high fidelity pcr  (TaKaRa)

 
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    Name:
    High Fidelity PCR EcoDry Premix
    Description:
    Advantage 2 Polymerase Mix is an optimized blend of PCR enzymes that is a proven balance of high yield and high fidelity with 3X higher fidelity than regular Taq for cDNA amplification and library construction With an automatic hot start feature our Advantage 2 Polymerase Mix can readily amplify a wide range of DNA templates including long templates up to 18 kb and complex genomic DNA up to 6kb The Advantage 2 Polymerase Mix includes two optimized buffers dNTPs need to be purchased separately
    Catalog Number:
    639282
    Price:
    None
    Size:
    24 Rxns
    Category:
    Advantage 2 Polymerase Mix Advantage 2 products High yield PCR PCR
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    Structured Review

    TaKaRa high fidelity pcr
    Expression analyses of <t>PRSS23</t> in human cell lines. Expression levels of PRSS23 as well as ERα were analyzed in eight different cell lines: MCF-7, BT-474, Hs.578t, MDA-MB-231, T-47D (all breast cancer), MCF-10A (mammary epithelial), RL95-2 (endometrial cancer), and Ca-SKi (cervical cancer) cell lines. A. Immunoblot analysis showed protein expression level of ERα in these human cell lines. B. <t>qRT-PCR</t> analysis showed relative gene expression of PRSS23 mRNA level. C. Immunoblot analysis showed protein expression level of PRSS23 and GAPDH in these human cell lines. The cell lysate was loaded 20 µg protein for each well in immunoblot anaylsis. qRT-PCR was performed in duplicate.
    Advantage 2 Polymerase Mix is an optimized blend of PCR enzymes that is a proven balance of high yield and high fidelity with 3X higher fidelity than regular Taq for cDNA amplification and library construction With an automatic hot start feature our Advantage 2 Polymerase Mix can readily amplify a wide range of DNA templates including long templates up to 18 kb and complex genomic DNA up to 6kb The Advantage 2 Polymerase Mix includes two optimized buffers dNTPs need to be purchased separately
    https://www.bioz.com/result/high fidelity pcr/product/TaKaRa
    Average 95 stars, based on 41 article reviews
    Price from $9.99 to $1999.99
    high fidelity pcr - by Bioz Stars, 2020-09
    95/100 stars

    Images

    1) Product Images from "Serine Protease PRSS23 Is Upregulated by Estrogen Receptor ? and Associated with Proliferation of Breast Cancer Cells"

    Article Title: Serine Protease PRSS23 Is Upregulated by Estrogen Receptor ? and Associated with Proliferation of Breast Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030397

    Expression analyses of PRSS23 in human cell lines. Expression levels of PRSS23 as well as ERα were analyzed in eight different cell lines: MCF-7, BT-474, Hs.578t, MDA-MB-231, T-47D (all breast cancer), MCF-10A (mammary epithelial), RL95-2 (endometrial cancer), and Ca-SKi (cervical cancer) cell lines. A. Immunoblot analysis showed protein expression level of ERα in these human cell lines. B. qRT-PCR analysis showed relative gene expression of PRSS23 mRNA level. C. Immunoblot analysis showed protein expression level of PRSS23 and GAPDH in these human cell lines. The cell lysate was loaded 20 µg protein for each well in immunoblot anaylsis. qRT-PCR was performed in duplicate.
    Figure Legend Snippet: Expression analyses of PRSS23 in human cell lines. Expression levels of PRSS23 as well as ERα were analyzed in eight different cell lines: MCF-7, BT-474, Hs.578t, MDA-MB-231, T-47D (all breast cancer), MCF-10A (mammary epithelial), RL95-2 (endometrial cancer), and Ca-SKi (cervical cancer) cell lines. A. Immunoblot analysis showed protein expression level of ERα in these human cell lines. B. qRT-PCR analysis showed relative gene expression of PRSS23 mRNA level. C. Immunoblot analysis showed protein expression level of PRSS23 and GAPDH in these human cell lines. The cell lysate was loaded 20 µg protein for each well in immunoblot anaylsis. qRT-PCR was performed in duplicate.

