takara dntp mixture  (TaKaRa)

 
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    Name:
    dNTP Mixture
    Description:
    Our dNTPs for PCR are 98 pure and are tested for quality control in a variety of applications Individual dNTPs are supplied at a concentration of 100 mM and can be diluted with water or buffer as needed The dNTP Set Cat 4025 provides one of each of the four deoxynucleotide triphosphates
    Catalog Number:
    4030
    Price:
    None
    Size:
    3 2 µmol Each 1 25 mL
    Category:
    dNTPs dNTPs Other PCR related products PCR
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    Structured Review

    TaKaRa takara dntp mixture
    Our dNTPs for PCR are 98 pure and are tested for quality control in a variety of applications Individual dNTPs are supplied at a concentration of 100 mM and can be diluted with water or buffer as needed The dNTP Set Cat 4025 provides one of each of the four deoxynucleotide triphosphates
    https://www.bioz.com/result/takara dntp mixture/product/TaKaRa
    Average 99 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    takara dntp mixture - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: A systematic study of the N-glycosylation sites of HIV-1 envelope protein on infectivity and antibody-mediated neutralization
    Article Snippet: Mutagenesis was performed using approximately 50 ng plasmid DNA template and 1 μL of 1 μM primer F (forward), R (reverse) were added into the PCR mixture which contain 10 μL 5X PrimeSTAR® buffer (Mg2+ plus) (TaKaRa, Dalian, China), 4 μL dNTP mixture (2.5 mM) and 0.5 μL PrimeSTAR®HS DNA polymerase (2.5 U/μL) (TaKaRa), then add ddH2 O to a total volume of 50 μL. .. Standard PCR and cloning procedure were used to obtain the mutant clones.

    Amplification:

    Article Title: Fluorescence Visual Detection of Herbal Product Substitutions at Terminal Herbal Markets by CCP-based FRET technique
    Article Snippet: .. PCR amplification was performed in a total volume of 25 μL containing 2.5 μL of 10× Fast Buffer I (Takara, China), 1 μL of dNTP Mixture (2.5 mol·L−1 each, Takara), 0.2 μL of SpeedStar HS Taq DNA polymerase (5 U·μL−1 , Takara), 0.2 μL each of the forward and reverse primers (10 μmol·L−1 , Sangon), and approximately 10 ng of genomic DNA. .. Thermal cycling was performed in a Veriti thermocycler with rapid PCR: an initial denaturation at 94 °C for 2 min, followed by 30 cycles of 5 s at 94 °C and 10 s at 60 °C, and a final extension of 2 min at 72 °C.

    Article Title: Age and gender distribution of Hepatitis C virus prevalence and genotypes of individuals of physical examination in WuHan, Central China
    Article Snippet: .. RT-PCR The cDNA was synthesized from viral RNA in a 20 μL reaction volume including 5 μL RNA and 1 μL Random primer at 65 °C for 6 min, then 4 μL Reaction Buffer, 2 μL dNTP Mixture, 1 μL RNase inhibitor, 1 μL Reverse transcriptase, 6 μL RNase Free H2 O (Takara, Dalian, China) at 42 °C for 60 min, followed by 72 °C for 7 s. HCV fragments were amplified using a PrimeSTAR Kit (Takara, Dalian, China) in a 20 μL reaction volume including cDNA 2 μL, primer 1 μL, mix 10 μL, 7 μL ddH2 O was performed with 1 cycle 42 °C for 5 min, 95 °C 3 min, followed by 40 cycles, each consisting of 94 °C 30 s, 56 °C 50 s, 72 °C 1 min, then 72 °C 10 min, 4 °C 10 min. All manufacturer protocols were followed. .. HCV genotyping analysis The PCR products of partial Core and NS5B were purified (ExpinGel SV, GeneAll Biotechnology, Seoul, Korea) and sequenced using an Applied Biosystems (ABI) PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit Version 3.1, the same PCR primers, and an ABI 3500 DX Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

    Article Title: Essential Domains for Ribonucleoprotein Complex Formation Required for Retrotransposition of Telomere-Specific Non-Long Terminal Repeat Retrotransposon SART1 †
    Article Snippet: .. After heat inactivation at 80°C for 20 min, 1 μl of the reaction mixture was amplified by PCR for 35 cycles of 98°C for 20 s, 60°C for 30 s, and 72°C for 30 s, with an initial denaturation at 96°C for 2 min and a final extension at 72°C for 5 min. PCR mixtures contained 200 μM concentrations of each dNTP, 2 mM MgCl2 , 50 mM KCl, 0.5 U of Ex- Taq (TaKaRa), and 0.5 μM concentrations of each primer (see Table S1 in the supplemental material). .. Purified SART1 complex was overlaid onto 3.3 ml of 10 to 40% glycerol gradient in the HG500 buffer.

    Article Title: Molecular characterization of hemotropic mycoplasmas (Mycoplasma ovis and ‘Candidatus Mycoplasma haemovis’) in sheep and goats in China
    Article Snippet: Nested PCR All samples were tested at least three times for hemoplasmas (M. ovis and ‘Candidatus M. haemovis’) DNA using a nested PCR assay that amplified an approximately 500 bp fragment of the 16S rRNA gene [ ]. .. First-round PCR was performed in a total volume of 25 μl, containing 2.5 μl of 10 × LA PCR Buffer II (Mg2+ plus), 4.0 μl of dNTP mixture (2.5 mM each dNTP), 0.5 μl of each A1 and A2 primer (25 μM), 1.25 U of LA Taq polymerase (TaKaRa, Dalian, China), 16.25 μl of double distilled water, and 1.0 μl of DNA template.

    Article Title: The Effect of Dietary Replacement of Ordinary Rice with Red Yeast Rice on Nutrient Utilization, Enteric Methane Emission and Rumen Archaeal Diversity in Goats
    Article Snippet: The total volume of the reaction mixture was 50 μL consisted of 200 nM of each primer, 5 μL of 2.5 mmol/L dNTP mixture, 5 μL of 10×Ex Taq buffer (20 mmol/L Mg2+ ; TaKaRa Inc., Dalian, China), 0.35 μg of template DNA, 2 mM of MgCl2 and four units of Taq DNA polymerase (Takara Inc., Dalian, China), approximately 37 μL milli-Q water. .. Three replicates of DNA extract from each sample were amplified and were mixed together.

    Article Title: Knockdown of Gli1 by small-interfering RNA enhances the effects of BCNU on the proliferation and apoptosis of glioma U251 cells
    Article Snippet: Thermal cycling amplification was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). .. The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each).

    DNA Synthesis:

    Article Title: Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease
    Article Snippet: .. Resumption of DNA Synthesis by DNA Polymerase after the Removal of CTNAs The hybridized oligonucleotides (400 fmol) were first treated with ERCC1-XPF (230 fmol) in 10 µL of 50 mM Tris-HCl buffer (pH 8.0), containing 2 mM MgCl2 , 0.5 mM DTT and 0.1 mg·mL−1 BSA, at 25 °C for 16 h. To the reaction mixture was added 5 µL of 30 mM Tris-HCl buffer (pH 7.9), containing 150 mM NaCl, 30 mM MgCl2 , 30 mM DTT, 300 µM dNTPs and the Klenow fragment of Escherichia coli DNA polymerase I, lacking the 3′–5′ exonuclease activity (KF− , 0.1 unit, Takara Bio). ..

