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    Jena Bioscience biotin 11 dctp
    Exoribonuclease activity of POLRMT is essential for priming DNA replication in vitro . (A) POLRMT E423P lacks the exoribonuclease activity. 5’-Biotin-labeled target ssRNA (0.05 µM) was incubated with recombinant POLRMT or POLRMT E423P (0.5 µM) in 10 µl reaction mix for indicated time (min) and separated on a denaturing polyacrylamide gel (20%). Arrowhead indicates the full-length target RNA. (B) Pseudo-first-order cleavage kinetics of 5’-Biotin-labeled target ssRNA by POLRMT and POLRMT E423P . The level of remaining RNA in each time point was measured, normalized to the initial level and plotted. Each data point represents the average of three independent experiments. Error bars represent sd. (C) RNA synthesis of POLRMT, POLRMT E423P (500 fmol) and T7 RNA polymerase (0.5 units) on M13mp18 ssDNA template. RNA products were separated on a denaturing polyacrylamide gel (10%). Molecular size markers are shown on the left. (D) Kinetics of Biotin-16-UTP incorporation in the in vitro transcription assay using POLRMT, POLRMT E423P (500 fmol) or T7 RNA polymerase (0.5 units) on ssDNA template. Each time point represents the average of three independent experiments. Error bars represent sd. A line of linear regression was plotted for each protein. (POLRMT, R 2 =0.98; POLRMT E423P , R 2 =0.84; T7 RNA polymerase, R 2 =0.96.) (E) The RNA-primed DNA synthesis assay was monitored in the presence of <t>Biotin-11-dCTP</t> for labeling of DNA products. Lane 1, POLRMT (500 fmol) and POL γ (300 fmol); Lane 2, POLRMT E423P (500 fmol) and POL γ (300 fmol); Lane 3, Control experiment using POL γ (300 fmol) only. Reactions were incubated for 1 h at 37 °C and the products were analyzed on a denaturing polyacrylamide gel (10%). Each data point represents the average of three independent experiments. Error bars represent sd.
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    Exoribonuclease activity of POLRMT is essential for priming DNA replication in vitro . (A) POLRMT E423P lacks the exoribonuclease activity. 5’-Biotin-labeled target ssRNA (0.05 µM) was incubated with recombinant POLRMT or POLRMT E423P (0.5 µM) in 10 µl reaction mix for indicated time (min) and separated on a denaturing polyacrylamide gel (20%). Arrowhead indicates the full-length target RNA. (B) Pseudo-first-order cleavage kinetics of 5’-Biotin-labeled target ssRNA by POLRMT and POLRMT E423P . The level of remaining RNA in each time point was measured, normalized to the initial level and plotted. Each data point represents the average of three independent experiments. Error bars represent sd. (C) RNA synthesis of POLRMT, POLRMT E423P (500 fmol) and T7 RNA polymerase (0.5 units) on M13mp18 ssDNA template. RNA products were separated on a denaturing polyacrylamide gel (10%). Molecular size markers are shown on the left. (D) Kinetics of Biotin-16-UTP incorporation in the in vitro transcription assay using POLRMT, POLRMT E423P (500 fmol) or T7 RNA polymerase (0.5 units) on ssDNA template. Each time point represents the average of three independent experiments. Error bars represent sd. A line of linear regression was plotted for each protein. (POLRMT, R 2 =0.98; POLRMT E423P , R 2 =0.84; T7 RNA polymerase, R 2 =0.96.) (E) The RNA-primed DNA synthesis assay was monitored in the presence of Biotin-11-dCTP for labeling of DNA products. Lane 1, POLRMT (500 fmol) and POL γ (300 fmol); Lane 2, POLRMT E423P (500 fmol) and POL γ (300 fmol); Lane 3, Control experiment using POL γ (300 fmol) only. Reactions were incubated for 1 h at 37 °C and the products were analyzed on a denaturing polyacrylamide gel (10%). Each data point represents the average of three independent experiments. Error bars represent sd.

    Journal: bioRxiv

    Article Title: The PPR domain of mitochondrial RNA polymerase is a ribonuclease required for mtDNA replication

    doi: 10.1101/2021.03.12.435139

    Figure Lengend Snippet: Exoribonuclease activity of POLRMT is essential for priming DNA replication in vitro . (A) POLRMT E423P lacks the exoribonuclease activity. 5’-Biotin-labeled target ssRNA (0.05 µM) was incubated with recombinant POLRMT or POLRMT E423P (0.5 µM) in 10 µl reaction mix for indicated time (min) and separated on a denaturing polyacrylamide gel (20%). Arrowhead indicates the full-length target RNA. (B) Pseudo-first-order cleavage kinetics of 5’-Biotin-labeled target ssRNA by POLRMT and POLRMT E423P . The level of remaining RNA in each time point was measured, normalized to the initial level and plotted. Each data point represents the average of three independent experiments. Error bars represent sd. (C) RNA synthesis of POLRMT, POLRMT E423P (500 fmol) and T7 RNA polymerase (0.5 units) on M13mp18 ssDNA template. RNA products were separated on a denaturing polyacrylamide gel (10%). Molecular size markers are shown on the left. (D) Kinetics of Biotin-16-UTP incorporation in the in vitro transcription assay using POLRMT, POLRMT E423P (500 fmol) or T7 RNA polymerase (0.5 units) on ssDNA template. Each time point represents the average of three independent experiments. Error bars represent sd. A line of linear regression was plotted for each protein. (POLRMT, R 2 =0.98; POLRMT E423P , R 2 =0.84; T7 RNA polymerase, R 2 =0.96.) (E) The RNA-primed DNA synthesis assay was monitored in the presence of Biotin-11-dCTP for labeling of DNA products. Lane 1, POLRMT (500 fmol) and POL γ (300 fmol); Lane 2, POLRMT E423P (500 fmol) and POL γ (300 fmol); Lane 3, Control experiment using POL γ (300 fmol) only. Reactions were incubated for 1 h at 37 °C and the products were analyzed on a denaturing polyacrylamide gel (10%). Each data point represents the average of three independent experiments. Error bars represent sd.

    Article Snippet: In vitro RNA-primed DNA replication The in vitro RNA-primed DNA replication assay was carried out using M13mp18 ssDNA as the template and in the presence of Biotin-11-dCTP (Jena Bioscience).

    Techniques: Activity Assay, In Vitro, Labeling, Incubation, Recombinant, DNA Synthesis