    Techniques Used: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR

    E 2 -activated ERα enhances PRSS23 expression in MCF-7 cells. A. MCF-7 cells were treated with 1 nM E 2 , 25 ppm ethanol, 5 µM Tam, and 0.5% dimethyl sulfoxide (DMSO) in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. The bar plots depicted the results of time-lapse profiling of PRSS23 mRNA levels at 6, 12, and 24 h. All experiments were performed in triplicate. The bars represent relative expression levels of PRSS23 after treatment, which was normalized to the level of 6 h-treated cells (mean ± S.E.M.). B. MDA-MB-231 cells were treated with 1 nM E 2 in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. Expression of PRSS23 (upper panel) and pS2 (lower panel) was evaluated by qRT-PCR at 0, 6, 12, and 24 h. The bars represented the gene expression levels of PRSS23 after treatment, which was normalized to the level of untreated cells (mean ± S.E.M.).
    Figure Legend Snippet: E 2 -activated ERα enhances PRSS23 expression in MCF-7 cells. A. MCF-7 cells were treated with 1 nM E 2 , 25 ppm ethanol, 5 µM Tam, and 0.5% dimethyl sulfoxide (DMSO) in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. The bar plots depicted the results of time-lapse profiling of PRSS23 mRNA levels at 6, 12, and 24 h. All experiments were performed in triplicate. The bars represent relative expression levels of PRSS23 after treatment, which was normalized to the level of 6 h-treated cells (mean ± S.E.M.). B. MDA-MB-231 cells were treated with 1 nM E 2 in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. Expression of PRSS23 (upper panel) and pS2 (lower panel) was evaluated by qRT-PCR at 0, 6, 12, and 24 h. The bars represented the gene expression levels of PRSS23 after treatment, which was normalized to the level of untreated cells (mean ± S.E.M.).

    Techniques Used: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR

    2) Product Images from "In Vivo Analysis of MEF2 Transcription Factors in Synapse Regulation and Neuronal Survival"

    Article Title: In Vivo Analysis of MEF2 Transcription Factors in Synapse Regulation and Neuronal Survival

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034863

    The loss of MEF2A/D does not alter basal synaptic transmission. ( A ) mEPSC frequency is unchanged upon deletion of Mef2a/d in hippocampal culture neurons using lentivirus expressing Cre recombinase (p > 0.05). The number of recordings is shown in the bar graph for the GFP and Cre infected neurons. Data is shown as mean ± SEM. ( B ) Lentivirus containing either GFP or Cre was infected at 4 DIV in hippocampal culture neurons and cells were harvested at 15–18 DIV, in parallel with the time of recordings. Quantitative PCR was used to confirm the deletion of Mef2a/d (*p
    Figure Legend Snippet: The loss of MEF2A/D does not alter basal synaptic transmission. ( A ) mEPSC frequency is unchanged upon deletion of Mef2a/d in hippocampal culture neurons using lentivirus expressing Cre recombinase (p > 0.05). The number of recordings is shown in the bar graph for the GFP and Cre infected neurons. Data is shown as mean ± SEM. ( B ) Lentivirus containing either GFP or Cre was infected at 4 DIV in hippocampal culture neurons and cells were harvested at 15–18 DIV, in parallel with the time of recordings. Quantitative PCR was used to confirm the deletion of Mef2a/d (*p

    Techniques Used: Transmission Assay, Expressing, Infection, Real-time Polymerase Chain Reaction

    Mef2a KO mice show normal behavior. ( A ) Left, PCR genotyping to distinguish different Mef2a alleles. Right, the bar graph shows expression levels of Mef2 isoforms in the hippocampus of 4 week old animals (n=3/genotype), detected by quantitative PCR. The fold change in RNA was calculated using the comparative Ct method, normalizing to GAPDH as a control. ( B ) Nissl staining of brain cross-sections from littermate control (CTL) and Mef2a KO mice at 2 months of age. Brain-specific deletion of MEF2A was achieved by crossing MEF2A loxP/loxP mice with mice harboring a transgene for hGFAP-Cre. ( C ) Mef2a KO (KO) mice show no significant differences in locomotor activity compared to littermate CTL mice as assessed by consecutive beam breaks over a two-hour period, although the number of beam breaks significantly decreased over time (F 23,184 =41.84, P
    Figure Legend Snippet: Mef2a KO mice show normal behavior. ( A ) Left, PCR genotyping to distinguish different Mef2a alleles. Right, the bar graph shows expression levels of Mef2 isoforms in the hippocampus of 4 week old animals (n=3/genotype), detected by quantitative PCR. The fold change in RNA was calculated using the comparative Ct method, normalizing to GAPDH as a control. ( B ) Nissl staining of brain cross-sections from littermate control (CTL) and Mef2a KO mice at 2 months of age. Brain-specific deletion of MEF2A was achieved by crossing MEF2A loxP/loxP mice with mice harboring a transgene for hGFAP-Cre. ( C ) Mef2a KO (KO) mice show no significant differences in locomotor activity compared to littermate CTL mice as assessed by consecutive beam breaks over a two-hour period, although the number of beam breaks significantly decreased over time (F 23,184 =41.84, P

