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InGex tgirt iii group ii intron rt
Tgirt Iii Group Ii Intron Rt, supplied by InGex, 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/tgirt iii group ii intron rt/product/InGex
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
tgirt iii group ii intron rt - by Bioz Stars, 2020-04
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Marker:

Article Title: A single nucleotide incorporation step limits human telomerase repeat addition activity
Article Snippet: The assay with TGIRT III group II intron RT (InGex) contained 50 units of enzyme, 1× reaction buffer (20 mM Tris–HCl, pH 7.5, 5 mM MgCl2 , 0.45 M NaCl, 5 mM DTT) and 10 μM dGTP, dGDP, or dGMP, and 0.165 μM α‐32 P‐dATP. .. The size marker was prepared in a 10 μl reaction containing 1× reaction buffer (100 mM sodium cacodylate, pH 6.8, 1 mM CoCl2 , and 0.1 mM DTT), 1 μM oligonucleotide as indicated, 10 units of terminal deoxynucleotidyl transferase (TdT, Affymetrix), and 0.165 μM α‐32 P‐dATP (3,000 Ci/mmol, 10 mCi/ml; PerkinElmer).

Ethanol Precipitation:

Article Title: A single nucleotide incorporation step limits human telomerase repeat addition activity
Article Snippet: The assay with TGIRT III group II intron RT (InGex) contained 50 units of enzyme, 1× reaction buffer (20 mM Tris–HCl, pH 7.5, 5 mM MgCl2 , 0.45 M NaCl, 5 mM DTT) and 10 μM dGTP, dGDP, or dGMP, and 0.165 μM α‐32 P‐dATP. .. Reactions were incubated at 30°C for 60 min and terminated by phenol/chloroform extraction, followed by ethanol precipitation.

Incubation:

Article Title: A single nucleotide incorporation step limits human telomerase repeat addition activity
Article Snippet: The assay with TGIRT III group II intron RT (InGex) contained 50 units of enzyme, 1× reaction buffer (20 mM Tris–HCl, pH 7.5, 5 mM MgCl2 , 0.45 M NaCl, 5 mM DTT) and 10 μM dGTP, dGDP, or dGMP, and 0.165 μM α‐32 P‐dATP. .. Reactions were incubated at 30°C for 60 min and terminated by phenol/chloroform extraction, followed by ethanol precipitation.

Activity Assay:

Article Title: A single nucleotide incorporation step limits human telomerase repeat addition activity
Article Snippet: Paragraph title: Activity assay for dNDP and dNMP incorporation ... The assay with TGIRT III group II intron RT (InGex) contained 50 units of enzyme, 1× reaction buffer (20 mM Tris–HCl, pH 7.5, 5 mM MgCl2 , 0.45 M NaCl, 5 mM DTT) and 10 μM dGTP, dGDP, or dGMP, and 0.165 μM α‐32 P‐dATP.

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    InGex tgirt enzyme
    <t>TGIRT</t> ssDNA-seq workflow. The target DNA (2–50 ng in this work) is treated with alkaline phosphatase to remove 3′ phosphates and heat denatured prior to DNA-seq library construction. The resulting ssDNAs with 3′ OH termini are then used for TGIRT template-switching DNA synthesis coupled to Illumina read 2 reverse (R2R) DNA-seq adapter addition. In this novel reaction, the <t>TGIRT-III</t> enzyme (InGex) binds first to a synthetic 34-bp R2 RNA/R2R DNA heteroduplex in which the R2R DNA primer has a single nucleotide 3′ overhang that can direct TGIRT template switching by base pairing to the 3′ nucleotide of a target DNA strand. For minimally biased library preparation, the 3′-overhang nucleotide is an equimolar mixture of A, C, G, and T (denoted N). A 3′-blocking group (C3 spacer; 3′SpC3) is attached to the end of the R2 RNA oligonucleotide to prevent template-switching to that RNA. After the TGIRT enzyme extends the DNA primer to produce a DNA copy of the target DNA strand with an R2R adapter seamlessly linked to its 5′ end, a 5′ adenylated (App) Illumina read 1 reverse (R1R) adapter is added to its 3′ end by single-stranded DNA ligation using a Thermostable 5′ AppDNA/RNA ligase (New England Biolabs). In the workflow shown, an unique molecular identifier (UMI) is positioned at the 5′ end of the R1R DNA oligonucleotide. A final PCR step adds flow cell capture sites and barcodes for Illumina sequencing and sample multiplexing.
    Tgirt Enzyme, supplied by InGex, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tgirt enzyme/product/InGex
    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    tgirt enzyme - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    93
    InGex tgirt iii group ii intron rt
    <t>TGIRT</t> ssDNA-seq workflow. The target DNA (2–50 ng in this work) is treated with alkaline phosphatase to remove 3′ phosphates and heat denatured prior to DNA-seq library construction. The resulting ssDNAs with 3′ OH termini are then used for TGIRT template-switching DNA synthesis coupled to Illumina read 2 reverse (R2R) DNA-seq adapter addition. In this novel reaction, the <t>TGIRT-III</t> enzyme (InGex) binds first to a synthetic 34-bp R2 RNA/R2R DNA heteroduplex in which the R2R DNA primer has a single nucleotide 3′ overhang that can direct TGIRT template switching by base pairing to the 3′ nucleotide of a target DNA strand. For minimally biased library preparation, the 3′-overhang nucleotide is an equimolar mixture of A, C, G, and T (denoted N). A 3′-blocking group (C3 spacer; 3′SpC3) is attached to the end of the R2 RNA oligonucleotide to prevent template-switching to that RNA. After the TGIRT enzyme extends the DNA primer to produce a DNA copy of the target DNA strand with an R2R adapter seamlessly linked to its 5′ end, a 5′ adenylated (App) Illumina read 1 reverse (R1R) adapter is added to its 3′ end by single-stranded DNA ligation using a Thermostable 5′ AppDNA/RNA ligase (New England Biolabs). In the workflow shown, an unique molecular identifier (UMI) is positioned at the 5′ end of the R1R DNA oligonucleotide. A final PCR step adds flow cell capture sites and barcodes for Illumina sequencing and sample multiplexing.
    Tgirt Iii Group Ii Intron Rt, supplied by InGex, 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/tgirt iii group ii intron rt/product/InGex
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tgirt iii group ii intron rt - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

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    TGIRT ssDNA-seq workflow. The target DNA (2–50 ng in this work) is treated with alkaline phosphatase to remove 3′ phosphates and heat denatured prior to DNA-seq library construction. The resulting ssDNAs with 3′ OH termini are then used for TGIRT template-switching DNA synthesis coupled to Illumina read 2 reverse (R2R) DNA-seq adapter addition. In this novel reaction, the TGIRT-III enzyme (InGex) binds first to a synthetic 34-bp R2 RNA/R2R DNA heteroduplex in which the R2R DNA primer has a single nucleotide 3′ overhang that can direct TGIRT template switching by base pairing to the 3′ nucleotide of a target DNA strand. For minimally biased library preparation, the 3′-overhang nucleotide is an equimolar mixture of A, C, G, and T (denoted N). A 3′-blocking group (C3 spacer; 3′SpC3) is attached to the end of the R2 RNA oligonucleotide to prevent template-switching to that RNA. After the TGIRT enzyme extends the DNA primer to produce a DNA copy of the target DNA strand with an R2R adapter seamlessly linked to its 5′ end, a 5′ adenylated (App) Illumina read 1 reverse (R1R) adapter is added to its 3′ end by single-stranded DNA ligation using a Thermostable 5′ AppDNA/RNA ligase (New England Biolabs). In the workflow shown, an unique molecular identifier (UMI) is positioned at the 5′ end of the R1R DNA oligonucleotide. A final PCR step adds flow cell capture sites and barcodes for Illumina sequencing and sample multiplexing.

    Journal: Scientific Reports

    Article Title: Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching

    doi: 10.1038/s41598-017-09064-w

    Figure Lengend Snippet: TGIRT ssDNA-seq workflow. The target DNA (2–50 ng in this work) is treated with alkaline phosphatase to remove 3′ phosphates and heat denatured prior to DNA-seq library construction. The resulting ssDNAs with 3′ OH termini are then used for TGIRT template-switching DNA synthesis coupled to Illumina read 2 reverse (R2R) DNA-seq adapter addition. In this novel reaction, the TGIRT-III enzyme (InGex) binds first to a synthetic 34-bp R2 RNA/R2R DNA heteroduplex in which the R2R DNA primer has a single nucleotide 3′ overhang that can direct TGIRT template switching by base pairing to the 3′ nucleotide of a target DNA strand. For minimally biased library preparation, the 3′-overhang nucleotide is an equimolar mixture of A, C, G, and T (denoted N). A 3′-blocking group (C3 spacer; 3′SpC3) is attached to the end of the R2 RNA oligonucleotide to prevent template-switching to that RNA. After the TGIRT enzyme extends the DNA primer to produce a DNA copy of the target DNA strand with an R2R adapter seamlessly linked to its 5′ end, a 5′ adenylated (App) Illumina read 1 reverse (R1R) adapter is added to its 3′ end by single-stranded DNA ligation using a Thermostable 5′ AppDNA/RNA ligase (New England Biolabs). In the workflow shown, an unique molecular identifier (UMI) is positioned at the 5′ end of the R1R DNA oligonucleotide. A final PCR step adds flow cell capture sites and barcodes for Illumina sequencing and sample multiplexing.

    Article Snippet: In the first step, the TGIRT enzyme (TGIRT-III, a derivative of the Geobacillus stearothermohilus GsI-IIC group II intron RT; InGex) binds to a short synthetic RNA template/DNA primer heteroduplex substrate in which the DNA primer contains a reverse complement of Illumina read 2 adapter sequence (R2R) and has a single-nucleotide 3′ DNA overhang (see Supplementary Table ).

    Techniques: DNA Sequencing, DNA Synthesis, Blocking Assay, DNA Ligation, Polymerase Chain Reaction, Flow Cytometry, Sequencing, Multiplexing

    Bisulfite sequencing of plasma cfDNA. Three separate TGIRT-seq libraries were each constructed from ~5 ng of bisulfite-treated plasma DNA from a healthy male individual, and sequenced to obtain TGIRT-seq datasets BPD1-3 (see Supplementary Table S4 ). The datasets were combined and analyzed to identify DNA methylation sites, as described in Methods. ( a ) Annotation of DNA methylation sites in the human genome and determination of DNA methylation densities by TGIRT-seq of bisulfite-treated plasma cfDNA. Tracks from the inner to the outer circle represent: (1) annotations of Type II biomarkers that are highly variable in methylation density across tissues; (2) annotations of Type I biomarkers that are highly tissue specific (color-coded); (3) bar graphs of methylation densities within the annotated regions based on TGIRT-seq of bisulfite-treated plasma cfDNA; (4) genome coordinates. ( b ) Tissues-of-origin of plasma cfDNA from a healthy male individual determined by TGIRT-seq of bisulfite-treated DNA. The pie chart shows the percent contributions of different tissues in combined datasets PB1-3 determined by quadratic programming 7 . Neutrophils are derived from myeloid precursors, and lymphocytes are a combination of T-cells and B-cells. Comparison of tissues-of-origin of plasma cfDNA determined as in panel ( b ) from the three independent datasets BPD1-3 are shown in Supplementary Fig. 11 . Pearson’s correlation coefficients were ≥0.96 for each pairwise combination of the three individual datasets.

    Journal: Scientific Reports

    Article Title: Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching

    doi: 10.1038/s41598-017-09064-w

    Figure Lengend Snippet: Bisulfite sequencing of plasma cfDNA. Three separate TGIRT-seq libraries were each constructed from ~5 ng of bisulfite-treated plasma DNA from a healthy male individual, and sequenced to obtain TGIRT-seq datasets BPD1-3 (see Supplementary Table S4 ). The datasets were combined and analyzed to identify DNA methylation sites, as described in Methods. ( a ) Annotation of DNA methylation sites in the human genome and determination of DNA methylation densities by TGIRT-seq of bisulfite-treated plasma cfDNA. Tracks from the inner to the outer circle represent: (1) annotations of Type II biomarkers that are highly variable in methylation density across tissues; (2) annotations of Type I biomarkers that are highly tissue specific (color-coded); (3) bar graphs of methylation densities within the annotated regions based on TGIRT-seq of bisulfite-treated plasma cfDNA; (4) genome coordinates. ( b ) Tissues-of-origin of plasma cfDNA from a healthy male individual determined by TGIRT-seq of bisulfite-treated DNA. The pie chart shows the percent contributions of different tissues in combined datasets PB1-3 determined by quadratic programming 7 . Neutrophils are derived from myeloid precursors, and lymphocytes are a combination of T-cells and B-cells. Comparison of tissues-of-origin of plasma cfDNA determined as in panel ( b ) from the three independent datasets BPD1-3 are shown in Supplementary Fig. 11 . Pearson’s correlation coefficients were ≥0.96 for each pairwise combination of the three individual datasets.

    Article Snippet: In the first step, the TGIRT enzyme (TGIRT-III, a derivative of the Geobacillus stearothermohilus GsI-IIC group II intron RT; InGex) binds to a short synthetic RNA template/DNA primer heteroduplex substrate in which the DNA primer contains a reverse complement of Illumina read 2 adapter sequence (R2R) and has a single-nucleotide 3′ DNA overhang (see Supplementary Table ).

    Techniques: Methylation Sequencing, Construct, DNA Methylation Assay, Methylation, Derivative Assay