    Positive Control:

    Article Title: Molecular characterization of hemotropic mycoplasmas (Mycoplasma ovis and ‘Candidatus Mycoplasma haemovis’) in sheep and goats in China
    Article Snippet: First-round PCR was performed in a total volume of 25 μl, containing 2.5 μl of 10 × LA PCR Buffer II (Mg2+ plus), 4.0 μl of dNTP mixture (2.5 mM each dNTP), 0.5 μl of each A1 and A2 primer (25 μM), 1.25 U of LA Taq polymerase (TaKaRa, Dalian, China), 16.25 μl of double distilled water, and 1.0 μl of DNA template. .. Cycling conditions were: 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 45 s, with a final extension at 72 °C for 7 min. A positive control for M. ovis from a naturally infected Boer goat preserved by our laboratory was used in all reactions, while double distilled water was used as a negative control.

    Synthesized:

    Article Title: Age and gender distribution of Hepatitis C virus prevalence and genotypes of individuals of physical examination in WuHan, Central China
    Article Snippet: .. RT-PCR The cDNA was synthesized from viral RNA in a 20 μL reaction volume including 5 μL RNA and 1 μL Random primer at 65 °C for 6 min, then 4 μL Reaction Buffer, 2 μL dNTP Mixture, 1 μL RNase inhibitor, 1 μL Reverse transcriptase, 6 μL RNase Free H2 O (Takara, Dalian, China) at 42 °C for 60 min, followed by 72 °C for 7 s. HCV fragments were amplified using a PrimeSTAR Kit (Takara, Dalian, China) in a 20 μL reaction volume including cDNA 2 μL, primer 1 μL, mix 10 μL, 7 μL ddH2 O was performed with 1 cycle 42 °C for 5 min, 95 °C 3 min, followed by 40 cycles, each consisting of 94 °C 30 s, 56 °C 50 s, 72 °C 1 min, then 72 °C 10 min, 4 °C 10 min. All manufacturer protocols were followed. .. HCV genotyping analysis The PCR products of partial Core and NS5B were purified (ExpinGel SV, GeneAll Biotechnology, Seoul, Korea) and sequenced using an Applied Biosystems (ABI) PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit Version 3.1, the same PCR primers, and an ABI 3500 DX Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

    Construct:

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer
    Article Snippet: The PCR amplifications were performed in reaction volumes of 50 μL containing 10 μL 5 × PrimeSTAR buffer (Mg2+ plus), 0.3 μm forward and reverse primers, 0.2 mm dNTP mixture, 4% DMSO, 0.025 U μL−1 PrimeSTAR HS DNA polymerase (Takara Bio), and 5 μL template genomic DNA using MJ‐Mini thermal cycler. .. The constructed reporter plasmids were transfected using Lipofectamine 3000 reagent (Thermo Fisher Scientific) into MCF10A‐ErbB2, MCF10A‐RafCAAX, or MCF10A‐neo cells, together with pGL4.74 Renilla luciferase plasmid (Promega).

    Real-time Polymerase Chain Reaction:

    Article Title: Knockdown of Gli1 by small-interfering RNA enhances the effects of BCNU on the proliferation and apoptosis of glioma U251 cells
    Article Snippet: Paragraph title: Semi-quantitative real-time polymerase chain reaction (semi-qRT-PCR) ... The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each).

    Incubation:

    Article Title: Essential Domains for Ribonucleoprotein Complex Formation Required for Retrotransposition of Telomere-Specific Non-Long Terminal Repeat Retrotransposon SART1 †
    Article Snippet: Ten nanograms of SART1 RNP was added to 40 μl of buffer and was incubated at 30°C for 60 min. .. After heat inactivation at 80°C for 20 min, 1 μl of the reaction mixture was amplified by PCR for 35 cycles of 98°C for 20 s, 60°C for 30 s, and 72°C for 30 s, with an initial denaturation at 96°C for 2 min and a final extension at 72°C for 5 min. PCR mixtures contained 200 μM concentrations of each dNTP, 2 mM MgCl2 , 50 mM KCl, 0.5 U of Ex- Taq (TaKaRa), and 0.5 μM concentrations of each primer (see Table S1 in the supplemental material).

    Luciferase:

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer
    Article Snippet: The PCR amplifications were performed in reaction volumes of 50 μL containing 10 μL 5 × PrimeSTAR buffer (Mg2+ plus), 0.3 μm forward and reverse primers, 0.2 mm dNTP mixture, 4% DMSO, 0.025 U μL−1 PrimeSTAR HS DNA polymerase (Takara Bio), and 5 μL template genomic DNA using MJ‐Mini thermal cycler. .. The constructed reporter plasmids were transfected using Lipofectamine 3000 reagent (Thermo Fisher Scientific) into MCF10A‐ErbB2, MCF10A‐RafCAAX, or MCF10A‐neo cells, together with pGL4.74 Renilla luciferase plasmid (Promega).

    Activity Assay:

    Article Title: Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease
    Article Snippet: .. Resumption of DNA Synthesis by DNA Polymerase after the Removal of CTNAs The hybridized oligonucleotides (400 fmol) were first treated with ERCC1-XPF (230 fmol) in 10 µL of 50 mM Tris-HCl buffer (pH 8.0), containing 2 mM MgCl2 , 0.5 mM DTT and 0.1 mg·mL−1 BSA, at 25 °C for 16 h. To the reaction mixture was added 5 µL of 30 mM Tris-HCl buffer (pH 7.9), containing 150 mM NaCl, 30 mM MgCl2 , 30 mM DTT, 300 µM dNTPs and the Klenow fragment of Escherichia coli DNA polymerase I, lacking the 3′–5′ exonuclease activity (KF− , 0.1 unit, Takara Bio). ..

    Reporter Assay:

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer
    Article Snippet: Paragraph title: Reporter plasmid construction and reporter assay ... The PCR amplifications were performed in reaction volumes of 50 μL containing 10 μL 5 × PrimeSTAR buffer (Mg2+ plus), 0.3 μm forward and reverse primers, 0.2 mm dNTP mixture, 4% DMSO, 0.025 U μL−1 PrimeSTAR HS DNA polymerase (Takara Bio), and 5 μL template genomic DNA using MJ‐Mini thermal cycler.

    High Performance Liquid Chromatography:

    Article Title: A new, but old, nucleoside analog: the first synthesis of 1-deaza-2?-deoxyguanosine and its properties as a nucleoside and as oligodeoxynucleotides
    Article Snippet: RP-HPLC analysis and purifications were performed on a Gilson HPLC system. .. AMV reverse transcriptase, M-MLV reverse transcriptase and dNTPs were purchased from Takara Biomedicals (Japan).

    Transfection:

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer
    Article Snippet: The PCR amplifications were performed in reaction volumes of 50 μL containing 10 μL 5 × PrimeSTAR buffer (Mg2+ plus), 0.3 μm forward and reverse primers, 0.2 mm dNTP mixture, 4% DMSO, 0.025 U μL−1 PrimeSTAR HS DNA polymerase (Takara Bio), and 5 μL template genomic DNA using MJ‐Mini thermal cycler. .. The constructed reporter plasmids were transfected using Lipofectamine 3000 reagent (Thermo Fisher Scientific) into MCF10A‐ErbB2, MCF10A‐RafCAAX, or MCF10A‐neo cells, together with pGL4.74 Renilla luciferase plasmid (Promega).