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Staining, CTL Assay, Activity Assay

    Brain-specific deletion of Mef2a and Mef2d causes impairments in motor coordination, as well as presynaptic release probability. ( A ) Detection of Mef2a and Mef2d transcripts by in situ hybridization in littermate CTL and Mef2a/d DKO (DKO) mice. Arrow indicates hippocampus. ( B ) Expression level of MEF2 transcription factors in the hippocampus of Mef2a/d DKO mice as detected by quantitative RT-PCR. RNA was isolated from the hippocampus of 4 week old animals (n=3/genotype), and then expression determined by quantitative PCR. The fold change in RNA was calculated using the comparative Ct method, normalizing to GAPDH as a control. ( C ) Mef2a/d DKO mice exhibit impaired motor coordination as assessed by falling off the accelerating rotarod faster than littermate CTLs (F 1,7 =27.64, P
    Figure Legend Snippet: Brain-specific deletion of Mef2a and Mef2d causes impairments in motor coordination, as well as presynaptic release probability. ( A ) Detection of Mef2a and Mef2d transcripts by in situ hybridization in littermate CTL and Mef2a/d DKO (DKO) mice. Arrow indicates hippocampus. ( B ) Expression level of MEF2 transcription factors in the hippocampus of Mef2a/d DKO mice as detected by quantitative RT-PCR. RNA was isolated from the hippocampus of 4 week old animals (n=3/genotype), and then expression determined by quantitative PCR. The fold change in RNA was calculated using the comparative Ct method, normalizing to GAPDH as a control. ( C ) Mef2a/d DKO mice exhibit impaired motor coordination as assessed by falling off the accelerating rotarod faster than littermate CTLs (F 1,7 =27.64, P

    Techniques Used: In Situ Hybridization, CTL Assay, Mouse Assay, Expressing, Quantitative RT-PCR, Isolation, Real-time Polymerase Chain Reaction

    3) Product Images from "Overexpression of SlOFP20 affects floral organ and pollen development"

    Article Title: Overexpression of SlOFP20 affects floral organ and pollen development

    Journal: Horticulture Research

    doi: 10.1038/s41438-019-0207-6

    Expression profile analysis of SlOFP20 in WT tomato plants by quantitative real-time PCR. a Expression levels of SlOFP20 genes in different tissues of WT tomato. Rt, root; St, stem; Yl, young leaf; Ml, mature leaf; Sl, senescent leaf; S, sepal; Fl, flower; IMG, immature green fruit; MG, mature fruit; b breaker fruit; B+4, 4 days after the breaker stage; B+7, 7 days after breaker stage. b Relative expression of SlOFP20 in the four-whorl floral organs of WT. Se, sepal; Pe, petal; St, stamen; Ca, carpel. c The expression levels of SlOFP20 in the WT and transgenic lines. Each value represents the mean ± SE of three replicates
    Figure Legend Snippet: Expression profile analysis of SlOFP20 in WT tomato plants by quantitative real-time PCR. a Expression levels of SlOFP20 genes in different tissues of WT tomato. Rt, root; St, stem; Yl, young leaf; Ml, mature leaf; Sl, senescent leaf; S, sepal; Fl, flower; IMG, immature green fruit; MG, mature fruit; b breaker fruit; B+4, 4 days after the breaker stage; B+7, 7 days after breaker stage. b Relative expression of SlOFP20 in the four-whorl floral organs of WT. Se, sepal; Pe, petal; St, stamen; Ca, carpel. c The expression levels of SlOFP20 in the WT and transgenic lines. Each value represents the mean ± SE of three replicates

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transgenic Assay

    4) Product Images from "cyclops encodes a nodal-related factor involved in midline signaling"

    Article Title: cyclops encodes a nodal-related factor involved in midline signaling

