taq polymerase  (Thermo Fisher)


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    Name:
    Taq DNA Polymerase
    Description:
    Thermo Scientific Taq DNA Polymerase is a highly thermostable DNA polymerase from the thermophilic bacterium Thermus aquaticus The enzyme catalyzes 5 →3 synthesis of DNA has no detectable 3 →5 exonuclease proofreading activity and possesses low 5 →3 exonuclease activity In addition Taq DNA Polymerase exhibits deoxynucleotidyl transferase activity which frequently results in the addition of extra adenines at the 3 end of PCR products Recombinant Taq DNA Polymerase is the ideal tool for standard PCR of templates 5 kb or shorter Highlights• Thermostable half life is more than 40 min at 95°C• Generates PCR products with 3 dA overhangs• Supplied with two buffers 10X Taq Buffer with KCl and 10X Taq Buffer with NH4 2SO4 The latter allows for PCR at wide range of magnesium concentrations and decreases unspecific priming• Incorporates modified nucleotides e g biotin digoxigenin fluorescently labeled nucleotides Applications• Routine PCR amplification of DNA fragments up to 5 kb• High throughput PCR• DNA labelingNote• The error rate of Taq DNA Polymerase in PCR is 2 2 x 10 5 errors per nt per cycle Accordingly the accuracy of PCR is 4 5 x 104 Accuracy is an inverse of the error rate and shows an average number of correct nucleotides incorporated before an error occurs • The 10X Taq Buffer without Detergent is recommended for microarray experiments
    Catalog Number:
    ep0401
    Price:
    None
    Category:
    Proteins Enzymes Peptides
    Applications:
    PCR|PCR & Real-Time PCR|Routine PCR
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    Structured Review

    Thermo Fisher taq polymerase
    Thermo Scientific Taq DNA Polymerase is a highly thermostable DNA polymerase from the thermophilic bacterium Thermus aquaticus The enzyme catalyzes 5 →3 synthesis of DNA has no detectable 3 →5 exonuclease proofreading activity and possesses low 5 →3 exonuclease activity In addition Taq DNA Polymerase exhibits deoxynucleotidyl transferase activity which frequently results in the addition of extra adenines at the 3 end of PCR products Recombinant Taq DNA Polymerase is the ideal tool for standard PCR of templates 5 kb or shorter Highlights• Thermostable half life is more than 40 min at 95°C• Generates PCR products with 3 dA overhangs• Supplied with two buffers 10X Taq Buffer with KCl and 10X Taq Buffer with NH4 2SO4 The latter allows for PCR at wide range of magnesium concentrations and decreases unspecific priming• Incorporates modified nucleotides e g biotin digoxigenin fluorescently labeled nucleotides Applications• Routine PCR amplification of DNA fragments up to 5 kb• High throughput PCR• DNA labelingNote• The error rate of Taq DNA Polymerase in PCR is 2 2 x 10 5 errors per nt per cycle Accordingly the accuracy of PCR is 4 5 x 104 Accuracy is an inverse of the error rate and shows an average number of correct nucleotides incorporated before an error occurs • The 10X Taq Buffer without Detergent is recommended for microarray experiments
    https://www.bioz.com/result/taq polymerase/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    taq polymerase - by Bioz Stars, 2021-03
    86/100 stars

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    Related Articles

    Quantitative RT-PCR:

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase
    Article Snippet: Both NEB and Thermo Fisher Taq DNA polymerases were also able to perform TaqMan RT-qPCR analysis of RNaseP armored RNA using the CDC assay . .. These results suggest that Taq DNA polymerase can support TaqMan RT-qPCR analyses of RNA in one-enzyme reactions. .. To further prove that the reverse transcriptase is inherent in Taq polymerase itself, we incubated RT-qPCR assays at 95 °C for 5 min prior to reverse transcription, which should inactivate any contaminating mesophilic reverse transcriptases ( Supplementary Figure 1).

