tumor-induced Spleens and Hepatic Metastasis
    Article Snippet: Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on tumor xenografts derived from cell lines injected in mice. mRNA was isolated using TRI-zol reagent (Invitrogen) from tumor cells removed from surgical specimens and xenografts from cell lines grown subcutaneously. .. Using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase Kit (Invitrogen, Cat # 1257-026), 5 μg of total RNA was primed reversed transcribed into cDNA in 50 μl reaction mixtures at 55° C for 30 min and was followed immediately by pre-denaturation consisting of one cycle at 94° C for 2 min. PCR amplification using the One-Step for 35 cycles of denaturing at 94° C for 15 sec, annealing at 60° C for 30 sec, and extension at 68° C for 2 min, followed by the final extension at 68° C for 5 minutes.

    Article Title: Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection
    Article Snippet: .. SUN1, SUN2, and LMNA cDNAs were derived from THP-1 total RNA using the SuperScript III One-Step RT-PCR system with Platinum Taq DNA polymerase (ThermoFisher Scientific) cloned into TopoTA pCR2.1 (ThermoFisher Scientific) and subcloned into pCSxW ( ). .. The SUN1 isoform cloned and used in the assays encoded 888 amino acids and represents a novel isoform with closest similarity to isoform ENST00000405266 but containing an additional exon (ENSE00003501736), which has been described in other alternatively spliced SUN1 isoforms (e.g., ENST00000429178.5) and with a described common single nucleotide polymorphism (SNP; rs6461378), leading to the H118Y amino acid change.

    Cell Culture:

    Article Title: Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth
    Article Snippet: Odontogenic-like phenotype differentiation by RT-PCR SHED (2×103 ) were cultured in 6-well plates with MTA, BD or CH conditioned medium for 1, 7, 14 and 21 days. .. RT-PCR was performed using 1 μg of RNA and SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instruction.

    Article Title: A subset of the diverse COG0523 family of putative metal chaperones is linked to zinc homeostasis in all kingdoms of life
    Article Snippet: Overnight cultures of ADP1 and Δzur:kan R cultured in Luria Broth was used to inoculate 5 ml culture of Luria Broth supplemented with 50 μM ZnSO4 to ensure repression of transcription by Zur. .. RT-PCR reactions were carried out with Superscript™ III One-Step RT-PCR System with Platinum® Taq High Fidelity (Invitrogen).

    Article Title: Oxidized galectin-1 reduces lipopolysaccharide-induced increase of proinflammatory cytokine mRNA in cultured macrophages
    Article Snippet: .. RT-PCR and real-time RT-PCR Total RNA was isolated from cultured peritoneal macrophages in each dish using an RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. cDNA was reverse transcribed and amplified using the SuperScript III One-Step RT-PCR system with platinum Taq DNA polymerase (Invitrogen). .. The primer pairs were synthesized by Invitrogen, and their sequences, PCR product sizes, and GenBank acc are shown below: Galectin-1: 5′-ATGGCCTGTGGTCTGGTCGCC-3′ and 5′-TCACTCAAAGGCCACACACTT-3′ (408 bp, NM_019904), GAPDH: 5′-TTCAACGGCACAGT CAAGG-3′ and 5′-CATGGACTGTGGTCATGAG-3′ (373 bp, NM_009084), IL-1β: 5′-GAAGCTGTGG CAGCTACCTATGTCT-3′ and 5′-CTCTGCTTGA GAGGTGCTGATGTAC-3′ (520 bp, NM_031512), IL-6: 5′-GACTGATGTTGTTGACAGCCACTGC-3′ and 5′-TAGCCACTCCTTCTGTGACTCTAACT-3′ (509 bp, NM_012589), and iNOS: 5′-GGAGAGATTTTTCACGA CACCC-3′ and 5′-CCATGCATAATTTGGACTTGCA-3′ (493 bp, NM_012611).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: HIV Nef-M1 Effects on Colorectal Cancer Growth in Tumor-induced Spleens and Hepatic Metastasis
    Article Snippet: .. Using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase Kit (Invitrogen, Cat # 1257-026), 5 μg of total RNA was primed reversed transcribed into cDNA in 50 μl reaction mixtures at 55° C for 30 min and was followed immediately by pre-denaturation consisting of one cycle at 94° C for 2 min. PCR amplification using the One-Step for 35 cycles of denaturing at 94° C for 15 sec, annealing at 60° C for 30 sec, and extension at 68° C for 2 min, followed by the final extension at 68° C for 5 minutes. ..

    Article Title: Direct Acting Antiviral Agents in Korean Patients with Chronic Hepatitis C and Hemophilia Who Are Treatment-Naïve or Treatment-Experienced
    Article Snippet: .. The extracted RNA was reverse transcribed and amplified by the PCR method using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, San Diego, CA, USA) with the pairs of primers as follows: sense (5872–5891) 5′AAGAGGCTCCACCAGTGGAT-3′ and antisense (6730–6749) 5′-CGCCGGAGCGTACCTGTGCA-3′. .. The targeted HCV genome was amplified by nested PCR using PrimeSTAR Max DNA Polymerase (TaKaRa), with the pairs of primers as follows: sense (5893–5912) 5′-AATGAGGACT GCTCCACGCC-3′ and antisense (6690–6709) 5′-GTG AAGAATTCGGGGGCCGG-3′.