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    ( A ) ndr2 RNA is present in wild-type (WT) but not homozygous cyc b16 embryos, as seen by in situ ). ( B ) ndr2 is deleted in cyc b16 DNA, visualized by PCR. Lanes: 1, ndr2 primer pair 1 + ndr2 plasmid; 2, ndr2 primer pair 1 + wild-type genomic DNA; 3, ndr2 primer pair 1 + cyc b16 homozygous genomic DNA; 4, ndr2 primer pair 2 + ndr2 plasmid; 5, ndr1 primers + ndr2 primer pair 2 + cyc b16 genomic DNA; 6, ndr1 primers + ndr1 plasmid.
    Figure Legend Snippet: ( A ) ndr2 RNA is present in wild-type (WT) but not homozygous cyc b16 embryos, as seen by in situ ). ( B ) ndr2 is deleted in cyc b16 DNA, visualized by PCR. Lanes: 1, ndr2 primer pair 1 + ndr2 plasmid; 2, ndr2 primer pair 1 + wild-type genomic DNA; 3, ndr2 primer pair 1 + cyc b16 homozygous genomic DNA; 4, ndr2 primer pair 2 + ndr2 plasmid; 5, ndr1 primers + ndr2 primer pair 2 + cyc b16 genomic DNA; 6, ndr1 primers + ndr1 plasmid.

    Techniques Used: In Situ, Polymerase Chain Reaction, Plasmid Preparation

    Related Articles

    Clone Assay:

    Article Title: The Mycobacterial LysR-Type Regulator OxyS Responds to Oxidative Stress and Negatively Regulates Expression of the Catalase-Peroxidase Gene
    Article Snippet: .. Cloning, expression and purification of recombinant proteins The M. tuberculosis oxyS gene was amplified from genomic DNA using the High Fidelity PCR system (TaKaRa) with appropriate primers and cloned into the pET28a over-expression vectors to produce recombinant vectors ( ). .. E. coli BL21 (DE3) cells transformed with the recombinant plasmid were grown at 37°C for 5 hours in 1 liter of LB medium containing 30 µg/ml−1 kanamycin, and at 20°C for 15 hours after 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added.

    Article Title: A GntR Family Transcription Factor in Streptococcus mutans Regulates Biofilm Formation and Expression of Multiple Sugar Transporter Genes
    Article Snippet: .. Cloning, Expression, and Purification of the Recombinant StsR Protein The S. mutans stsR gene coding sequence was amplified with primers SMU.1193F and SMU.1193R from S. mutans genomic DNA, using the high-fidelity PCR system (TakaRa). .. The recombinant vector pETstsR was produced by cloning the stsR coding sequence into the pET28a expression vector using the restriction sites listed in Supplementary Table .

    Amplification:

    Article Title: The Mycobacterial LysR-Type Regulator OxyS Responds to Oxidative Stress and Negatively Regulates Expression of the Catalase-Peroxidase Gene
    Article Snippet: .. Cloning, expression and purification of recombinant proteins The M. tuberculosis oxyS gene was amplified from genomic DNA using the High Fidelity PCR system (TaKaRa) with appropriate primers and cloned into the pET28a over-expression vectors to produce recombinant vectors ( ). .. E. coli BL21 (DE3) cells transformed with the recombinant plasmid were grown at 37°C for 5 hours in 1 liter of LB medium containing 30 µg/ml−1 kanamycin, and at 20°C for 15 hours after 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added.

    Article Title: A GntR Family Transcription Factor in Streptococcus mutans Regulates Biofilm Formation and Expression of Multiple Sugar Transporter Genes
    Article Snippet: .. Cloning, Expression, and Purification of the Recombinant StsR Protein The S. mutans stsR gene coding sequence was amplified with primers SMU.1193F and SMU.1193R from S. mutans genomic DNA, using the high-fidelity PCR system (TakaRa). .. The recombinant vector pETstsR was produced by cloning the stsR coding sequence into the pET28a expression vector using the restriction sites listed in Supplementary Table .

    Purification:

    Article Title: The Mycobacterial LysR-Type Regulator OxyS Responds to Oxidative Stress and Negatively Regulates Expression of the Catalase-Peroxidase Gene
    Article Snippet: .. Cloning, expression and purification of recombinant proteins The M. tuberculosis oxyS gene was amplified from genomic DNA using the High Fidelity PCR system (TaKaRa) with appropriate primers and cloned into the pET28a over-expression vectors to produce recombinant vectors ( ). .. E. coli BL21 (DE3) cells transformed with the recombinant plasmid were grown at 37°C for 5 hours in 1 liter of LB medium containing 30 µg/ml−1 kanamycin, and at 20°C for 15 hours after 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added.