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase
    Article Snippet: Similar to the NEB enzyme, Thermo Fisher Taq DNA polymerase also demonstrated better activity in Gen 6 A buffer and was able to detect viral genomic RNA using all three N gene assays, albeit with a higher detection limit ( ). .. Both NEB and Thermo Fisher Taq DNA polymerases were also able to perform TaqMan RT-qPCR analysis of RNaseP armored RNA using the CDC assay . .. These results suggest that Taq DNA polymerase can support TaqMan RT-qPCR analyses of RNA in one-enzyme reactions.

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase
    Article Snippet: Both NEB and Thermo Fisher Taq DNA polymerases were also able to perform TaqMan RT-qPCR analysis of RNaseP armored RNA using the CDC assay ( and ). .. These results suggest that Taq DNA polymerase can support TaqMan RT-qPCR analyses of RNA in one-enzyme reactions. .. To further prove that the reverse transcriptase is inherent in Taq polymerase itself, we incubated RT-qPCR assays at 95 °C for 5 min prior to reverse transcription, which should inactivate any contaminating mesophilic reverse transcriptases ( ).

    CDC Assay:

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase
    Article Snippet: Similar to the NEB enzyme, Thermo Fisher Taq DNA polymerase also demonstrated better activity in Gen 6 A buffer and was able to detect viral genomic RNA using all three N gene assays, albeit with a higher detection limit ( ). .. Both NEB and Thermo Fisher Taq DNA polymerases were also able to perform TaqMan RT-qPCR analysis of RNaseP armored RNA using the CDC assay . .. These results suggest that Taq DNA polymerase can support TaqMan RT-qPCR analyses of RNA in one-enzyme reactions.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV
    Article Snippet: .. In this study, we evaluated several one-step RT-PCR kits and a Taq DNA polymerase for the contamination of MLV-related genomes and found that the test kit and the Taq DNA polymerase from Invitrogen were contaminated with MLV-related genomes. .. The findings in the present study indicate that contaminating nucleic acids in the test kits can potentially produce false-positive PCR results in studies of XMRV and other MLV-related viruses.

    Recombinant:

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
    Article Snippet: A low copy plasmid pHSG576 was initially used to clone Taq DNA pol/TBD and enabled the activity of the polymerase to be assessed by in vivo complementation of the E.coli strain JS200 polAts as previously reported ( ). .. Six histidine codons were introduced onto the N-terminus of the Taq DNA polymerase and Taq DNA pol/TBD gene and the recombinant genes were cloned in frame into the NdeI site of the pLEX vector (Invitrogen). .. 5′–3′ Exonuclease-deficient mutants which lack the first 235 amino acids of the N-terminal were likewise generated.

    Clone Assay:

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
    Article Snippet: A low copy plasmid pHSG576 was initially used to clone Taq DNA pol/TBD and enabled the activity of the polymerase to be assessed by in vivo complementation of the E.coli strain JS200 polAts as previously reported ( ). .. Six histidine codons were introduced onto the N-terminus of the Taq DNA polymerase and Taq DNA pol/TBD gene and the recombinant genes were cloned in frame into the NdeI site of the pLEX vector (Invitrogen). .. 5′–3′ Exonuclease-deficient mutants which lack the first 235 amino acids of the N-terminal were likewise generated.

    Plasmid Preparation:

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
    Article Snippet: A low copy plasmid pHSG576 was initially used to clone Taq DNA pol/TBD and enabled the activity of the polymerase to be assessed by in vivo complementation of the E.coli strain JS200 polAts as previously reported ( ). .. Six histidine codons were introduced onto the N-terminus of the Taq DNA polymerase and Taq DNA pol/TBD gene and the recombinant genes were cloned in frame into the NdeI site of the pLEX vector (Invitrogen). .. 5′–3′ Exonuclease-deficient mutants which lack the first 235 amino acids of the N-terminal were likewise generated.

    Polymerase Chain Reaction:

    Article Title: Activation of alternative Jdp2 promoters and functional protein isoforms in T-cell lymphomas by retroviral insertion mutagenesis
    Article Snippet: Two μg total RNA was reverse transcribed using the First Strand cDNA Synthesis Kit (Fermentas) following the manufacturer's recommendation with the provided poly(dT) oligo. .. To look for alternative splicing between published exons 1a through 1d, PCR reactions were done with forward primers Exon1a-154 (5′-tgggcaccgcgcctgcagcag-3′), Exon1b-148 (5′-ggaggagcgcgagcat-3′), Exon1c-70 (5′-gctctggctgggttaggagggaac-3′) or Exon1d-150 (5′-cagctgcctctctccatctt-3′) and reverse primer Intron2-87 (5′-tccttcgctcttcttcctcgtctagctt-3′) using 1/500 of the cDNA (corresponding to 4 ng of total RNA) per PCR reaction (Taq polymerase, Invitrogen). .. Each PCR reaction consisted of an initial melting step at 95°C for 5 min followed by 35 cycles of amplification, each consisting of a 30 s melting step at 95°C, annealing at 55–60°C for 30 s and elongation at 72°C for 45 s. A final elongation step at 72°C was done for 10 min. Quantitative real-time PCR (QRT-PCR) was done using Platinum SYBR Green QRT-PCR SuperMix UDG (Invitrogen) following the manufacturer's recommendations.

    Purification:

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase
    Article Snippet: Robust amplification curves were generated and as few as 10 copies/μL (30 copies total) of the RNA template could be detected when Gen 6 A buffer was used. .. To confirm this novel application of Taq DNA polymerase, we tested two different commercial sources of Taq DNA polymerase, NEB and Thermo Fisher, using additional RNA templates and TaqMan RT-qPCR assays: (i) CDC N1, N2, and N3 TaqMan RT-qPCR assays of SARS-CoV-2 genomic RNA purified from infected cells (ATCC) and (ii) CDC RNaseP TaqMan RT-qPCR assay of RNaseP armored RNA. .. Similar to our results with armored N gene RNA, the NEB Taq DNA polymerase was able to perform TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA with all three CDC assays ( and ).

    Infection:

    Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase
    Article Snippet: Robust amplification curves were generated and as few as 10 copies/μL (30 copies total) of the RNA template could be detected when Gen 6 A buffer was used. .. To confirm this novel application of Taq DNA polymerase, we tested two different commercial sources of Taq DNA polymerase, NEB and Thermo Fisher, using additional RNA templates and TaqMan RT-qPCR assays: (i) CDC N1, N2, and N3 TaqMan RT-qPCR assays of SARS-CoV-2 genomic RNA purified from infected cells (ATCC) and (ii) CDC RNaseP TaqMan RT-qPCR assay of RNaseP armored RNA. .. Similar to our results with armored N gene RNA, the NEB Taq DNA polymerase was able to perform TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA with all three CDC assays ( and ).