    Sequencing:

    Article Title: The Effect of Dietary Replacement of Ordinary Rice with Red Yeast Rice on Nutrient Utilization, Enteric Methane Emission and Rumen Archaeal Diversity in Goats
    Article Snippet: The total volume of the reaction mixture was 50 μL consisted of 200 nM of each primer, 5 μL of 2.5 mmol/L dNTP mixture, 5 μL of 10×Ex Taq buffer (20 mmol/L Mg2+ ; TaKaRa Inc., Dalian, China), 0.35 μg of template DNA, 2 mM of MgCl2 and four units of Taq DNA polymerase (Takara Inc., Dalian, China), approximately 37 μL milli-Q water. .. The PCR products were sent to Macrogen Inc (Seoul, South Korea) and were throughput sequenced on the Illumina MiSeq 300PE Sequencing Platform.

    Infection:

    Article Title: Molecular characterization of hemotropic mycoplasmas (Mycoplasma ovis and ‘Candidatus Mycoplasma haemovis’) in sheep and goats in China
    Article Snippet: First-round PCR was performed in a total volume of 25 μl, containing 2.5 μl of 10 × LA PCR Buffer II (Mg2+ plus), 4.0 μl of dNTP mixture (2.5 mM each dNTP), 0.5 μl of each A1 and A2 primer (25 μM), 1.25 U of LA Taq polymerase (TaKaRa, Dalian, China), 16.25 μl of double distilled water, and 1.0 μl of DNA template. .. Cycling conditions were: 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 45 s, with a final extension at 72 °C for 7 min. A positive control for M. ovis from a naturally infected Boer goat preserved by our laboratory was used in all reactions, while double distilled water was used as a negative control.

    Imaging:

    Article Title: Knockdown of Gli1 by small-interfering RNA enhances the effects of BCNU on the proliferation and apoptosis of glioma U251 cells
    Article Snippet: The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). .. PCR amplification conditions for Gli1, Bcl-2, cyclin D1, and Bax were as follows: initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 56°C for 30 sec, annealing at 60°C for 60 sec, and elongation at 72°C for 60 sec; and final elongation at 72°C for 7 min. PCR amplification conditions for β-actin were: initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 95°C for 30 sec, annealing at 59°C for 30 sec, and elongation at 72°C for 90 sec; and final elongation at 72°C for 7 min. After amplification, the PCR products were loaded to 2% (W/V) agarose gel (1× TBE buffer) containing 0.5 μg/ml bromoethane for electrophoresis, followed by imaging with ultraviolet projection reflectometer (ChemiDoc MP, Bio-Rad, Hercules, CA, USA).

    Polymerase Chain Reaction:

    Article Title: Fluorescence Visual Detection of Herbal Product Substitutions at Terminal Herbal Markets by CCP-based FRET technique
    Article Snippet: .. PCR amplification was performed in a total volume of 25 μL containing 2.5 μL of 10× Fast Buffer I (Takara, China), 1 μL of dNTP Mixture (2.5 mol·L−1 each, Takara), 0.2 μL of SpeedStar HS Taq DNA polymerase (5 U·μL−1 , Takara), 0.2 μL each of the forward and reverse primers (10 μmol·L−1 , Sangon), and approximately 10 ng of genomic DNA. .. Thermal cycling was performed in a Veriti thermocycler with rapid PCR: an initial denaturation at 94 °C for 2 min, followed by 30 cycles of 5 s at 94 °C and 10 s at 60 °C, and a final extension of 2 min at 72 °C.

    Article Title: A systematic study of the N-glycosylation sites of HIV-1 envelope protein on infectivity and antibody-mediated neutralization
    Article Snippet: .. Mutagenesis was performed using approximately 50 ng plasmid DNA template and 1 μL of 1 μM primer F (forward), R (reverse) were added into the PCR mixture which contain 10 μL 5X PrimeSTAR® buffer (Mg2+ plus) (TaKaRa, Dalian, China), 4 μL dNTP mixture (2.5 mM) and 0.5 μL PrimeSTAR®HS DNA polymerase (2.5 U/μL) (TaKaRa), then add ddH2 O to a total volume of 50 μL. .. Standard PCR and cloning procedure were used to obtain the mutant clones.

    Article Title: Essential Domains for Ribonucleoprotein Complex Formation Required for Retrotransposition of Telomere-Specific Non-Long Terminal Repeat Retrotransposon SART1 †
    Article Snippet: .. After heat inactivation at 80°C for 20 min, 1 μl of the reaction mixture was amplified by PCR for 35 cycles of 98°C for 20 s, 60°C for 30 s, and 72°C for 30 s, with an initial denaturation at 96°C for 2 min and a final extension at 72°C for 5 min. PCR mixtures contained 200 μM concentrations of each dNTP, 2 mM MgCl2 , 50 mM KCl, 0.5 U of Ex- Taq (TaKaRa), and 0.5 μM concentrations of each primer (see Table S1 in the supplemental material). .. Purified SART1 complex was overlaid onto 3.3 ml of 10 to 40% glycerol gradient in the HG500 buffer.

    Article Title: Molecular characterization of hemotropic mycoplasmas (Mycoplasma ovis and ‘Candidatus Mycoplasma haemovis’) in sheep and goats in China
    Article Snippet: .. First-round PCR was performed in a total volume of 25 μl, containing 2.5 μl of 10 × LA PCR Buffer II (Mg2+ plus), 4.0 μl of dNTP mixture (2.5 mM each dNTP), 0.5 μl of each A1 and A2 primer (25 μM), 1.25 U of LA Taq polymerase (TaKaRa, Dalian, China), 16.25 μl of double distilled water, and 1.0 μl of DNA template. .. Cycling conditions were: 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 61 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 7 min. Second-round nested PCR contained 2.5 μl of 10 × PCR Buffer, 2.0 μl of dNTP mixture (2.5 mM each dNTP), 0.5 μl of each B1 and B2 primer (25 μM), 0.75 U of r Taq polymerase (TaKaRa), 18.35 μl of double distilled water, and 1.0 μl of the first-round PCR product (diluted 10-fold).

    Article Title: The Effect of Dietary Replacement of Ordinary Rice with Red Yeast Rice on Nutrient Utilization, Enteric Methane Emission and Rumen Archaeal Diversity in Goats
    Article Snippet: The total volume of the reaction mixture was 50 μL consisted of 200 nM of each primer, 5 μL of 2.5 mmol/L dNTP mixture, 5 μL of 10×Ex Taq buffer (20 mmol/L Mg2+ ; TaKaRa Inc., Dalian, China), 0.35 μg of template DNA, 2 mM of MgCl2 and four units of Taq DNA polymerase (Takara Inc., Dalian, China), approximately 37 μL milli-Q water. .. The amplicons were purified using a PCR Clean-Up system (Promega, Madison, USA) with a purification kit (QIAGEN, Australia), and were quantified using QuantiFlour™ -ST fluorometer (Promega, Madison, USA).