    Article Title: A GntR Family Transcription Factor in Streptococcus mutans Regulates Biofilm Formation and Expression of Multiple Sugar Transporter Genes
    Article Snippet: .. Cloning, Expression, and Purification of the Recombinant StsR Protein The S. mutans stsR gene coding sequence was amplified with primers SMU.1193F and SMU.1193R from S. mutans genomic DNA, using the high-fidelity PCR system (TakaRa). .. The recombinant vector pETstsR was produced by cloning the stsR coding sequence into the pET28a expression vector using the restriction sites listed in Supplementary Table .

    Polymerase Chain Reaction:

    Article Title: High-pressure EPR spectroscopy studies of the E. coli lipopolysaccharide transport proteins LptA and LptC
    Article Snippet: .. Unique cysteine mutations were introduced into the naturally cysteine-free soluble domain of LptC (amino acids 24–191) or mature LptA (amino acids 28–185) using High Fidelity PCR EcoDry Premix (Clontech, Mountain View, CA) and verified (Retrogen, San Diego, CA) as described previously [ , ] or by gene synthesis (GenScript, Piscataway, NJ). .. The LptC protein was expressed from a pET28b (Novagen, EMD Millipore, Billerica, MA) vector with an N-terminal 6xHis tag in E. coli BL21(DE3) cells, and the LptA proteins were expressed from a pET21b vector with a C-terminal 6xHis tag in E. coli BL21(DE3)pLysS cells using cobalt affinity chromatography (Clontech, Mountain View, CA).

    Article Title: Characterization of and lipopolysaccharide binding to the E. coliLpt C protein dimer
    Article Snippet: .. Single cysteine mutations were introduced into the LptC soluble domain (amino acids 24–191) using High Fidelity PCR EcoDry Premix (Clontech, Mountain View, CA) and verified by sequencing (Retrogen, San Diego, CA) as described previously., LptC protein was expressed in E. coli BL21(DE3) cells from a pET28b (Novagen, EMD Millipore, Billerica, MA) vector with an N‐terminal 6xHis tag. .. Purified protein was spin labeled at the introduced cysteine using a 50‐fold excess of the sulfhydryl‐specific spin probe, 2,2,5,5‐tetramethylpyrroline‐3‐yl‐methanethiosulfonate spin label (Toronto Research Chemicals, New York, ON) to generate the R1 side chain prior to eluting from the cobalt resin (Clontech, Mountain View, CA), as described previously., Protein was concentrated using Microcon YM‐10 centrifugal concentrators, and concentrations were determined using the Thermo Scientific Pierce BCA Protein Assay Kit (Rockford, IL) with lysozyme (14 kDa) as the protein standard.

    Article Title: Tissue Inhibitor of Metalloprotease-2 (TIMP-2): Bioprocess Development, Physicochemical, Biochemical, and Biological Characterization of Highly Expressed Recombinant Protein
    Article Snippet: .. The PCR was performed using the pT2MO1 cDNA as a template with the forward 5’-ATAGTCGACATGGGAGCTGCTGCTCGCAC-3’ and reverse 5’-GACGGTATCGATAAGCTTTCAATGGTGATGGTGATGATGCTTGTCGTCGTCATCTGGGTCCTCGATGTCGAGAAA-3’ primers using High Fidelity PCR EcoDryTM Premix (Clontech). .. The amplified cDNA fragment was inserted into the pcDNA 3.3 Topo TA vector using a Topo TA cloning kit (both from Themo Fisher Scientific).

    Article Title: Multi-population genomic analysis of malaria parasites indicates local selection and differentiation at the gdv1 locus regulating sexual development
    Article Snippet: .. PCR amplifications were performed using TaKaRa high fidelity PCR Premix (Clontech, UK) in a volume of 5 ul, with 35 cycles of 95 °C for 30 seconds denaturation, 64.5 °C for 15 seconds annealing, and 70 °C for 3 minutes elongation, following which PCR products were separated on 1% agarose gels and stained with ethidium bromide for scoring of allelic product sizes. .. For RNA analysis by reverse transcription followed by quantitative polymerase chain reaction amplification (RT-qPCR), peripheral blood samples of 81 patients with clinical P. falciparum malaria infections were studied, 48 sampled at Pikine in Senegal, and 33 sampled at Kintampo in Ghana, with each sample containing at least 100 μL packed red blood cells cryopreserved within four hours of collection.

    Article Title: Disruption of LptA oligomerization and affinity of the LptA–LptC interaction
    Article Snippet: .. Single cysteine and double alanine mutations were generated using High Fidelity PCR EcoDry Premix (Clontech, Mountain View, CA). ..