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    Thermo Fisher taq dna polymerase
    Targeted sequencing library preparation method . (a) Overview of the assay. (b) Specific preparation steps: (1) genomic <t>DNA</t> is digested using Mse I restriction endonuclease. (2) Then, genomic DNA fragments are circularized using thermostable DNA ligase and <t>Taq</t> DNA polymerase for 5' editing. Pool of oligonucleotides targeting 5' and 3' ends of the DNA fragments and vector oligonucleotide are used for targeted DNA capture. (3) After circularization, regular Illumina sequencing library can be prepared by PCR. (4) PCR amplified library fragments are similar to regular Illumina library constructs and anneal to immobilized primers on the flow cell. (5) Additionally, circular constructs can be directly sequenced as the adapted genomic DNA circles incorporate all DNA components required for library immobilization and sequencing. (c) Molecular structures of vector oligonucleotide and capture oligonucleotides.
    Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerase/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
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    taq dna polymerase - by Bioz Stars, 2021-03
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    97
    Thermo Fisher platinum taq high fidelity dna polymerase
    Analysis of the recombinant ANGPTL7/CDT6 plasmid (pNC1) in primary human trabecular meshwork cells. Two primary HTM cell lines (HTM-55 and HTM-69) were nucleofector-transfected with either ANGPTL7/CDT6 plasmid <t>DNA</t> or mock-transfected, and assayed at 72-h post-transfection. (A) Normalized ANGPTL7/CDT6 cDNA in the treated cells versus the mock-transfected cells. Top panel : representative C T logarithmic curve of the hybridizations of ANGPTL7/CDT6 and endogenous 18S cDNAs from treated and mock-transfected cells with their corresponding <t>Taq</t> Man probes. Bottom panel : fold change of ANGPTL7/CDT6 cDNA in treated versus mock-transfected, normalized to 18S and expressed as fold change mean ± range ( n = 3 * P
    Platinum Taq High Fidelity Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/platinum taq high fidelity dna polymerase/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    platinum taq high fidelity dna polymerase - by Bioz Stars, 2021-03
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    Targeted sequencing library preparation method . (a) Overview of the assay. (b) Specific preparation steps: (1) genomic DNA is digested using Mse I restriction endonuclease. (2) Then, genomic DNA fragments are circularized using thermostable DNA ligase and Taq DNA polymerase for 5' editing. Pool of oligonucleotides targeting 5' and 3' ends of the DNA fragments and vector oligonucleotide are used for targeted DNA capture. (3) After circularization, regular Illumina sequencing library can be prepared by PCR. (4) PCR amplified library fragments are similar to regular Illumina library constructs and anneal to immobilized primers on the flow cell. (5) Additionally, circular constructs can be directly sequenced as the adapted genomic DNA circles incorporate all DNA components required for library immobilization and sequencing. (c) Molecular structures of vector oligonucleotide and capture oligonucleotides.

    Journal: BMC Biotechnology

    Article Title: Targeted sequencing library preparation by genomic DNA circularization

    doi: 10.1186/1472-6750-11-122

    Figure Lengend Snippet: Targeted sequencing library preparation method . (a) Overview of the assay. (b) Specific preparation steps: (1) genomic DNA is digested using Mse I restriction endonuclease. (2) Then, genomic DNA fragments are circularized using thermostable DNA ligase and Taq DNA polymerase for 5' editing. Pool of oligonucleotides targeting 5' and 3' ends of the DNA fragments and vector oligonucleotide are used for targeted DNA capture. (3) After circularization, regular Illumina sequencing library can be prepared by PCR. (4) PCR amplified library fragments are similar to regular Illumina library constructs and anneal to immobilized primers on the flow cell. (5) Additionally, circular constructs can be directly sequenced as the adapted genomic DNA circles incorporate all DNA components required for library immobilization and sequencing. (c) Molecular structures of vector oligonucleotide and capture oligonucleotides.

    Article Snippet: Circularization experiments were carried out using Ampligase thermostable ligase (Epicentre) and Taq DNA polymerase (Invitrogen) was used for 5' flap processing.

    Techniques: Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Construct, Flow Cytometry

    Identification of Jdp2 intron 2 mRNAs. ( A ) Ethidium bromide-stained agarose gel showing representative PCR products with linker-specific forward primer on 5′ RACE cDNA from tumor 1161 using different reverse primers in Jdp2 (oligos 96, 46 and 86, lanes 2, 3 and 4, respectively) and Actb exon 3 (lane 5); in lane 1 no cDNA template was added. ( B ) and ( C ) Schematic structure of the alternative Jdp2 exon 1e through 1k as found by 5′ RACE in tumor tissue (B) and normal tissue (C). Positions of exon 1-specific splice donor sites relative to exon 3 are given in base pairs. Putative start codons (M) in frame with the ORF of Jdp2 are indicated, while an asterisk indicates that no ORF is present in frame with Jdp2. ( D ) Protein structure of Jdp2 as generated from exon 1a through 1d, and predicted Jdp2 isoforms generated from exon 1e, 1f, 1i and 1j. The INHAT domain as well as the basic DNA binding domain (DBD) and the leucine zipper (ZIP) regions are indicated. Methionines are indicated (M) and the N-terminal peptides are shown for the isoforms.

    Journal: Nucleic Acids Research

    Article Title: Activation of alternative Jdp2 promoters and functional protein isoforms in T-cell lymphomas by retroviral insertion mutagenesis

    doi: 10.1093/nar/gkp469

    Figure Lengend Snippet: Identification of Jdp2 intron 2 mRNAs. ( A ) Ethidium bromide-stained agarose gel showing representative PCR products with linker-specific forward primer on 5′ RACE cDNA from tumor 1161 using different reverse primers in Jdp2 (oligos 96, 46 and 86, lanes 2, 3 and 4, respectively) and Actb exon 3 (lane 5); in lane 1 no cDNA template was added. ( B ) and ( C ) Schematic structure of the alternative Jdp2 exon 1e through 1k as found by 5′ RACE in tumor tissue (B) and normal tissue (C). Positions of exon 1-specific splice donor sites relative to exon 3 are given in base pairs. Putative start codons (M) in frame with the ORF of Jdp2 are indicated, while an asterisk indicates that no ORF is present in frame with Jdp2. ( D ) Protein structure of Jdp2 as generated from exon 1a through 1d, and predicted Jdp2 isoforms generated from exon 1e, 1f, 1i and 1j. The INHAT domain as well as the basic DNA binding domain (DBD) and the leucine zipper (ZIP) regions are indicated. Methionines are indicated (M) and the N-terminal peptides are shown for the isoforms.

    Article Snippet: To look for alternative splicing between published exons 1a through 1d, PCR reactions were done with forward primers Exon1a-154 (5′-tgggcaccgcgcctgcagcag-3′), Exon1b-148 (5′-ggaggagcgcgagcat-3′), Exon1c-70 (5′-gctctggctgggttaggagggaac-3′) or Exon1d-150 (5′-cagctgcctctctccatctt-3′) and reverse primer Intron2-87 (5′-tccttcgctcttcttcctcgtctagctt-3′) using 1/500 of the cDNA (corresponding to 4 ng of total RNA) per PCR reaction (Taq polymerase, Invitrogen).

    Techniques: Staining, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Generated, Binding Assay

    Jdp2 isoforms are differentially expressed in the normal tissue. ( A – C ) QRT-PCR was done in triplicates as described for Figure 5 on the indicated BALB/c mouse tissues to detect full length (A), exon 1e-3-4 (B) and exon 1f-3-4 (C) mRNA. Expression signal is shown as normalized to the geometric mean of Actb and Tbp (black bars) or normalized to total RNA (white bars) and is shown as fold difference to thymus. The figures are representative of two–three experiments. ( D ) Western blotting on 0.2 μM PVDF membranes using polyclonal anti-Jdp2 and, subsequently, anti-β-Actin and anti-H2B antibody on crude protein extracts from the same panel of BALB/c tissue. Open and closed arrows indicate the position of full length and isoform Jdp2.

    Journal: Nucleic Acids Research

    Article Title: Activation of alternative Jdp2 promoters and functional protein isoforms in T-cell lymphomas by retroviral insertion mutagenesis

    doi: 10.1093/nar/gkp469

    Figure Lengend Snippet: Jdp2 isoforms are differentially expressed in the normal tissue. ( A – C ) QRT-PCR was done in triplicates as described for Figure 5 on the indicated BALB/c mouse tissues to detect full length (A), exon 1e-3-4 (B) and exon 1f-3-4 (C) mRNA. Expression signal is shown as normalized to the geometric mean of Actb and Tbp (black bars) or normalized to total RNA (white bars) and is shown as fold difference to thymus. The figures are representative of two–three experiments. ( D ) Western blotting on 0.2 μM PVDF membranes using polyclonal anti-Jdp2 and, subsequently, anti-β-Actin and anti-H2B antibody on crude protein extracts from the same panel of BALB/c tissue. Open and closed arrows indicate the position of full length and isoform Jdp2.

    Article Snippet: To look for alternative splicing between published exons 1a through 1d, PCR reactions were done with forward primers Exon1a-154 (5′-tgggcaccgcgcctgcagcag-3′), Exon1b-148 (5′-ggaggagcgcgagcat-3′), Exon1c-70 (5′-gctctggctgggttaggagggaac-3′) or Exon1d-150 (5′-cagctgcctctctccatctt-3′) and reverse primer Intron2-87 (5′-tccttcgctcttcttcctcgtctagctt-3′) using 1/500 of the cDNA (corresponding to 4 ng of total RNA) per PCR reaction (Taq polymerase, Invitrogen).

    Techniques: Quantitative RT-PCR, Expressing, Western Blot

    Correlation of intragenic provirus insertion and appearance of Jdp2 intron 2 including transcripts. ( A ) Schematic representation of the localization of the Northern blot probe and QRT-PCR amplicons E2-E3, E2-E4, I2-E4, E1e-E4 and E1f-E4 for Jdp2 mRNA detection with exons shown as boxes and coding sequence in black. ( B and C ) Northern blotting on total RNA from thymus tumors from a subset of animals with integration in clusters D (B) and B (C) according to the retroviral tagging data. Control samples (Ctrl) are thymic tumors from retrovirus-infected animals of the same cohort in which no integrations were found by retroviral tagging. The distance between integration and the beginning of Jdp2 exon 3 for each tumor is shown above the lanes. Ethidium bromide staining of ribosomal bands 28S and 18S was used to evaluate even loading and RNA integrity. ( D and E ) QRT-PCR on a subset of D tumors and B tumors amplifying either Refseq Jdp2 mRNA (E2-E3) (D) or intron 2-including alternative mRNA (I2-E4) (E). The signal was normalized to Tbp and shown as fold difference to the average of control tumor samples (Ctrl).

    Journal: Nucleic Acids Research

    Article Title: Activation of alternative Jdp2 promoters and functional protein isoforms in T-cell lymphomas by retroviral insertion mutagenesis

    doi: 10.1093/nar/gkp469

    Figure Lengend Snippet: Correlation of intragenic provirus insertion and appearance of Jdp2 intron 2 including transcripts. ( A ) Schematic representation of the localization of the Northern blot probe and QRT-PCR amplicons E2-E3, E2-E4, I2-E4, E1e-E4 and E1f-E4 for Jdp2 mRNA detection with exons shown as boxes and coding sequence in black. ( B and C ) Northern blotting on total RNA from thymus tumors from a subset of animals with integration in clusters D (B) and B (C) according to the retroviral tagging data. Control samples (Ctrl) are thymic tumors from retrovirus-infected animals of the same cohort in which no integrations were found by retroviral tagging. The distance between integration and the beginning of Jdp2 exon 3 for each tumor is shown above the lanes. Ethidium bromide staining of ribosomal bands 28S and 18S was used to evaluate even loading and RNA integrity. ( D and E ) QRT-PCR on a subset of D tumors and B tumors amplifying either Refseq Jdp2 mRNA (E2-E3) (D) or intron 2-including alternative mRNA (I2-E4) (E). The signal was normalized to Tbp and shown as fold difference to the average of control tumor samples (Ctrl).

    Article Snippet: To look for alternative splicing between published exons 1a through 1d, PCR reactions were done with forward primers Exon1a-154 (5′-tgggcaccgcgcctgcagcag-3′), Exon1b-148 (5′-ggaggagcgcgagcat-3′), Exon1c-70 (5′-gctctggctgggttaggagggaac-3′) or Exon1d-150 (5′-cagctgcctctctccatctt-3′) and reverse primer Intron2-87 (5′-tccttcgctcttcttcctcgtctagctt-3′) using 1/500 of the cDNA (corresponding to 4 ng of total RNA) per PCR reaction (Taq polymerase, Invitrogen).

    Techniques: Northern Blot, Quantitative RT-PCR, Sequencing, Infection, Staining

    Analysis of the recombinant ANGPTL7/CDT6 plasmid (pNC1) in primary human trabecular meshwork cells. Two primary HTM cell lines (HTM-55 and HTM-69) were nucleofector-transfected with either ANGPTL7/CDT6 plasmid DNA or mock-transfected, and assayed at 72-h post-transfection. (A) Normalized ANGPTL7/CDT6 cDNA in the treated cells versus the mock-transfected cells. Top panel : representative C T logarithmic curve of the hybridizations of ANGPTL7/CDT6 and endogenous 18S cDNAs from treated and mock-transfected cells with their corresponding Taq Man probes. Bottom panel : fold change of ANGPTL7/CDT6 cDNA in treated versus mock-transfected, normalized to 18S and expressed as fold change mean ± range ( n = 3 * P

    Journal: Genes to cells : devoted to molecular & cellular mechanisms

    Article Title: Evidence for a role of angiopoietin-like 7 (ANGPTL7) in extracellular matrix formation of the human trabecular meshwork: implications for glaucoma

    doi: 10.1111/j.1365-2443.2010.01483.x

    Figure Lengend Snippet: Analysis of the recombinant ANGPTL7/CDT6 plasmid (pNC1) in primary human trabecular meshwork cells. Two primary HTM cell lines (HTM-55 and HTM-69) were nucleofector-transfected with either ANGPTL7/CDT6 plasmid DNA or mock-transfected, and assayed at 72-h post-transfection. (A) Normalized ANGPTL7/CDT6 cDNA in the treated cells versus the mock-transfected cells. Top panel : representative C T logarithmic curve of the hybridizations of ANGPTL7/CDT6 and endogenous 18S cDNAs from treated and mock-transfected cells with their corresponding Taq Man probes. Bottom panel : fold change of ANGPTL7/CDT6 cDNA in treated versus mock-transfected, normalized to 18S and expressed as fold change mean ± range ( n = 3 * P

    Article Snippet: PCR was carried out in a 50-μL reaction mixture containing 5 μL 10× high-fidelity PCR buffer, 1 μL dNTP (10 μM each), 2 μL MgSO4 (50 μM), 4 μL primers (5 μM each), 1 μL template cDNA and 1 μL Platinum® Taq High Fidelity DNA Polymerase (5 U) (Invitrogen).

    Techniques: Recombinant, Plasmid Preparation, Transfection

    Daf1 expression in murine fibroblasts, B cells, and macrophages. A , Constitutive Daf1 mRNA level. Total RNA was extracted from the indicated cell lines and reverse transcribed. PCR was then performed using both Daf1- and β-actin-specific primers for the indicated cycle number. PCR products were run on agarose gel and visualized with ethidium bromide. L represents DNA ladder. B , A genomic fragment corresponding to the Daf1 gene 5′ flanking region (−2408/+85 from translational start site) was cloned into the promoterless vector pGL3 encoding firefly luciferase. Either this construct or the pGL3 control vector was transiently transfected into the indicated cell line along with the transfection efficiency control plasmid pRL-TK encoding the Renilla luciferase. Luciferase activities were measured 48 h posttransfection. Data (mean ± SD) have been normalized relative to the Renilla luciferase activity.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Constitutive Expression of Murine Decay-Accelerating Factor 1 Is Controlled by the Transcription Factor Sp1 1

    doi:

    Figure Lengend Snippet: Daf1 expression in murine fibroblasts, B cells, and macrophages. A , Constitutive Daf1 mRNA level. Total RNA was extracted from the indicated cell lines and reverse transcribed. PCR was then performed using both Daf1- and β-actin-specific primers for the indicated cycle number. PCR products were run on agarose gel and visualized with ethidium bromide. L represents DNA ladder. B , A genomic fragment corresponding to the Daf1 gene 5′ flanking region (−2408/+85 from translational start site) was cloned into the promoterless vector pGL3 encoding firefly luciferase. Either this construct or the pGL3 control vector was transiently transfected into the indicated cell line along with the transfection efficiency control plasmid pRL-TK encoding the Renilla luciferase. Luciferase activities were measured 48 h posttransfection. Data (mean ± SD) have been normalized relative to the Renilla luciferase activity.

    Article Snippet: Reverse transcription was then performed using SuperScript III followed by PCR amplification of the cDNA 5′ end using Platinum DNA Polymerase High Fidelity (Invitrogen Life Technologies).

    Techniques: Expressing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Clone Assay, Plasmid Preparation, Luciferase, Construct, Transfection, Activity Assay