    Article Title: A subset of the diverse COG0523 family of putative metal chaperones is linked to zinc homeostasis in all kingdoms of life
    Article Snippet: .. RT-PCR reactions were carried out with Superscript™ III One-Step RT-PCR System with Platinum® Taq High Fidelity (Invitrogen). ..

    Article Title: Prevalence of resistance-associated substitutions to direct-acting antiviral agents in hemodialysis and renal transplant patients infected with hepatitis C virus
    Article Snippet: .. NS3, NS5A and NS5B amplification Viral RNA was extracted from 200 µL of each plasma sample using the QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany) according to manufacturer recommendations. cDNA synthesis and the first round of PCR of the NS3, NS5A and NS5B genes were performed using the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA). .. Next, a nested PCR was performed using Platinum Taq DNA Polymerase (Thermo Fisher Scientific).

    Article Title: Mechanisms Associated with HIV-1 Resistance to Acyclovir by the V75I Mutation in Reverse Transcriptase *
    Article Snippet: .. HIV-1 RNA was reverse-transcribed into cDNA, and a 2878-bp nucleotide fragment encompassing protease and reverse transcriptase was amplified in an outer PCR using SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Invitrogen) and outer primers AV190-1 and CR1 ( ). .. A 2853-bp nucleotide fragment was amplified using Expand High Fidelity PCR System (Roche Diagnostics) and the inner primers AV190-2 and CR2.

    Article Title: Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection
    Article Snippet: .. SUN1, SUN2, and LMNA cDNAs were derived from THP-1 total RNA using the SuperScript III One-Step RT-PCR system with Platinum Taq DNA polymerase (ThermoFisher Scientific) cloned into TopoTA pCR2.1 (ThermoFisher Scientific) and subcloned into pCSxW ( ). .. The SUN1 isoform cloned and used in the assays encoded 888 amino acids and represents a novel isoform with closest similarity to isoform ENST00000405266 but containing an additional exon (ENSE00003501736), which has been described in other alternatively spliced SUN1 isoforms (e.g., ENST00000429178.5) and with a described common single nucleotide polymorphism (SNP; rs6461378), leading to the H118Y amino acid change.

    Article Title: Characterisation of the canine faecal virome in healthy dogs and dogs with acute diarrhoea using shotgun metagenomics
    Article Snippet: .. RT-PCR was performed with SuperScript III One-Step RT-PCR System with Platinum Taq (Invitrogen™ ). .. PCR conditions used were: 45°C for 60 min and 95°C for 5 min, 35 cycle of 94°C for 40 sec, 55°C for 1min and 72°C for 5 min, and a final elongation step of 72°C for 5 min, followed by final hold at 4°C.

    Article Title: Oxidized galectin-1 reduces lipopolysaccharide-induced increase of proinflammatory cytokine mRNA in cultured macrophages
    Article Snippet: .. RT-PCR and real-time RT-PCR Total RNA was isolated from cultured peritoneal macrophages in each dish using an RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. cDNA was reverse transcribed and amplified using the SuperScript III One-Step RT-PCR system with platinum Taq DNA polymerase (Invitrogen). .. The primer pairs were synthesized by Invitrogen, and their sequences, PCR product sizes, and GenBank acc are shown below: Galectin-1: 5′-ATGGCCTGTGGTCTGGTCGCC-3′ and 5′-TCACTCAAAGGCCACACACTT-3′ (408 bp, NM_019904), GAPDH: 5′-TTCAACGGCACAGT CAAGG-3′ and 5′-CATGGACTGTGGTCATGAG-3′ (373 bp, NM_009084), IL-1β: 5′-GAAGCTGTGG CAGCTACCTATGTCT-3′ and 5′-CTCTGCTTGA GAGGTGCTGATGTAC-3′ (520 bp, NM_031512), IL-6: 5′-GACTGATGTTGTTGACAGCCACTGC-3′ and 5′-TAGCCACTCCTTCTGTGACTCTAACT-3′ (509 bp, NM_012589), and iNOS: 5′-GGAGAGATTTTTCACGA CACCC-3′ and 5′-CCATGCATAATTTGGACTTGCA-3′ (493 bp, NM_012611).

    Article Title: Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239?nef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients
    Article Snippet: .. Next, four overlapping PCR amplicons which span the entire SIVmac239 coding sequence were amplified by using the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity (Invitrogen, Carlsbad, CA). .. These products were gel purified and fragmented with a Nextera DNA sample prep kit (Epicentre, Madison, WI).

    Article Title: Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica
    Article Snippet: .. PCR amplification was carried out using the SuperScript III One-Step RT-PCR System, Platinum Taq DNA Polymerase (Invitrogen) and either the primers ZNC1iD_3 and ZNC1iR_4 for the amplification of ZNC1 cDNA or HIS1F and HIS1R for the amplification of HIS1 cDNA. .. The cDNA was amplified using the following parameters: 1 cycle of 30 min at 60°C, 2 min at 94°C; 30 cycles of 1 min at 96°C, 1 min at 55°C, 1 min at 72°C; and 1 cycle of 7 min at 72°C.

    Article Title: Prevalence of hepatitis C virus (HCV) variants resistant to NS5A inhibitors in naïve patients infected with HCV genotype 1 in Tunisia
    Article Snippet: .. After reverse transcription (RT) using the SuperScript® III One-Step RT-PCR system with Platinum® Taq High Fidelity DNA Polymerase (Invitrogen, Cergy-Pontoise, France), a nested-polymerase chain reaction (nested-PCR) was performed with AmpliTaq Gold DNA Polymerase (Applied Biosystem, Courtaboeuf, France). .. HCV RNA was amplified as previously described using primers 1a-NS5A-F0 (5’-GACATCTGGGACTGGATATGYGA -3’; nt 6276–6298) and 1a-NS5A-R0 (5’- GTCCAGGWRTARGACATYGAGCA-3’; nt 7599–7621) for subtype 1a and 1b-NS5A-F0 (5’- GAYGTTTGGGAYTGGATATGCAC-3’; nt 6276–6298) and 1b-NS5A-R0 (5’- GTCCAYGWRTARGACATYGAGCA -3’; nt 7599–7621) for subtype 1b.

    Article Title: Successful immunotherapy induces previously unidentified allergen-specific CD4+ T-cell subsets
    Article Snippet: .. For single sorted cells, RT-PCR was performed directly in a 96-well PCR plate (ABI) containing lysis buffer (Invitrogen) by using SuperScript III One-Step RT-PCR System with PlatinumTaq (CellDirect kit, Invitrogen). .. PreAmp was performed on a thermocycler using the TaqMan PreAmp Master Mix Kit (Invitrogen) added to cDNA and 0.2× pooled Taqman assays.

    Imaging:

    Article Title: Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica
    Article Snippet: PCR amplification was carried out using the SuperScript III One-Step RT-PCR System, Platinum Taq DNA Polymerase (Invitrogen) and either the primers ZNC1iD_3 and ZNC1iR_4 for the amplification of ZNC1 cDNA or HIS1F and HIS1R for the amplification of HIS1 cDNA. .. The results were documented using the Gene Genius Imaging System (Syngene) and analyzed with Quantity One software, v4.6.9 (Bio-Rad).

    Polymerase Chain Reaction:

    Article Title: HIV Nef-M1 Effects on Colorectal Cancer Growth in Tumor-induced Spleens and Hepatic Metastasis
    Article Snippet: .. Using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase Kit (Invitrogen, Cat # 1257-026), 5 μg of total RNA was primed reversed transcribed into cDNA in 50 μl reaction mixtures at 55° C for 30 min and was followed immediately by pre-denaturation consisting of one cycle at 94° C for 2 min. PCR amplification using the One-Step for 35 cycles of denaturing at 94° C for 15 sec, annealing at 60° C for 30 sec, and extension at 68° C for 2 min, followed by the final extension at 68° C for 5 minutes. ..

    Article Title: Direct Acting Antiviral Agents in Korean Patients with Chronic Hepatitis C and Hemophilia Who Are Treatment-Naïve or Treatment-Experienced
    Article Snippet: .. The extracted RNA was reverse transcribed and amplified by the PCR method using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, San Diego, CA, USA) with the pairs of primers as follows: sense (5872–5891) 5′AAGAGGCTCCACCAGTGGAT-3′ and antisense (6730–6749) 5′-CGCCGGAGCGTACCTGTGCA-3′. .. The targeted HCV genome was amplified by nested PCR using PrimeSTAR Max DNA Polymerase (TaKaRa), with the pairs of primers as follows: sense (5893–5912) 5′-AATGAGGACT GCTCCACGCC-3′ and antisense (6690–6709) 5′-GTG AAGAATTCGGGGGCCGG-3′.

    Article Title: Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth
    Article Snippet: RT-PCR was performed using 1 μg of RNA and SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instruction. .. The thermal cycling process consisted of reverse transcription for 30 minutes at 55°C and 2 minutes at 94°C, followed by 35 cycles of PCR amplification.

    Article Title: Prevalence of resistance-associated substitutions to direct-acting antiviral agents in hemodialysis and renal transplant patients infected with hepatitis C virus
    Article Snippet: .. NS3, NS5A and NS5B amplification Viral RNA was extracted from 200 µL of each plasma sample using the QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany) according to manufacturer recommendations. cDNA synthesis and the first round of PCR of the NS3, NS5A and NS5B genes were performed using the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA). .. Next, a nested PCR was performed using Platinum Taq DNA Polymerase (Thermo Fisher Scientific).

    Article Title: Mechanisms Associated with HIV-1 Resistance to Acyclovir by the V75I Mutation in Reverse Transcriptase *
    Article Snippet: .. HIV-1 RNA was reverse-transcribed into cDNA, and a 2878-bp nucleotide fragment encompassing protease and reverse transcriptase was amplified in an outer PCR using SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Invitrogen) and outer primers AV190-1 and CR1 ( ). .. A 2853-bp nucleotide fragment was amplified using Expand High Fidelity PCR System (Roche Diagnostics) and the inner primers AV190-2 and CR2.