    Article Title: The Mycobacterial LysR-Type Regulator OxyS Responds to Oxidative Stress and Negatively Regulates Expression of the Catalase-Peroxidase Gene
    Article Snippet: .. Cloning, expression and purification of recombinant proteins The M. tuberculosis oxyS gene was amplified from genomic DNA using the High Fidelity PCR system (TaKaRa) with appropriate primers and cloned into the pET28a over-expression vectors to produce recombinant vectors ( ). .. E. coli BL21 (DE3) cells transformed with the recombinant plasmid were grown at 37°C for 5 hours in 1 liter of LB medium containing 30 µg/ml−1 kanamycin, and at 20°C for 15 hours after 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added.

    Article Title: A GntR Family Transcription Factor in Streptococcus mutans Regulates Biofilm Formation and Expression of Multiple Sugar Transporter Genes
    Article Snippet: .. Cloning, Expression, and Purification of the Recombinant StsR Protein The S. mutans stsR gene coding sequence was amplified with primers SMU.1193F and SMU.1193R from S. mutans genomic DNA, using the high-fidelity PCR system (TakaRa). .. The recombinant vector pETstsR was produced by cloning the stsR coding sequence into the pET28a expression vector using the restriction sites listed in Supplementary Table .

    Generated:

    Article Title: Disruption of LptA oligomerization and affinity of the LptA–LptC interaction
    Article Snippet: .. Single cysteine and double alanine mutations were generated using High Fidelity PCR EcoDry Premix (Clontech, Mountain View, CA). ..

    Expressing:

    Article Title: The Mycobacterial LysR-Type Regulator OxyS Responds to Oxidative Stress and Negatively Regulates Expression of the Catalase-Peroxidase Gene
    Article Snippet: .. Cloning, expression and purification of recombinant proteins The M. tuberculosis oxyS gene was amplified from genomic DNA using the High Fidelity PCR system (TaKaRa) with appropriate primers and cloned into the pET28a over-expression vectors to produce recombinant vectors ( ). .. E. coli BL21 (DE3) cells transformed with the recombinant plasmid were grown at 37°C for 5 hours in 1 liter of LB medium containing 30 µg/ml−1 kanamycin, and at 20°C for 15 hours after 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added.

    Article Title: A GntR Family Transcription Factor in Streptococcus mutans Regulates Biofilm Formation and Expression of Multiple Sugar Transporter Genes
    Article Snippet: .. Cloning, Expression, and Purification of the Recombinant StsR Protein The S. mutans stsR gene coding sequence was amplified with primers SMU.1193F and SMU.1193R from S. mutans genomic DNA, using the high-fidelity PCR system (TakaRa). .. The recombinant vector pETstsR was produced by cloning the stsR coding sequence into the pET28a expression vector using the restriction sites listed in Supplementary Table .

    Sequencing:

    Article Title: Characterization of and lipopolysaccharide binding to the E. coliLpt C protein dimer
    Article Snippet: .. Single cysteine mutations were introduced into the LptC soluble domain (amino acids 24–191) using High Fidelity PCR EcoDry Premix (Clontech, Mountain View, CA) and verified by sequencing (Retrogen, San Diego, CA) as described previously., LptC protein was expressed in E. coli BL21(DE3) cells from a pET28b (Novagen, EMD Millipore, Billerica, MA) vector with an N‐terminal 6xHis tag. .. Purified protein was spin labeled at the introduced cysteine using a 50‐fold excess of the sulfhydryl‐specific spin probe, 2,2,5,5‐tetramethylpyrroline‐3‐yl‐methanethiosulfonate spin label (Toronto Research Chemicals, New York, ON) to generate the R1 side chain prior to eluting from the cobalt resin (Clontech, Mountain View, CA), as described previously., Protein was concentrated using Microcon YM‐10 centrifugal concentrators, and concentrations were determined using the Thermo Scientific Pierce BCA Protein Assay Kit (Rockford, IL) with lysozyme (14 kDa) as the protein standard.

    Article Title: A GntR Family Transcription Factor in Streptococcus mutans Regulates Biofilm Formation and Expression of Multiple Sugar Transporter Genes
    Article Snippet: .. Cloning, Expression, and Purification of the Recombinant StsR Protein The S. mutans stsR gene coding sequence was amplified with primers SMU.1193F and SMU.1193R from S. mutans genomic DNA, using the high-fidelity PCR system (TakaRa). .. The recombinant vector pETstsR was produced by cloning the stsR coding sequence into the pET28a expression vector using the restriction sites listed in Supplementary Table .