    Article Title: Knockdown of Gli1 by small-interfering RNA enhances the effects of BCNU on the proliferation and apoptosis of glioma U251 cells
    Article Snippet: Thermal cycling amplification was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). .. The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each).

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer
    Article Snippet: .. The PCR amplifications were performed in reaction volumes of 50 μL containing 10 μL 5 × PrimeSTAR buffer (Mg2+ plus), 0.3 μm forward and reverse primers, 0.2 mm dNTP mixture, 4% DMSO, 0.025 U μL−1 PrimeSTAR HS DNA polymerase (Takara Bio), and 5 μL template genomic DNA using MJ‐Mini thermal cycler. .. The thermal cycling conditions were as follows: 30 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 5 s, and extension at 72 °C for 20 s. The final PCR products were inserted into pGL4.14 vector (Promega) digested with Eco RV (Roche).

    Mutagenesis:

    Article Title: Fluorescence Visual Detection of Herbal Product Substitutions at Terminal Herbal Markets by CCP-based FRET technique
    Article Snippet: PCR amplification PCR was performed to amplify gene fragments that included the mutation sites of interest. .. PCR amplification was performed in a total volume of 25 μL containing 2.5 μL of 10× Fast Buffer I (Takara, China), 1 μL of dNTP Mixture (2.5 mol·L−1 each, Takara), 0.2 μL of SpeedStar HS Taq DNA polymerase (5 U·μL−1 , Takara), 0.2 μL each of the forward and reverse primers (10 μmol·L−1 , Sangon), and approximately 10 ng of genomic DNA.

    Article Title: A systematic study of the N-glycosylation sites of HIV-1 envelope protein on infectivity and antibody-mediated neutralization
    Article Snippet: .. Mutagenesis was performed using approximately 50 ng plasmid DNA template and 1 μL of 1 μM primer F (forward), R (reverse) were added into the PCR mixture which contain 10 μL 5X PrimeSTAR® buffer (Mg2+ plus) (TaKaRa, Dalian, China), 4 μL dNTP mixture (2.5 mM) and 0.5 μL PrimeSTAR®HS DNA polymerase (2.5 U/μL) (TaKaRa), then add ddH2 O to a total volume of 50 μL. .. Standard PCR and cloning procedure were used to obtain the mutant clones.

    Size-exclusion Chromatography:

    Article Title: Knockdown of Gli1 by small-interfering RNA enhances the effects of BCNU on the proliferation and apoptosis of glioma U251 cells
    Article Snippet: The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). .. PCR amplification conditions for Gli1, Bcl-2, cyclin D1, and Bax were as follows: initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 56°C for 30 sec, annealing at 60°C for 60 sec, and elongation at 72°C for 60 sec; and final elongation at 72°C for 7 min. PCR amplification conditions for β-actin were: initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 95°C for 30 sec, annealing at 59°C for 30 sec, and elongation at 72°C for 90 sec; and final elongation at 72°C for 7 min. After amplification, the PCR products were loaded to 2% (W/V) agarose gel (1× TBE buffer) containing 0.5 μg/ml bromoethane for electrophoresis, followed by imaging with ultraviolet projection reflectometer (ChemiDoc MP, Bio-Rad, Hercules, CA, USA).

    Purification:

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication
    Article Snippet: Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara). .. Most of the proteins described above were purified according to a previously described method except for yeast inorganic pyrophosphatase, ribonuclease inhibitor, and T7 RNA polymerase.

    Article Title: Essential Domains for Ribonucleoprotein Complex Formation Required for Retrotransposition of Telomere-Specific Non-Long Terminal Repeat Retrotransposon SART1 †
    Article Snippet: In the in vitro rescue experiment (shown in Fig. ) 50 ng of WT SART1 ORF1p, which was purified in parallel, was simultaneously added to the buffer. .. After heat inactivation at 80°C for 20 min, 1 μl of the reaction mixture was amplified by PCR for 35 cycles of 98°C for 20 s, 60°C for 30 s, and 72°C for 30 s, with an initial denaturation at 96°C for 2 min and a final extension at 72°C for 5 min. PCR mixtures contained 200 μM concentrations of each dNTP, 2 mM MgCl2 , 50 mM KCl, 0.5 U of Ex- Taq (TaKaRa), and 0.5 μM concentrations of each primer (see Table S1 in the supplemental material).

    Article Title: The Effect of Dietary Replacement of Ordinary Rice with Red Yeast Rice on Nutrient Utilization, Enteric Methane Emission and Rumen Archaeal Diversity in Goats
    Article Snippet: Analysis of rumen arechaeal composition The total DNAs in the rumen liquid were extracted and purified using the method described previously [ ]. .. The total volume of the reaction mixture was 50 μL consisted of 200 nM of each primer, 5 μL of 2.5 mmol/L dNTP mixture, 5 μL of 10×Ex Taq buffer (20 mmol/L Mg2+ ; TaKaRa Inc., Dalian, China), 0.35 μg of template DNA, 2 mM of MgCl2 and four units of Taq DNA polymerase (Takara Inc., Dalian, China), approximately 37 μL milli-Q water.

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer
    Article Snippet: Reporter plasmid construction and reporter assay Genomic DNA was purified from MCF10A cells by using DNeasy Blood & Tissue kit (Qiagen). .. The PCR amplifications were performed in reaction volumes of 50 μL containing 10 μL 5 × PrimeSTAR buffer (Mg2+ plus), 0.3 μm forward and reverse primers, 0.2 mm dNTP mixture, 4% DMSO, 0.025 U μL−1 PrimeSTAR HS DNA polymerase (Takara Bio), and 5 μL template genomic DNA using MJ‐Mini thermal cycler.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Age and gender distribution of Hepatitis C virus prevalence and genotypes of individuals of physical examination in WuHan, Central China
    Article Snippet: .. RT-PCR The cDNA was synthesized from viral RNA in a 20 μL reaction volume including 5 μL RNA and 1 μL Random primer at 65 °C for 6 min, then 4 μL Reaction Buffer, 2 μL dNTP Mixture, 1 μL RNase inhibitor, 1 μL Reverse transcriptase, 6 μL RNase Free H2 O (Takara, Dalian, China) at 42 °C for 60 min, followed by 72 °C for 7 s. HCV fragments were amplified using a PrimeSTAR Kit (Takara, Dalian, China) in a 20 μL reaction volume including cDNA 2 μL, primer 1 μL, mix 10 μL, 7 μL ddH2 O was performed with 1 cycle 42 °C for 5 min, 95 °C 3 min, followed by 40 cycles, each consisting of 94 °C 30 s, 56 °C 50 s, 72 °C 1 min, then 72 °C 10 min, 4 °C 10 min. All manufacturer protocols were followed. .. HCV genotyping analysis The PCR products of partial Core and NS5B were purified (ExpinGel SV, GeneAll Biotechnology, Seoul, Korea) and sequenced using an Applied Biosystems (ABI) PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit Version 3.1, the same PCR primers, and an ABI 3500 DX Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

    Article Title: Knockdown of Gli1 by small-interfering RNA enhances the effects of BCNU on the proliferation and apoptosis of glioma U251 cells
    Article Snippet: Thermal cycling amplification was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). .. The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each).