    Article Title: Effects of Inner Nuclear Membrane Proteins SUN1/UNC-84A and SUN2/UNC-84B on the Early Steps of HIV-1 Infection
    Article Snippet: SUN1, SUN2, and LMNA cDNAs were derived from THP-1 total RNA using the SuperScript III One-Step RT-PCR system with Platinum Taq DNA polymerase (ThermoFisher Scientific) cloned into TopoTA pCR2.1 (ThermoFisher Scientific) and subcloned into pCSxW ( ). .. The LULL1, LBR, NET26, NET39, and LUMA cDNAs were a gift from Rose Goodchild (KU Leuven) and Barbara Klupp ( ) and were cloned into pCSxW following PCR amplification.

    Article Title: Characterisation of the canine faecal virome in healthy dogs and dogs with acute diarrhoea using shotgun metagenomics
    Article Snippet: RT-PCR was performed with SuperScript III One-Step RT-PCR System with Platinum Taq (Invitrogen™ ). .. PCR conditions used were: 45°C for 60 min and 95°C for 5 min, 35 cycle of 94°C for 40 sec, 55°C for 1min and 72°C for 5 min, and a final elongation step of 72°C for 5 min, followed by final hold at 4°C.

    Article Title: Oxidized galectin-1 reduces lipopolysaccharide-induced increase of proinflammatory cytokine mRNA in cultured macrophages
    Article Snippet: RT-PCR and real-time RT-PCR Total RNA was isolated from cultured peritoneal macrophages in each dish using an RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. cDNA was reverse transcribed and amplified using the SuperScript III One-Step RT-PCR system with platinum Taq DNA polymerase (Invitrogen). .. The primer pairs were synthesized by Invitrogen, and their sequences, PCR product sizes, and GenBank acc are shown below: Galectin-1: 5′-ATGGCCTGTGGTCTGGTCGCC-3′ and 5′-TCACTCAAAGGCCACACACTT-3′ (408 bp, NM_019904), GAPDH: 5′-TTCAACGGCACAGT CAAGG-3′ and 5′-CATGGACTGTGGTCATGAG-3′ (373 bp, NM_009084), IL-1β: 5′-GAAGCTGTGG CAGCTACCTATGTCT-3′ and 5′-CTCTGCTTGA GAGGTGCTGATGTAC-3′ (520 bp, NM_031512), IL-6: 5′-GACTGATGTTGTTGACAGCCACTGC-3′ and 5′-TAGCCACTCCTTCTGTGACTCTAACT-3′ (509 bp, NM_012589), and iNOS: 5′-GGAGAGATTTTTCACGA CACCC-3′ and 5′-CCATGCATAATTTGGACTTGCA-3′ (493 bp, NM_012611).

    Article Title: Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239?nef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients
    Article Snippet: .. Next, four overlapping PCR amplicons which span the entire SIVmac239 coding sequence were amplified by using the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity (Invitrogen, Carlsbad, CA). .. These products were gel purified and fragmented with a Nextera DNA sample prep kit (Epicentre, Madison, WI).

    Article Title: Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica
    Article Snippet: .. PCR amplification was carried out using the SuperScript III One-Step RT-PCR System, Platinum Taq DNA Polymerase (Invitrogen) and either the primers ZNC1iD_3 and ZNC1iR_4 for the amplification of ZNC1 cDNA or HIS1F and HIS1R for the amplification of HIS1 cDNA. .. The cDNA was amplified using the following parameters: 1 cycle of 30 min at 60°C, 2 min at 94°C; 30 cycles of 1 min at 96°C, 1 min at 55°C, 1 min at 72°C; and 1 cycle of 7 min at 72°C.

    Article Title: Role of Host-Driven Mutagenesis in Determining Genome Evolution of Sigma Virus (DMelSV; Rhabdoviridae) in Drosophila melanogaster
    Article Snippet: .. Reverse Transcription and PCR About 6,370 consecutive nucleotides of the viral genome (partial N, G, M, X, P, and partial L genes) were amplified from each fly in four overlapping pieces 1–2 kb in length using the Superscript III one-step RT-PCR system with Platinum® Taq High Fidelity and following the manufacturer’s standard protocol ( http://www.thermofisher.com/us/en/home.html ; last accessed August 29, 2016). .. Libraries were made from each sample, following the standard manufacturer’s protocol for SOLiDTM sequencing.

    Article Title: Successful immunotherapy induces previously unidentified allergen-specific CD4+ T-cell subsets
    Article Snippet: .. For single sorted cells, RT-PCR was performed directly in a 96-well PCR plate (ABI) containing lysis buffer (Invitrogen) by using SuperScript III One-Step RT-PCR System with PlatinumTaq (CellDirect kit, Invitrogen). .. PreAmp was performed on a thermocycler using the TaqMan PreAmp Master Mix Kit (Invitrogen) added to cDNA and 0.2× pooled Taqman assays.

    Injection:

    Article Title: HIV Nef-M1 Effects on Colorectal Cancer Growth in Tumor-induced Spleens and Hepatic Metastasis
    Article Snippet: Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on tumor xenografts derived from cell lines injected in mice. mRNA was isolated using TRI-zol reagent (Invitrogen) from tumor cells removed from surgical specimens and xenografts from cell lines grown subcutaneously. .. Using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase Kit (Invitrogen, Cat # 1257-026), 5 μg of total RNA was primed reversed transcribed into cDNA in 50 μl reaction mixtures at 55° C for 30 min and was followed immediately by pre-denaturation consisting of one cycle at 94° C for 2 min. PCR amplification using the One-Step for 35 cycles of denaturing at 94° C for 15 sec, annealing at 60° C for 30 sec, and extension at 68° C for 2 min, followed by the final extension at 68° C for 5 minutes.

    Isolation:

    Article Title: HIV Nef-M1 Effects on Colorectal Cancer Growth in Tumor-induced Spleens and Hepatic Metastasis
    Article Snippet: Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on tumor xenografts derived from cell lines injected in mice. mRNA was isolated using TRI-zol reagent (Invitrogen) from tumor cells removed from surgical specimens and xenografts from cell lines grown subcutaneously. .. Using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase Kit (Invitrogen, Cat # 1257-026), 5 μg of total RNA was primed reversed transcribed into cDNA in 50 μl reaction mixtures at 55° C for 30 min and was followed immediately by pre-denaturation consisting of one cycle at 94° C for 2 min. PCR amplification using the One-Step for 35 cycles of denaturing at 94° C for 15 sec, annealing at 60° C for 30 sec, and extension at 68° C for 2 min, followed by the final extension at 68° C for 5 minutes.

    Article Title: Oxidized galectin-1 reduces lipopolysaccharide-induced increase of proinflammatory cytokine mRNA in cultured macrophages
    Article Snippet: .. RT-PCR and real-time RT-PCR Total RNA was isolated from cultured peritoneal macrophages in each dish using an RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. cDNA was reverse transcribed and amplified using the SuperScript III One-Step RT-PCR system with platinum Taq DNA polymerase (Invitrogen). .. The primer pairs were synthesized by Invitrogen, and their sequences, PCR product sizes, and GenBank acc are shown below: Galectin-1: 5′-ATGGCCTGTGGTCTGGTCGCC-3′ and 5′-TCACTCAAAGGCCACACACTT-3′ (408 bp, NM_019904), GAPDH: 5′-TTCAACGGCACAGT CAAGG-3′ and 5′-CATGGACTGTGGTCATGAG-3′ (373 bp, NM_009084), IL-1β: 5′-GAAGCTGTGG CAGCTACCTATGTCT-3′ and 5′-CTCTGCTTGA GAGGTGCTGATGTAC-3′ (520 bp, NM_031512), IL-6: 5′-GACTGATGTTGTTGACAGCCACTGC-3′ and 5′-TAGCCACTCCTTCTGTGACTCTAACT-3′ (509 bp, NM_012589), and iNOS: 5′-GGAGAGATTTTTCACGA CACCC-3′ and 5′-CCATGCATAATTTGGACTTGCA-3′ (493 bp, NM_012611).

    Article Title: Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239?nef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients
    Article Snippet: Viral RNA was isolated from cell-free plasma by using a QIAmp MinElute Virus Spin kit (Qiagen, Valencia, CA). .. Next, four overlapping PCR amplicons which span the entire SIVmac239 coding sequence were amplified by using the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity (Invitrogen, Carlsbad, CA).

    Size-exclusion Chromatography:

    Article Title: HIV Nef-M1 Effects on Colorectal Cancer Growth in Tumor-induced Spleens and Hepatic Metastasis
    Article Snippet: .. Using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase Kit (Invitrogen, Cat # 1257-026), 5 μg of total RNA was primed reversed transcribed into cDNA in 50 μl reaction mixtures at 55° C for 30 min and was followed immediately by pre-denaturation consisting of one cycle at 94° C for 2 min. PCR amplification using the One-Step for 35 cycles of denaturing at 94° C for 15 sec, annealing at 60° C for 30 sec, and extension at 68° C for 2 min, followed by the final extension at 68° C for 5 minutes. ..

    Article Title: Characterisation of the canine faecal virome in healthy dogs and dogs with acute diarrhoea using shotgun metagenomics
    Article Snippet: RT-PCR was performed with SuperScript III One-Step RT-PCR System with Platinum Taq (Invitrogen™ ). .. PCR conditions used were: 45°C for 60 min and 95°C for 5 min, 35 cycle of 94°C for 40 sec, 55°C for 1min and 72°C for 5 min, and a final elongation step of 72°C for 5 min, followed by final hold at 4°C.

    Mouse Assay:

    Article Title: HIV Nef-M1 Effects on Colorectal Cancer Growth in Tumor-induced Spleens and Hepatic Metastasis
    Article Snippet: Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on tumor xenografts derived from cell lines injected in mice. mRNA was isolated using TRI-zol reagent (Invitrogen) from tumor cells removed from surgical specimens and xenografts from cell lines grown subcutaneously. .. Using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase Kit (Invitrogen, Cat # 1257-026), 5 μg of total RNA was primed reversed transcribed into cDNA in 50 μl reaction mixtures at 55° C for 30 min and was followed immediately by pre-denaturation consisting of one cycle at 94° C for 2 min. PCR amplification using the One-Step for 35 cycles of denaturing at 94° C for 15 sec, annealing at 60° C for 30 sec, and extension at 68° C for 2 min, followed by the final extension at 68° C for 5 minutes.

    Sequencing:

    Article Title: Direct Acting Antiviral Agents in Korean Patients with Chronic Hepatitis C and Hemophilia Who Are Treatment-Naïve or Treatment-Experienced
    Article Snippet: The viral resistance testing on NS5A was performed on baseline samples for all patients and samples at the first time of virologic failure in patients with viral breakthrough by direct sequencing method. .. The extracted RNA was reverse transcribed and amplified by the PCR method using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, San Diego, CA, USA) with the pairs of primers as follows: sense (5872–5891) 5′AAGAGGCTCCACCAGTGGAT-3′ and antisense (6730–6749) 5′-CGCCGGAGCGTACCTGTGCA-3′.

    Article Title: Characterisation of the canine faecal virome in healthy dogs and dogs with acute diarrhoea using shotgun metagenomics
    Article Snippet: Paragraph title: Sequencing of canine astrovirus genome ... RT-PCR was performed with SuperScript III One-Step RT-PCR System with Platinum Taq (Invitrogen™ ).

    Article Title: Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239?nef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients
    Article Snippet: .. Next, four overlapping PCR amplicons which span the entire SIVmac239 coding sequence were amplified by using the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity (Invitrogen, Carlsbad, CA). .. These products were gel purified and fragmented with a Nextera DNA sample prep kit (Epicentre, Madison, WI).

    Article Title: Role of Host-Driven Mutagenesis in Determining Genome Evolution of Sigma Virus (DMelSV; Rhabdoviridae) in Drosophila melanogaster
    Article Snippet: Reverse Transcription and PCR About 6,370 consecutive nucleotides of the viral genome (partial N, G, M, X, P, and partial L genes) were amplified from each fly in four overlapping pieces 1–2 kb in length using the Superscript III one-step RT-PCR system with Platinum® Taq High Fidelity and following the manufacturer’s standard protocol ( http://www.thermofisher.com/us/en/home.html ; last accessed August 29, 2016). .. Libraries were made from each sample, following the standard manufacturer’s protocol for SOLiDTM sequencing.

    Staining:

    Article Title: Mechanisms Associated with HIV-1 Resistance to Acyclovir by the V75I Mutation in Reverse Transcriptase *
    Article Snippet: HIV-1 RNA was reverse-transcribed into cDNA, and a 2878-bp nucleotide fragment encompassing protease and reverse transcriptase was amplified in an outer PCR using SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Invitrogen) and outer primers AV190-1 and CR1 ( ). .. Amplification products were separated on a 1% agarose gel and visualized by ethidium bromide staining.

    Article Title: Characterisation of the canine faecal virome in healthy dogs and dogs with acute diarrhoea using shotgun metagenomics
    Article Snippet: RT-PCR was performed with SuperScript III One-Step RT-PCR System with Platinum Taq (Invitrogen™ ). .. PCR products were run on a 1.2% agarose TBE gel stained with RedSafe™ nucleic acid staining solution (iNtRON Biotechnology).

    Article Title: Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica
    Article Snippet: PCR amplification was carried out using the SuperScript III One-Step RT-PCR System, Platinum Taq DNA Polymerase (Invitrogen) and either the primers ZNC1iD_3 and ZNC1iR_4 for the amplification of ZNC1 cDNA or HIS1F and HIS1R for the amplification of HIS1 cDNA. .. Aliquots from each reaction were analyzed by electrophoresis on 2% agarose gels and staining with ethidium bromide.

    Nested PCR:

    Article Title: Direct Acting Antiviral Agents in Korean Patients with Chronic Hepatitis C and Hemophilia Who Are Treatment-Naïve or Treatment-Experienced
    Article Snippet: The extracted RNA was reverse transcribed and amplified by the PCR method using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, San Diego, CA, USA) with the pairs of primers as follows: sense (5872–5891) 5′AAGAGGCTCCACCAGTGGAT-3′ and antisense (6730–6749) 5′-CGCCGGAGCGTACCTGTGCA-3′. .. The targeted HCV genome was amplified by nested PCR using PrimeSTAR Max DNA Polymerase (TaKaRa), with the pairs of primers as follows: sense (5893–5912) 5′-AATGAGGACT GCTCCACGCC-3′ and antisense (6690–6709) 5′-GTG AAGAATTCGGGGGCCGG-3′.

    Article Title: Prevalence of resistance-associated substitutions to direct-acting antiviral agents in hemodialysis and renal transplant patients infected with hepatitis C virus
    Article Snippet: NS3, NS5A and NS5B amplification Viral RNA was extracted from 200 µL of each plasma sample using the QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany) according to manufacturer recommendations. cDNA synthesis and the first round of PCR of the NS3, NS5A and NS5B genes were performed using the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA). .. Next, a nested PCR was performed using Platinum Taq DNA Polymerase (Thermo Fisher Scientific).

    Article Title: Prevalence of hepatitis C virus (HCV) variants resistant to NS5A inhibitors in naïve patients infected with HCV genotype 1 in Tunisia
    Article Snippet: .. After reverse transcription (RT) using the SuperScript® III One-Step RT-PCR system with Platinum® Taq High Fidelity DNA Polymerase (Invitrogen, Cergy-Pontoise, France), a nested-polymerase chain reaction (nested-PCR) was performed with AmpliTaq Gold DNA Polymerase (Applied Biosystem, Courtaboeuf, France). .. HCV RNA was amplified as previously described using primers 1a-NS5A-F0 (5’-GACATCTGGGACTGGATATGYGA -3’; nt 6276–6298) and 1a-NS5A-R0 (5’- GTCCAGGWRTARGACATYGAGCA-3’; nt 7599–7621) for subtype 1a and 1b-NS5A-F0 (5’- GAYGTTTGGGAYTGGATATGCAC-3’; nt 6276–6298) and 1b-NS5A-R0 (5’- GTCCAYGWRTARGACATYGAGCA -3’; nt 7599–7621) for subtype 1b.

    Agarose Gel Electrophoresis:

    Article Title: Mechanisms Associated with HIV-1 Resistance to Acyclovir by the V75I Mutation in Reverse Transcriptase *
    Article Snippet: HIV-1 RNA was reverse-transcribed into cDNA, and a 2878-bp nucleotide fragment encompassing protease and reverse transcriptase was amplified in an outer PCR using SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Invitrogen) and outer primers AV190-1 and CR1 ( ). .. Amplification products were separated on a 1% agarose gel and visualized by ethidium bromide staining.

    Purification:

    Article Title: Direct Acting Antiviral Agents in Korean Patients with Chronic Hepatitis C and Hemophilia Who Are Treatment-Naïve or Treatment-Experienced
    Article Snippet: The extracted RNA was reverse transcribed and amplified by the PCR method using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, San Diego, CA, USA) with the pairs of primers as follows: sense (5872–5891) 5′AAGAGGCTCCACCAGTGGAT-3′ and antisense (6730–6749) 5′-CGCCGGAGCGTACCTGTGCA-3′. .. The PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN) and sequenced using an automated DNA sequencer (3730xl DNA Analyzer; Applied Biosystems, Foster City, CA, USA).

    Article Title: Mechanisms Associated with HIV-1 Resistance to Acyclovir by the V75I Mutation in Reverse Transcriptase *
    Article Snippet: HIV-1 RNA was reverse-transcribed into cDNA, and a 2878-bp nucleotide fragment encompassing protease and reverse transcriptase was amplified in an outer PCR using SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Invitrogen) and outer primers AV190-1 and CR1 ( ). .. PCR products were purified with Microspin S-400 (GE Healthcare).

    Article Title: Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239?nef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients
    Article Snippet: Next, four overlapping PCR amplicons which span the entire SIVmac239 coding sequence were amplified by using the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity (Invitrogen, Carlsbad, CA). .. These products were gel purified and fragmented with a Nextera DNA sample prep kit (Epicentre, Madison, WI).

    Software:

    Article Title: Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica
    Article Snippet: PCR amplification was carried out using the SuperScript III One-Step RT-PCR System, Platinum Taq DNA Polymerase (Invitrogen) and either the primers ZNC1iD_3 and ZNC1iR_4 for the amplification of ZNC1 cDNA or HIS1F and HIS1R for the amplification of HIS1 cDNA. .. The results were documented using the Gene Genius Imaging System (Syngene) and analyzed with Quantity One software, v4.6.9 (Bio-Rad).

    Multiplex Assay:

    Article Title: Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239?nef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients
    Article Snippet: Next, four overlapping PCR amplicons which span the entire SIVmac239 coding sequence were amplified by using the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity (Invitrogen, Carlsbad, CA). .. Multiplex identifier (MID) tags and adapter sequences for Roche 454 pyrosequencing were added.

    Sample Prep:

    Article Title: Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239?nef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients
    Article Snippet: Next, four overlapping PCR amplicons which span the entire SIVmac239 coding sequence were amplified by using the SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity (Invitrogen, Carlsbad, CA). .. These products were gel purified and fragmented with a Nextera DNA sample prep kit (Epicentre, Madison, WI).

    Spectrophotometry:

    Article Title: Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth
    Article Snippet: Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instruction, and RNA concentration was measured using Nanodrop 2000C spectrophotometer (Wilmington, DE 19810, USA). .. RT-PCR was performed using 1 μg of RNA and SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instruction.

    Concentration Assay:

    Article Title: Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth
    Article Snippet: Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instruction, and RNA concentration was measured using Nanodrop 2000C spectrophotometer (Wilmington, DE 19810, USA). .. RT-PCR was performed using 1 μg of RNA and SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instruction.

    Lysis:

    Article Title: Successful immunotherapy induces previously unidentified allergen-specific CD4+ T-cell subsets
    Article Snippet: .. For single sorted cells, RT-PCR was performed directly in a 96-well PCR plate (ABI) containing lysis buffer (Invitrogen) by using SuperScript III One-Step RT-PCR System with PlatinumTaq (CellDirect kit, Invitrogen). .. PreAmp was performed on a thermocycler using the TaqMan PreAmp Master Mix Kit (Invitrogen) added to cDNA and 0.2× pooled Taqman assays.

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    Thermo Fisher superscript iii rt pcr platinum taq high fidelity kit
    ZIKV insertional mutant library screening protocol and deep sequencing results. (A) Representative images of individual mutants from the library of ZIKV cDNA clones (blue), each bearing a single 15-nucleotide insertion, represented by a colored line (not drawn to scale). (B) 293T cells were transfected with the mutant plasmid library to select for mutant genomes that could initiate RNA replication and virus release. (C) Forty-eight hours after transfection, the supernatant was collected and passaged on Vero cells to select mutants that retained the ability to spread. (D) At 48 h postinfection, the supernatant was passaged once more on Vero cells to select for the fittest mutants. At the time of supernatant collection, RNAs from all <t>three</t> of the cell populations were subjected to <t>RT-PCR</t> to amplify the viral genomes. The subsequent RT-PCR products were then deep sequenced. (E) Raw number of deep sequence reads with inserts at each location for the input plasmid library. The ZIKV genome schematic and bars in the graph show structural genes in red, nonstructural genes in blue, UTRs in black, and the intron used to stabilize the plasmid in bacteria in yellow. This intron was not present in the rescued viruses. Alternating gray and white shading within each graph differentiates the genomic regions. (F to H) Frequencies of insertions at each location following normalization to the representation in the input library after plasmid DNA transfection (F), infected cell passage 1 (G), and infected cell passage 2 (H), with a schematic of the genome shown below. Numbering along the x axis indicates the nucleotide position in the ZIKV genome, while the y axis gives the log raw number of reads (E) or the log fold change over input (F to H), with horizontal dotted lines representing the cutoff of the mutants enriched two standard deviations above the average. Values are averages for three independent screens (see Materials and Methods for details).
    Superscript Iii Rt Pcr Platinum Taq High Fidelity Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ZIKV insertional mutant library screening protocol and deep sequencing results. (A) Representative images of individual mutants from the library of ZIKV cDNA clones (blue), each bearing a single 15-nucleotide insertion, represented by a colored line (not drawn to scale). (B) 293T cells were transfected with the mutant plasmid library to select for mutant genomes that could initiate RNA replication and virus release. (C) Forty-eight hours after transfection, the supernatant was collected and passaged on Vero cells to select mutants that retained the ability to spread. (D) At 48 h postinfection, the supernatant was passaged once more on Vero cells to select for the fittest mutants. At the time of supernatant collection, RNAs from all three of the cell populations were subjected to RT-PCR to amplify the viral genomes. The subsequent RT-PCR products were then deep sequenced. (E) Raw number of deep sequence reads with inserts at each location for the input plasmid library. The ZIKV genome schematic and bars in the graph show structural genes in red, nonstructural genes in blue, UTRs in black, and the intron used to stabilize the plasmid in bacteria in yellow. This intron was not present in the rescued viruses. Alternating gray and white shading within each graph differentiates the genomic regions. (F to H) Frequencies of insertions at each location following normalization to the representation in the input library after plasmid DNA transfection (F), infected cell passage 1 (G), and infected cell passage 2 (H), with a schematic of the genome shown below. Numbering along the x axis indicates the nucleotide position in the ZIKV genome, while the y axis gives the log raw number of reads (E) or the log fold change over input (F to H), with horizontal dotted lines representing the cutoff of the mutants enriched two standard deviations above the average. Values are averages for three independent screens (see Materials and Methods for details).

    Journal: Journal of Virology

    Article Title: Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion

    doi: 10.1128/JVI.00698-17

    Figure Lengend Snippet: ZIKV insertional mutant library screening protocol and deep sequencing results. (A) Representative images of individual mutants from the library of ZIKV cDNA clones (blue), each bearing a single 15-nucleotide insertion, represented by a colored line (not drawn to scale). (B) 293T cells were transfected with the mutant plasmid library to select for mutant genomes that could initiate RNA replication and virus release. (C) Forty-eight hours after transfection, the supernatant was collected and passaged on Vero cells to select mutants that retained the ability to spread. (D) At 48 h postinfection, the supernatant was passaged once more on Vero cells to select for the fittest mutants. At the time of supernatant collection, RNAs from all three of the cell populations were subjected to RT-PCR to amplify the viral genomes. The subsequent RT-PCR products were then deep sequenced. (E) Raw number of deep sequence reads with inserts at each location for the input plasmid library. The ZIKV genome schematic and bars in the graph show structural genes in red, nonstructural genes in blue, UTRs in black, and the intron used to stabilize the plasmid in bacteria in yellow. This intron was not present in the rescued viruses. Alternating gray and white shading within each graph differentiates the genomic regions. (F to H) Frequencies of insertions at each location following normalization to the representation in the input library after plasmid DNA transfection (F), infected cell passage 1 (G), and infected cell passage 2 (H), with a schematic of the genome shown below. Numbering along the x axis indicates the nucleotide position in the ZIKV genome, while the y axis gives the log raw number of reads (E) or the log fold change over input (F to H), with horizontal dotted lines representing the cutoff of the mutants enriched two standard deviations above the average. Values are averages for three independent screens (see Materials and Methods for details).

    Article Snippet: The viral RNA was then amplified in three segments by utilizing a SuperScript III RT-PCR Platinum Taq High Fidelity kit (Thermo Fisher Scientific).

    Techniques: Mutagenesis, Library Screening, Sequencing, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Infection