    Staining:

    Article Title: Multi-population genomic analysis of malaria parasites indicates local selection and differentiation at the gdv1 locus regulating sexual development
    Article Snippet: .. PCR amplifications were performed using TaKaRa high fidelity PCR Premix (Clontech, UK) in a volume of 5 ul, with 35 cycles of 95 °C for 30 seconds denaturation, 64.5 °C for 15 seconds annealing, and 70 °C for 3 minutes elongation, following which PCR products were separated on 1% agarose gels and stained with ethidium bromide for scoring of allelic product sizes. .. For RNA analysis by reverse transcription followed by quantitative polymerase chain reaction amplification (RT-qPCR), peripheral blood samples of 81 patients with clinical P. falciparum malaria infections were studied, 48 sampled at Pikine in Senegal, and 33 sampled at Kintampo in Ghana, with each sample containing at least 100 μL packed red blood cells cryopreserved within four hours of collection.

    Recombinant:

    Article Title: The Mycobacterial LysR-Type Regulator OxyS Responds to Oxidative Stress and Negatively Regulates Expression of the Catalase-Peroxidase Gene
    Article Snippet: .. Cloning, expression and purification of recombinant proteins The M. tuberculosis oxyS gene was amplified from genomic DNA using the High Fidelity PCR system (TaKaRa) with appropriate primers and cloned into the pET28a over-expression vectors to produce recombinant vectors ( ). .. E. coli BL21 (DE3) cells transformed with the recombinant plasmid were grown at 37°C for 5 hours in 1 liter of LB medium containing 30 µg/ml−1 kanamycin, and at 20°C for 15 hours after 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added.

    Article Title: A GntR Family Transcription Factor in Streptococcus mutans Regulates Biofilm Formation and Expression of Multiple Sugar Transporter Genes
    Article Snippet: .. Cloning, Expression, and Purification of the Recombinant StsR Protein The S. mutans stsR gene coding sequence was amplified with primers SMU.1193F and SMU.1193R from S. mutans genomic DNA, using the high-fidelity PCR system (TakaRa). .. The recombinant vector pETstsR was produced by cloning the stsR coding sequence into the pET28a expression vector using the restriction sites listed in Supplementary Table .

    Over Expression:

    Article Title: The Mycobacterial LysR-Type Regulator OxyS Responds to Oxidative Stress and Negatively Regulates Expression of the Catalase-Peroxidase Gene
    Article Snippet: .. Cloning, expression and purification of recombinant proteins The M. tuberculosis oxyS gene was amplified from genomic DNA using the High Fidelity PCR system (TaKaRa) with appropriate primers and cloned into the pET28a over-expression vectors to produce recombinant vectors ( ). .. E. coli BL21 (DE3) cells transformed with the recombinant plasmid were grown at 37°C for 5 hours in 1 liter of LB medium containing 30 µg/ml−1 kanamycin, and at 20°C for 15 hours after 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added.

    Plasmid Preparation:

    Article Title: Characterization of and lipopolysaccharide binding to the E. coliLpt C protein dimer
    Article Snippet: .. Single cysteine mutations were introduced into the LptC soluble domain (amino acids 24–191) using High Fidelity PCR EcoDry Premix (Clontech, Mountain View, CA) and verified by sequencing (Retrogen, San Diego, CA) as described previously., LptC protein was expressed in E. coli BL21(DE3) cells from a pET28b (Novagen, EMD Millipore, Billerica, MA) vector with an N‐terminal 6xHis tag. .. Purified protein was spin labeled at the introduced cysteine using a 50‐fold excess of the sulfhydryl‐specific spin probe, 2,2,5,5‐tetramethylpyrroline‐3‐yl‐methanethiosulfonate spin label (Toronto Research Chemicals, New York, ON) to generate the R1 side chain prior to eluting from the cobalt resin (Clontech, Mountain View, CA), as described previously., Protein was concentrated using Microcon YM‐10 centrifugal concentrators, and concentrations were determined using the Thermo Scientific Pierce BCA Protein Assay Kit (Rockford, IL) with lysozyme (14 kDa) as the protein standard.