    Staining:

    Article Title: Molecular characterization of hemotropic mycoplasmas (Mycoplasma ovis and ‘Candidatus Mycoplasma haemovis’) in sheep and goats in China
    Article Snippet: First-round PCR was performed in a total volume of 25 μl, containing 2.5 μl of 10 × LA PCR Buffer II (Mg2+ plus), 4.0 μl of dNTP mixture (2.5 mM each dNTP), 0.5 μl of each A1 and A2 primer (25 μM), 1.25 U of LA Taq polymerase (TaKaRa, Dalian, China), 16.25 μl of double distilled water, and 1.0 μl of DNA template. .. Secondary PCR products were examined by 1% agarose gel electrophoresis and visualized after GelRed (Biotium Inc., Hayward, CA) staining.

    Nested PCR:

    Article Title: Molecular characterization of hemotropic mycoplasmas (Mycoplasma ovis and ‘Candidatus Mycoplasma haemovis’) in sheep and goats in China
    Article Snippet: Paragraph title: Nested PCR ... First-round PCR was performed in a total volume of 25 μl, containing 2.5 μl of 10 × LA PCR Buffer II (Mg2+ plus), 4.0 μl of dNTP mixture (2.5 mM each dNTP), 0.5 μl of each A1 and A2 primer (25 μM), 1.25 U of LA Taq polymerase (TaKaRa, Dalian, China), 16.25 μl of double distilled water, and 1.0 μl of DNA template.

    Plasmid Preparation:

    Article Title: A systematic study of the N-glycosylation sites of HIV-1 envelope protein on infectivity and antibody-mediated neutralization
    Article Snippet: .. Mutagenesis was performed using approximately 50 ng plasmid DNA template and 1 μL of 1 μM primer F (forward), R (reverse) were added into the PCR mixture which contain 10 μL 5X PrimeSTAR® buffer (Mg2+ plus) (TaKaRa, Dalian, China), 4 μL dNTP mixture (2.5 mM) and 0.5 μL PrimeSTAR®HS DNA polymerase (2.5 U/μL) (TaKaRa), then add ddH2 O to a total volume of 50 μL. .. Standard PCR and cloning procedure were used to obtain the mutant clones.

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication
    Article Snippet: .. Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara). .. Most of the proteins described above were purified according to a previously described method except for yeast inorganic pyrophosphatase, ribonuclease inhibitor, and T7 RNA polymerase.

    Article Title: Essential Domains for Ribonucleoprotein Complex Formation Required for Retrotransposition of Telomere-Specific Non-Long Terminal Repeat Retrotransposon SART1 †
    Article Snippet: The in vitro TPRT reaction buffer included 50 mM HEPES-KOH, pH 7.9, 3 mM MgCl2 , 10 mM dithiothreitol, 0.01% bovine serum albumin (BSA), 0.5 mM concentrations of each deoxynucleoside triphosphate (dNTP), and 50 ng of target plasmid containing the Bombyx telomeric repeats. .. After heat inactivation at 80°C for 20 min, 1 μl of the reaction mixture was amplified by PCR for 35 cycles of 98°C for 20 s, 60°C for 30 s, and 72°C for 30 s, with an initial denaturation at 96°C for 2 min and a final extension at 72°C for 5 min. PCR mixtures contained 200 μM concentrations of each dNTP, 2 mM MgCl2 , 50 mM KCl, 0.5 U of Ex- Taq (TaKaRa), and 0.5 μM concentrations of each primer (see Table S1 in the supplemental material).

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer
    Article Snippet: Paragraph title: Reporter plasmid construction and reporter assay ... The PCR amplifications were performed in reaction volumes of 50 μL containing 10 μL 5 × PrimeSTAR buffer (Mg2+ plus), 0.3 μm forward and reverse primers, 0.2 mm dNTP mixture, 4% DMSO, 0.025 U μL−1 PrimeSTAR HS DNA polymerase (Takara Bio), and 5 μL template genomic DNA using MJ‐Mini thermal cycler.

    Electrophoresis:

    Article Title: Knockdown of Gli1 by small-interfering RNA enhances the effects of BCNU on the proliferation and apoptosis of glioma U251 cells
    Article Snippet: The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). .. PCR amplification conditions for Gli1, Bcl-2, cyclin D1, and Bax were as follows: initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 56°C for 30 sec, annealing at 60°C for 60 sec, and elongation at 72°C for 60 sec; and final elongation at 72°C for 7 min. PCR amplification conditions for β-actin were: initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 95°C for 30 sec, annealing at 59°C for 30 sec, and elongation at 72°C for 90 sec; and final elongation at 72°C for 7 min. After amplification, the PCR products were loaded to 2% (W/V) agarose gel (1× TBE buffer) containing 0.5 μg/ml bromoethane for electrophoresis, followed by imaging with ultraviolet projection reflectometer (ChemiDoc MP, Bio-Rad, Hercules, CA, USA).

    Negative Control:

    Article Title: Molecular characterization of hemotropic mycoplasmas (Mycoplasma ovis and ‘Candidatus Mycoplasma haemovis’) in sheep and goats in China
    Article Snippet: First-round PCR was performed in a total volume of 25 μl, containing 2.5 μl of 10 × LA PCR Buffer II (Mg2+ plus), 4.0 μl of dNTP mixture (2.5 mM each dNTP), 0.5 μl of each A1 and A2 primer (25 μM), 1.25 U of LA Taq polymerase (TaKaRa, Dalian, China), 16.25 μl of double distilled water, and 1.0 μl of DNA template. .. Cycling conditions were: 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 45 s, with a final extension at 72 °C for 7 min. A positive control for M. ovis from a naturally infected Boer goat preserved by our laboratory was used in all reactions, while double distilled water was used as a negative control.

    Agarose Gel Electrophoresis:

    Article Title: Molecular characterization of hemotropic mycoplasmas (Mycoplasma ovis and ‘Candidatus Mycoplasma haemovis’) in sheep and goats in China
    Article Snippet: First-round PCR was performed in a total volume of 25 μl, containing 2.5 μl of 10 × LA PCR Buffer II (Mg2+ plus), 4.0 μl of dNTP mixture (2.5 mM each dNTP), 0.5 μl of each A1 and A2 primer (25 μM), 1.25 U of LA Taq polymerase (TaKaRa, Dalian, China), 16.25 μl of double distilled water, and 1.0 μl of DNA template. .. Secondary PCR products were examined by 1% agarose gel electrophoresis and visualized after GelRed (Biotium Inc., Hayward, CA) staining.

    Article Title: Knockdown of Gli1 by small-interfering RNA enhances the effects of BCNU on the proliferation and apoptosis of glioma U251 cells
    Article Snippet: The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). .. PCR amplification conditions for Gli1, Bcl-2, cyclin D1, and Bax were as follows: initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 56°C for 30 sec, annealing at 60°C for 60 sec, and elongation at 72°C for 60 sec; and final elongation at 72°C for 7 min. PCR amplification conditions for β-actin were: initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 95°C for 30 sec, annealing at 59°C for 30 sec, and elongation at 72°C for 90 sec; and final elongation at 72°C for 7 min. After amplification, the PCR products were loaded to 2% (W/V) agarose gel (1× TBE buffer) containing 0.5 μg/ml bromoethane for electrophoresis, followed by imaging with ultraviolet projection reflectometer (ChemiDoc MP, Bio-Rad, Hercules, CA, USA).

    In Vitro:

    Article Title: Essential Domains for Ribonucleoprotein Complex Formation Required for Retrotransposition of Telomere-Specific Non-Long Terminal Repeat Retrotransposon SART1 †
    Article Snippet: Paragraph title: In vitro TPRT reaction. ... After heat inactivation at 80°C for 20 min, 1 μl of the reaction mixture was amplified by PCR for 35 cycles of 98°C for 20 s, 60°C for 30 s, and 72°C for 30 s, with an initial denaturation at 96°C for 2 min and a final extension at 72°C for 5 min. PCR mixtures contained 200 μM concentrations of each dNTP, 2 mM MgCl2 , 50 mM KCl, 0.5 U of Ex- Taq (TaKaRa), and 0.5 μM concentrations of each primer (see Table S1 in the supplemental material).

    Column Chromatography:

    Article Title: A new, but old, nucleoside analog: the first synthesis of 1-deaza-2?-deoxyguanosine and its properties as a nucleoside and as oligodeoxynucleotides
    Article Snippet: Silica gels used for column chromatography were Wakogel C-200 (particle size 75–150 µm) (Wako Pure Chemical Industries, Japan) or ICN Silica 60Å (particle size 63–200 µm) (ICN Biomedicals, Belgium). .. AMV reverse transcriptase, M-MLV reverse transcriptase and dNTPs were purchased from Takara Biomedicals (Japan).

    Produced:

    Article Title: Small Molecular Contaminant and Microorganism Can Be Simultaneously Detected Based on Nanobody-Phage: Using Carcinogen Aflatoxin and Its Main Fungal Aspergillus Section Flavi spp. in Stored Maize for Demonstration
    Article Snippet: DNA polymerase (iTaq), Mg2+ , dNTPs, 6× loading buffer, and DNA marker were bought from Takara Bio (Beijing, China). .. The anti-aflatoxins monoclonal antibody 1C11 (mAb 1C11) and V2–5 phage displaying nanobody specific for 1C11 were produced by our team ( ; ).

    Thin Layer Chromatography:

    Article Title: A new, but old, nucleoside analog: the first synthesis of 1-deaza-2?-deoxyguanosine and its properties as a nucleoside and as oligodeoxynucleotides
    Article Snippet: TLC was done on Merck silica gel 60F254 precoated plates (Merck, Germany). .. AMV reverse transcriptase, M-MLV reverse transcriptase and dNTPs were purchased from Takara Biomedicals (Japan).

    Marker:

    Article Title: Small Molecular Contaminant and Microorganism Can Be Simultaneously Detected Based on Nanobody-Phage: Using Carcinogen Aflatoxin and Its Main Fungal Aspergillus Section Flavi spp. in Stored Maize for Demonstration
    Article Snippet: .. DNA polymerase (iTaq), Mg2+ , dNTPs, 6× loading buffer, and DNA marker were bought from Takara Bio (Beijing, China). .. The anti-aflatoxins monoclonal antibody 1C11 (mAb 1C11) and V2–5 phage displaying nanobody specific for 1C11 were produced by our team ( ; ).

    Lysis:

    Article Title: ErbB2 signaling epigenetically suppresses micro RNA‐205 transcription via Ras/Raf/ MEK/ ERK pathway in breast cancer
    Article Snippet: The PCR amplifications were performed in reaction volumes of 50 μL containing 10 μL 5 × PrimeSTAR buffer (Mg2+ plus), 0.3 μm forward and reverse primers, 0.2 mm dNTP mixture, 4% DMSO, 0.025 U μL−1 PrimeSTAR HS DNA polymerase (Takara Bio), and 5 μL template genomic DNA using MJ‐Mini thermal cycler. .. At 36 h post‐transfection, cells were lysed in Passive Lysis Buffer (Promega), and firefly and Renilla luciferase reporter activities were determined.

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    TaKaRa telomeric repeat amplification protocol trap lig assay dntp mix
    The Pt-tripod binds the intramolecular hybrid-1 human <t>telomeric</t> G-quadruplex and inhibits telomerase activity. a The chemical structure of the Pt-tripod with proton numbering. b The folding topology of the hybrid-1 human telomeric G-quadruplex adopted by Tel26 in K + solution 44 . Deep gray box = ( syn ) guanine, light gray box = ( anti ) guanine. c Imino proton regions of the 1D 1 H NMR titration spectra of Tel26 interacting with Pt-tripod in 100 mM K + , pH 7.0 solution, at 25 °C. Ratios of Pt-tripod/Tel26 are shown in the spectra. The imino assignments are labeled. Peaks arising from the new complexes are marked with asterisks. d CD spectra of the Tel26 titrated by the Pt-tripod in 100 mM K + solution. Ratios of Pt-tripod/Tel26 are shown in the spectra. e The Pt-tripod showed excellent telomerase inhibition property by the <t>TRAP-LIG</t> assay. The band labeled as ITAS is an internal control primer. The IC 50 value is determined to be 1.22 ± 0.10 μM. Error value represents the standard deviation, n = 3
    Telomeric Repeat Amplification Protocol Trap Lig Assay Dntp Mix, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/telomeric repeat amplification protocol trap lig assay dntp mix/product/TaKaRa
    Average 93 stars, based on 1 article reviews
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    telomeric repeat amplification protocol trap lig assay dntp mix - by Bioz Stars, 2020-04
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    99
    TaKaRa dntps
    Primer extension from the CTNA-blocked termini by the Klenow fragment of Escherichia coli DNA polymerase I, with or without its proofreading 3′–5′ exonuclease activity (KF + or KF − , respectively, from Takara Bio, Inc., Shiga, Japan), in the ( A ) presence or ( B ) absence of <t>dNTPs.</t> ( A ) The 32 P-labeled oligonucleotides, 32 P-d(TCCGTTGAAGCCTGCTTT)X, where X represents no added nucleoside (OH, lanes 1–3), 2’-deoxyadenosine (lanes 4–6), acyclovir (ACV, lanes 7–9), abacavir (ABC, lanes 10–12), carbovir (CBV, lanes 13–15) or lamivudine ((−)3TC, lanes 16–18), were hybridized with their complementary strands, d(CTCGTCAGCTANAAAGCAGGCTTCAACGGA), where N represents A (for ABC and an oligonucleotide without CTNAs), G (for A and (−)3TC) or C (for ACV and CBV). Each substrate was incubated at 37 °C for 10 min, in the absence (lanes 1, 4, 7, 10, 13 and 16) or presence of KF − (0.1 unit, lanes 2, 5, 8, 11, 14 and 17) or KF + (0.1 unit, lanes 3, 6, 9, 12, 15 and 18), in 10 mM Tris-HCl buffer (pH 7.9) containing 50 mM <t>NaCl,</t> 10 mM MgCl 2 , 10 mM DTT and 100 µM dNTPs; ( B ) The 32 P-labeled substrates were incubated with KF + at 37 °C for the indicated incubation time, in the same reaction buffer without dNTPs.
    Dntps, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1675 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dntps/product/TaKaRa
    Average 99 stars, based on 1675 article reviews
    Price from $9.99 to $1999.99
    dntps - by Bioz Stars, 2020-04
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    86
    TaKaRa pcr mixture iii
    Affect of residual RT reagents on subsequent reactions. The number of cDNA molecules was evaluated by qPCR. Error bars are independent amplification replicates. ( a ) The residual RT reagents in a poly(A)-tailing reaction mixture containing bead-supported cDNA made biases large and amplification factors small (red column). The difficulty was overcome by washing beads (green column). ( b ) The addition of RT reagents to purified model cDNAs free in a poly(A)-tailing reaction mixture made biases large and amplification factors small as well. After purifying model cDNA with a purification kit, RT reagents were added to cDNA (red column) or not added (green column). ( c ) RT reagents did not affect the 2nd-strand cDNA synthesis and <t>PCR.</t> ( d ) Amplification efficiency was strongly affected by the presence of SuperScript <t>III.</t> The 11 components in an RT reagent kit were, respectively, added to 11 purified RT samples before poly(A) tailing. The 1st PCR products were evaluated by qPCR. Although the original amounts of samples were the same, the product produced by adding SuperScript III was small.
    Pcr Mixture Iii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The Pt-tripod binds the intramolecular hybrid-1 human telomeric G-quadruplex and inhibits telomerase activity. a The chemical structure of the Pt-tripod with proton numbering. b The folding topology of the hybrid-1 human telomeric G-quadruplex adopted by Tel26 in K + solution 44 . Deep gray box = ( syn ) guanine, light gray box = ( anti ) guanine. c Imino proton regions of the 1D 1 H NMR titration spectra of Tel26 interacting with Pt-tripod in 100 mM K + , pH 7.0 solution, at 25 °C. Ratios of Pt-tripod/Tel26 are shown in the spectra. The imino assignments are labeled. Peaks arising from the new complexes are marked with asterisks. d CD spectra of the Tel26 titrated by the Pt-tripod in 100 mM K + solution. Ratios of Pt-tripod/Tel26 are shown in the spectra. e The Pt-tripod showed excellent telomerase inhibition property by the TRAP-LIG assay. The band labeled as ITAS is an internal control primer. The IC 50 value is determined to be 1.22 ± 0.10 μM. Error value represents the standard deviation, n = 3

    Journal: Nature Communications

    Article Title: Solution structures of multiple G-quadruplex complexes induced by a platinum(II)-based tripod reveal dynamic binding

    doi: 10.1038/s41467-018-05810-4

    Figure Lengend Snippet: The Pt-tripod binds the intramolecular hybrid-1 human telomeric G-quadruplex and inhibits telomerase activity. a The chemical structure of the Pt-tripod with proton numbering. b The folding topology of the hybrid-1 human telomeric G-quadruplex adopted by Tel26 in K + solution 44 . Deep gray box = ( syn ) guanine, light gray box = ( anti ) guanine. c Imino proton regions of the 1D 1 H NMR titration spectra of Tel26 interacting with Pt-tripod in 100 mM K + , pH 7.0 solution, at 25 °C. Ratios of Pt-tripod/Tel26 are shown in the spectra. The imino assignments are labeled. Peaks arising from the new complexes are marked with asterisks. d CD spectra of the Tel26 titrated by the Pt-tripod in 100 mM K + solution. Ratios of Pt-tripod/Tel26 are shown in the spectra. e The Pt-tripod showed excellent telomerase inhibition property by the TRAP-LIG assay. The band labeled as ITAS is an internal control primer. The IC 50 value is determined to be 1.22 ± 0.10 μM. Error value represents the standard deviation, n = 3

    Article Snippet: Telomeric repeat amplification protocol (TRAP-LIG) assay dNTP mix, RNase inhibitor, and Taq polymerase were purchased from TaKaRa Biotechnology.

    Techniques: Activity Assay, Nuclear Magnetic Resonance, Titration, Labeling, Inhibition, Standard Deviation

    Primer extension from the CTNA-blocked termini by the Klenow fragment of Escherichia coli DNA polymerase I, with or without its proofreading 3′–5′ exonuclease activity (KF + or KF − , respectively, from Takara Bio, Inc., Shiga, Japan), in the ( A ) presence or ( B ) absence of dNTPs. ( A ) The 32 P-labeled oligonucleotides, 32 P-d(TCCGTTGAAGCCTGCTTT)X, where X represents no added nucleoside (OH, lanes 1–3), 2’-deoxyadenosine (lanes 4–6), acyclovir (ACV, lanes 7–9), abacavir (ABC, lanes 10–12), carbovir (CBV, lanes 13–15) or lamivudine ((−)3TC, lanes 16–18), were hybridized with their complementary strands, d(CTCGTCAGCTANAAAGCAGGCTTCAACGGA), where N represents A (for ABC and an oligonucleotide without CTNAs), G (for A and (−)3TC) or C (for ACV and CBV). Each substrate was incubated at 37 °C for 10 min, in the absence (lanes 1, 4, 7, 10, 13 and 16) or presence of KF − (0.1 unit, lanes 2, 5, 8, 11, 14 and 17) or KF + (0.1 unit, lanes 3, 6, 9, 12, 15 and 18), in 10 mM Tris-HCl buffer (pH 7.9) containing 50 mM NaCl, 10 mM MgCl 2 , 10 mM DTT and 100 µM dNTPs; ( B ) The 32 P-labeled substrates were incubated with KF + at 37 °C for the indicated incubation time, in the same reaction buffer without dNTPs.

    Journal: Molecules

    Article Title: Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease

    doi: 10.3390/molecules21060766

    Figure Lengend Snippet: Primer extension from the CTNA-blocked termini by the Klenow fragment of Escherichia coli DNA polymerase I, with or without its proofreading 3′–5′ exonuclease activity (KF + or KF − , respectively, from Takara Bio, Inc., Shiga, Japan), in the ( A ) presence or ( B ) absence of dNTPs. ( A ) The 32 P-labeled oligonucleotides, 32 P-d(TCCGTTGAAGCCTGCTTT)X, where X represents no added nucleoside (OH, lanes 1–3), 2’-deoxyadenosine (lanes 4–6), acyclovir (ACV, lanes 7–9), abacavir (ABC, lanes 10–12), carbovir (CBV, lanes 13–15) or lamivudine ((−)3TC, lanes 16–18), were hybridized with their complementary strands, d(CTCGTCAGCTANAAAGCAGGCTTCAACGGA), where N represents A (for ABC and an oligonucleotide without CTNAs), G (for A and (−)3TC) or C (for ACV and CBV). Each substrate was incubated at 37 °C for 10 min, in the absence (lanes 1, 4, 7, 10, 13 and 16) or presence of KF − (0.1 unit, lanes 2, 5, 8, 11, 14 and 17) or KF + (0.1 unit, lanes 3, 6, 9, 12, 15 and 18), in 10 mM Tris-HCl buffer (pH 7.9) containing 50 mM NaCl, 10 mM MgCl 2 , 10 mM DTT and 100 µM dNTPs; ( B ) The 32 P-labeled substrates were incubated with KF + at 37 °C for the indicated incubation time, in the same reaction buffer without dNTPs.

    Article Snippet: Resumption of DNA Synthesis by DNA Polymerase after the Removal of CTNAs The hybridized oligonucleotides (400 fmol) were first treated with ERCC1-XPF (230 fmol) in 10 µL of 50 mM Tris-HCl buffer (pH 8.0), containing 2 mM MgCl2 , 0.5 mM DTT and 0.1 mg·mL−1 BSA, at 25 °C for 16 h. To the reaction mixture was added 5 µL of 30 mM Tris-HCl buffer (pH 7.9), containing 150 mM NaCl, 30 mM MgCl2 , 30 mM DTT, 300 µM dNTPs and the Klenow fragment of Escherichia coli DNA polymerase I, lacking the 3′–5′ exonuclease activity (KF− , 0.1 unit, Takara Bio).

    Techniques: Activity Assay, Labeling, Incubation

    Repair of the CTNA-containing oligonucleotides by human ERCC1-XPF endonuclease and DNA polymerase. Panels A and B represent the reaction scheme and the results, respectively. First, the 32 P-labeled substrates (400 fmol) were incubated at 25 °C for 16 h, in the absence (lanes 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21 and 22) or presence (the other lanes) of ERCC1-XPF (230 fmol), in 10 µL of 50 mM Tris-HCl buffer (pH 8.0) containing 2 mM MgCl 2 , 0.5 mM DTT and 0.1 mg·mL −1 BSA. Then, a polymerase reaction mixture (5 µL, containing 30 mM Tris-HCl, (pH 7.9), 150 mM NaCl, 30 mM MgCl 2 , 30 mM DTT, 300 µM dNTPs and 0.1 unit of KF − for even lanes) or 5 mM EDTA (5 µL for odd lanes) was added to the reaction mixture, and the total reaction mixtures (15 µL) were incubated at 37 °C for 10 min. The cleavage sites observed with ERCC1-XPF are indicated by black triangles. The fully extended products were quantified, and the values are shown.

    Journal: Molecules

    Article Title: Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease

    doi: 10.3390/molecules21060766

    Figure Lengend Snippet: Repair of the CTNA-containing oligonucleotides by human ERCC1-XPF endonuclease and DNA polymerase. Panels A and B represent the reaction scheme and the results, respectively. First, the 32 P-labeled substrates (400 fmol) were incubated at 25 °C for 16 h, in the absence (lanes 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21 and 22) or presence (the other lanes) of ERCC1-XPF (230 fmol), in 10 µL of 50 mM Tris-HCl buffer (pH 8.0) containing 2 mM MgCl 2 , 0.5 mM DTT and 0.1 mg·mL −1 BSA. Then, a polymerase reaction mixture (5 µL, containing 30 mM Tris-HCl, (pH 7.9), 150 mM NaCl, 30 mM MgCl 2 , 30 mM DTT, 300 µM dNTPs and 0.1 unit of KF − for even lanes) or 5 mM EDTA (5 µL for odd lanes) was added to the reaction mixture, and the total reaction mixtures (15 µL) were incubated at 37 °C for 10 min. The cleavage sites observed with ERCC1-XPF are indicated by black triangles. The fully extended products were quantified, and the values are shown.

    Article Snippet: Resumption of DNA Synthesis by DNA Polymerase after the Removal of CTNAs The hybridized oligonucleotides (400 fmol) were first treated with ERCC1-XPF (230 fmol) in 10 µL of 50 mM Tris-HCl buffer (pH 8.0), containing 2 mM MgCl2 , 0.5 mM DTT and 0.1 mg·mL−1 BSA, at 25 °C for 16 h. To the reaction mixture was added 5 µL of 30 mM Tris-HCl buffer (pH 7.9), containing 150 mM NaCl, 30 mM MgCl2 , 30 mM DTT, 300 µM dNTPs and the Klenow fragment of Escherichia coli DNA polymerase I, lacking the 3′–5′ exonuclease activity (KF− , 0.1 unit, Takara Bio).

    Techniques: Labeling, Incubation

    Gli1, cyclin D1, Bcl-2 and Bax mRNA levels in blank, Lipofectamine, siRNA-Gli1, BCNU and combination groups. Semi-quantitative real-time polymerase chain reaction was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl 2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). M, markers; 1, blank group; 2, Lipofectamine group; 3, BCNU group; 4, siRNA-Gli1 group; 5, combination group.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Knockdown of Gli1 by small-interfering RNA enhances the effects of BCNU on the proliferation and apoptosis of glioma U251 cells

    doi:

    Figure Lengend Snippet: Gli1, cyclin D1, Bcl-2 and Bax mRNA levels in blank, Lipofectamine, siRNA-Gli1, BCNU and combination groups. Semi-quantitative real-time polymerase chain reaction was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl 2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). M, markers; 1, blank group; 2, Lipofectamine group; 3, BCNU group; 4, siRNA-Gli1 group; 5, combination group.

    Article Snippet: The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each).

    Techniques: Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Affect of residual RT reagents on subsequent reactions. The number of cDNA molecules was evaluated by qPCR. Error bars are independent amplification replicates. ( a ) The residual RT reagents in a poly(A)-tailing reaction mixture containing bead-supported cDNA made biases large and amplification factors small (red column). The difficulty was overcome by washing beads (green column). ( b ) The addition of RT reagents to purified model cDNAs free in a poly(A)-tailing reaction mixture made biases large and amplification factors small as well. After purifying model cDNA with a purification kit, RT reagents were added to cDNA (red column) or not added (green column). ( c ) RT reagents did not affect the 2nd-strand cDNA synthesis and PCR. ( d ) Amplification efficiency was strongly affected by the presence of SuperScript III. The 11 components in an RT reagent kit were, respectively, added to 11 purified RT samples before poly(A) tailing. The 1st PCR products were evaluated by qPCR. Although the original amounts of samples were the same, the product produced by adding SuperScript III was small.

    Journal: Nucleic Acids Research

    Article Title: Non-biased and efficient global amplification of a single-cell cDNA library

    doi: 10.1093/nar/gkt965

    Figure Lengend Snippet: Affect of residual RT reagents on subsequent reactions. The number of cDNA molecules was evaluated by qPCR. Error bars are independent amplification replicates. ( a ) The residual RT reagents in a poly(A)-tailing reaction mixture containing bead-supported cDNA made biases large and amplification factors small (red column). The difficulty was overcome by washing beads (green column). ( b ) The addition of RT reagents to purified model cDNAs free in a poly(A)-tailing reaction mixture made biases large and amplification factors small as well. After purifying model cDNA with a purification kit, RT reagents were added to cDNA (red column) or not added (green column). ( c ) RT reagents did not affect the 2nd-strand cDNA synthesis and PCR. ( d ) Amplification efficiency was strongly affected by the presence of SuperScript III. The 11 components in an RT reagent kit were, respectively, added to 11 purified RT samples before poly(A) tailing. The 1st PCR products were evaluated by qPCR. Although the original amounts of samples were the same, the product produced by adding SuperScript III was small.

    Article Snippet: Another 15 cycles of PCR was then performed on the four fractions, each containing 1 μl of the 50 -μl purified PCR products together with 49 μl of PCR mixture III (5-μl 10 × EX Taq Buffer, 0.25-mM dNTP, 1-μM AUP1, 1-μM AUP2 and 2.5-U TaKaRa ExTaqTM HS).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Purification, Polymerase Chain Reaction, Produced