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    TaKaRa high fidelity pcr
    Expression analyses of <t>PRSS23</t> in human cell lines. Expression levels of PRSS23 as well as ERα were analyzed in eight different cell lines: MCF-7, BT-474, Hs.578t, MDA-MB-231, T-47D (all breast cancer), MCF-10A (mammary epithelial), RL95-2 (endometrial cancer), and Ca-SKi (cervical cancer) cell lines. A. Immunoblot analysis showed protein expression level of ERα in these human cell lines. B. <t>qRT-PCR</t> analysis showed relative gene expression of PRSS23 mRNA level. C. Immunoblot analysis showed protein expression level of PRSS23 and GAPDH in these human cell lines. The cell lysate was loaded 20 µg protein for each well in immunoblot anaylsis. qRT-PCR was performed in duplicate.
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    Expression analyses of PRSS23 in human cell lines. Expression levels of PRSS23 as well as ERα were analyzed in eight different cell lines: MCF-7, BT-474, Hs.578t, MDA-MB-231, T-47D (all breast cancer), MCF-10A (mammary epithelial), RL95-2 (endometrial cancer), and Ca-SKi (cervical cancer) cell lines. A. Immunoblot analysis showed protein expression level of ERα in these human cell lines. B. qRT-PCR analysis showed relative gene expression of PRSS23 mRNA level. C. Immunoblot analysis showed protein expression level of PRSS23 and GAPDH in these human cell lines. The cell lysate was loaded 20 µg protein for each well in immunoblot anaylsis. qRT-PCR was performed in duplicate.

    Journal: PLoS ONE

    Article Title: Serine Protease PRSS23 Is Upregulated by Estrogen Receptor ? and Associated with Proliferation of Breast Cancer Cells

    doi: 10.1371/journal.pone.0030397

    Figure Lengend Snippet: Expression analyses of PRSS23 in human cell lines. Expression levels of PRSS23 as well as ERα were analyzed in eight different cell lines: MCF-7, BT-474, Hs.578t, MDA-MB-231, T-47D (all breast cancer), MCF-10A (mammary epithelial), RL95-2 (endometrial cancer), and Ca-SKi (cervical cancer) cell lines. A. Immunoblot analysis showed protein expression level of ERα in these human cell lines. B. qRT-PCR analysis showed relative gene expression of PRSS23 mRNA level. C. Immunoblot analysis showed protein expression level of PRSS23 and GAPDH in these human cell lines. The cell lysate was loaded 20 µg protein for each well in immunoblot anaylsis. qRT-PCR was performed in duplicate.

    Article Snippet: The open-reading frame of PRSS23 was amplified by high-fidelity PCR (primers are listed in ) and cloned into the p IRES-ZsGreen1 vector (Clontech).

    Techniques: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR

    E 2 -activated ERα enhances PRSS23 expression in MCF-7 cells. A. MCF-7 cells were treated with 1 nM E 2 , 25 ppm ethanol, 5 µM Tam, and 0.5% dimethyl sulfoxide (DMSO) in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. The bar plots depicted the results of time-lapse profiling of PRSS23 mRNA levels at 6, 12, and 24 h. All experiments were performed in triplicate. The bars represent relative expression levels of PRSS23 after treatment, which was normalized to the level of 6 h-treated cells (mean ± S.E.M.). B. MDA-MB-231 cells were treated with 1 nM E 2 in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. Expression of PRSS23 (upper panel) and pS2 (lower panel) was evaluated by qRT-PCR at 0, 6, 12, and 24 h. The bars represented the gene expression levels of PRSS23 after treatment, which was normalized to the level of untreated cells (mean ± S.E.M.).

    Journal: PLoS ONE

    Article Title: Serine Protease PRSS23 Is Upregulated by Estrogen Receptor ? and Associated with Proliferation of Breast Cancer Cells

    doi: 10.1371/journal.pone.0030397

    Figure Lengend Snippet: E 2 -activated ERα enhances PRSS23 expression in MCF-7 cells. A. MCF-7 cells were treated with 1 nM E 2 , 25 ppm ethanol, 5 µM Tam, and 0.5% dimethyl sulfoxide (DMSO) in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. The bar plots depicted the results of time-lapse profiling of PRSS23 mRNA levels at 6, 12, and 24 h. All experiments were performed in triplicate. The bars represent relative expression levels of PRSS23 after treatment, which was normalized to the level of 6 h-treated cells (mean ± S.E.M.). B. MDA-MB-231 cells were treated with 1 nM E 2 in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. Expression of PRSS23 (upper panel) and pS2 (lower panel) was evaluated by qRT-PCR at 0, 6, 12, and 24 h. The bars represented the gene expression levels of PRSS23 after treatment, which was normalized to the level of untreated cells (mean ± S.E.M.).

    Article Snippet: The open-reading frame of PRSS23 was amplified by high-fidelity PCR (primers are listed in ) and cloned into the p IRES-ZsGreen1 vector (Clontech).

    Techniques: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR