e coli poly a polymerase  (New England Biolabs)


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    Name:
    E coli Poly A Polymerase
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    E coli Poly A Polymerase 500 units
    Catalog Number:
    m0276l
    Price:
    292
    Size:
    500 units
    Category:
    DNA Polymerases
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    New England Biolabs e coli poly a polymerase
    E coli Poly A Polymerase
    E coli Poly A Polymerase 500 units
    https://www.bioz.com/result/e coli poly a polymerase/product/New England Biolabs
    Average 99 stars, based on 32 article reviews
    Price from $9.99 to $1999.99
    e coli poly a polymerase - by Bioz Stars, 2020-09
    99/100 stars

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    1) Product Images from "A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation"

    Article Title: A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.011132

    QL47 inhibits eukaryotic but not prokaryotic protein synthesis. A , E. coli cells carrying the pUA66- rrnB plasmid that constitutively expresses GFP ( 24 ) were treated with DMSO, 250 μg/ml G418, or 50 μ m QL47. The intracellular GFP fluorescence signal was then measured continuously for 14 h at 37 °C. The signal obtained from growth medium was subtracted, and data are presented as means ± S.D. of 12 experimental replicates. One representative experiment is shown from two independent experiments. B , analysis of in vitro translation assays performed in rabbit reticulocyte lysates, yeast cell lysates, or a reconstituted E. coli cell-free synthesis system (PURExpress®). Translation in rabbit reticulocyte lysates was performed in the presence of DMSO, 30 μg/ml CHX, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed reporter DV subgenomic RNA was used as a template, and the luciferase signal was measured after 90-min incubation at 30 °C. Data are presented as means normalized to DMSO ± S.D. of four experimental replicates. Translation in yeast cell lysates was performed in the presence of DMSO, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed vesicular stomatitis virus (VSV) RNA bearing a luciferase reporter gene ( 44 ) was used as a template, and the luciferase signal was measured after 2-h incubation at 25 °C. Data are presented as means normalized to DMSO ± S.D. of three experimental replicates. Translation in a reconstituted E. coli cell-free synthesis system (PURExpress®) was performed in the presence of DMSO, 250 μg/ml G418, 100 μ m QL47, or 100 μ m compound 14. A plasmid expressing GFP under control of a T7 promoter was used as a template. After 1-h incubation at 37 °C, the total protein content was analyzed by Western blotting. The reporter protein was detected using a GFP antibody, and its abundance was normalized to the loading control (histidine tag). Data are presented as means normalized to DMSO ± S.D. of two technical replicates. One representative experiment is shown from four (rabbit reticulocyte lysates) or two (yeast cell lysates and E. coli cell-free synthesis system) independent experiments. Asterisks indicate that the differences between experimental samples and the DMSO-treated control samples are statistically significant when compared using unpaired t test: ***, p
    Figure Legend Snippet: QL47 inhibits eukaryotic but not prokaryotic protein synthesis. A , E. coli cells carrying the pUA66- rrnB plasmid that constitutively expresses GFP ( 24 ) were treated with DMSO, 250 μg/ml G418, or 50 μ m QL47. The intracellular GFP fluorescence signal was then measured continuously for 14 h at 37 °C. The signal obtained from growth medium was subtracted, and data are presented as means ± S.D. of 12 experimental replicates. One representative experiment is shown from two independent experiments. B , analysis of in vitro translation assays performed in rabbit reticulocyte lysates, yeast cell lysates, or a reconstituted E. coli cell-free synthesis system (PURExpress®). Translation in rabbit reticulocyte lysates was performed in the presence of DMSO, 30 μg/ml CHX, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed reporter DV subgenomic RNA was used as a template, and the luciferase signal was measured after 90-min incubation at 30 °C. Data are presented as means normalized to DMSO ± S.D. of four experimental replicates. Translation in yeast cell lysates was performed in the presence of DMSO, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed vesicular stomatitis virus (VSV) RNA bearing a luciferase reporter gene ( 44 ) was used as a template, and the luciferase signal was measured after 2-h incubation at 25 °C. Data are presented as means normalized to DMSO ± S.D. of three experimental replicates. Translation in a reconstituted E. coli cell-free synthesis system (PURExpress®) was performed in the presence of DMSO, 250 μg/ml G418, 100 μ m QL47, or 100 μ m compound 14. A plasmid expressing GFP under control of a T7 promoter was used as a template. After 1-h incubation at 37 °C, the total protein content was analyzed by Western blotting. The reporter protein was detected using a GFP antibody, and its abundance was normalized to the loading control (histidine tag). Data are presented as means normalized to DMSO ± S.D. of two technical replicates. One representative experiment is shown from four (rabbit reticulocyte lysates) or two (yeast cell lysates and E. coli cell-free synthesis system) independent experiments. Asterisks indicate that the differences between experimental samples and the DMSO-treated control samples are statistically significant when compared using unpaired t test: ***, p

    Techniques Used: Plasmid Preparation, Fluorescence, In Vitro, Luciferase, Incubation, Expressing, Western Blot

    2) Product Images from "A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation"

    Article Title: A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.011132

    QL47 inhibits protein synthesis in vitro . A , SDS-PAGE analysis of in vitro translations performed in rabbit reticulocyte lysates for 90 min at 30 °C in the presence of precharged FluoroTect TM lysine tRNA and DMSO, 40 μ m QL47, or 30 μg/ml CHX. An in vitro transcribed reporter RNA bearing the EMCV IRES and a luciferase ( Luc ) reporter gene ( 42 ) was used as a template. One representative experiment is shown from three independent experiments. The abundance of neosynthesized fluorescent proteins was measured and is graphically presented on the right . Data are presented as means normalized to DMSO ± S.D. of two independent experiments. B , rabbit reticulocyte lysates were pretreated with DMSO or 25 μ m proteasome inhibitor lactacystin for 15 min at room temperature. Subsequently, an in vitro transcribed reporter DV subgenomic RNA ( 40 ) was added, and lysates were incubated in the presence of DMSO or 40 μ m of the indicated small molecules for 90 min at 30 °C. The luciferase signal was measured, and data are presented as means ± S.D. of two technical replicates. One representative experiment is shown from two independent experiments. RLU , relative light units. C , translation of in vitro transcribed reporter DV subgenomic RNA in rabbit reticulocyte lysates was performed for 90 min at 30 °C in the presence of DMSO, 30 μg/ml CHX, or 40 μ m of QL47 and the indicated analogs. The luciferase signal was measured, and data are presented as means ± S.D. of two technical replicates. One representative experiment is shown from three independent experiments.
    Figure Legend Snippet: QL47 inhibits protein synthesis in vitro . A , SDS-PAGE analysis of in vitro translations performed in rabbit reticulocyte lysates for 90 min at 30 °C in the presence of precharged FluoroTect TM lysine tRNA and DMSO, 40 μ m QL47, or 30 μg/ml CHX. An in vitro transcribed reporter RNA bearing the EMCV IRES and a luciferase ( Luc ) reporter gene ( 42 ) was used as a template. One representative experiment is shown from three independent experiments. The abundance of neosynthesized fluorescent proteins was measured and is graphically presented on the right . Data are presented as means normalized to DMSO ± S.D. of two independent experiments. B , rabbit reticulocyte lysates were pretreated with DMSO or 25 μ m proteasome inhibitor lactacystin for 15 min at room temperature. Subsequently, an in vitro transcribed reporter DV subgenomic RNA ( 40 ) was added, and lysates were incubated in the presence of DMSO or 40 μ m of the indicated small molecules for 90 min at 30 °C. The luciferase signal was measured, and data are presented as means ± S.D. of two technical replicates. One representative experiment is shown from two independent experiments. RLU , relative light units. C , translation of in vitro transcribed reporter DV subgenomic RNA in rabbit reticulocyte lysates was performed for 90 min at 30 °C in the presence of DMSO, 30 μg/ml CHX, or 40 μ m of QL47 and the indicated analogs. The luciferase signal was measured, and data are presented as means ± S.D. of two technical replicates. One representative experiment is shown from three independent experiments.

    Techniques Used: In Vitro, SDS Page, Luciferase, Incubation

    QL47 inhibits protein synthesis in live cells. A , chemical structures of QL47 and the derivatives YKL-04-085, QL47R, and YKL-03-109 (compound 14) ( 6 , 9 ). QL47R is an inactive analog due to replacement of the cysteine-reactive acrylamide moiety with a nonreactive propyl amide. Compound 14 is a negative control compound with significantly diminished antiviral activity. B , HEK293T cells were transfected with an in vitro transcribed reporter DV subgenomic RNA bearing the virus's seven nonstructural genes ( NS1–NS5 ) as well as a NanoLuc®-proline, glutamate, serine, threonine ( NlucP ) luciferase reporter gene. The intracellular reporter activity was immediately measured for 1 h at 37 °C. Cells were treated with DMSO or 2 μ m QL47 at 1 h post-transfection, and the intracellular luciferase signal was then measured continuously for 3 h at 37 °C. The luciferase signal obtained from mock-transfected cells was subtracted, and data are presented as means ± S.D. of three experimental replicates. One representative experiment is shown from two independent experiments. RLU , relative light units. C , measurement of puromycin incorporation in nascent cellular proteins. Huh7 cells were treated with DMSO, 50 μg/ml CHX, or 2 μ m QL47 for 1 h. The cells were next pulse-labeled with 1 μ m puromycin for 30 min, and their cellular contents were analyzed by Western blotting. Prematurely terminated polypeptides were detected using a puromycin-specific antibody. Their abundance was normalized to the loading control (tubulin) and is presented as a percentage of the DMSO-treated control samples. One representative experiment is shown from two independent experiments. D , metabolic labeling of cellular proteins with radiolabeled amino acids. Huh7 cells were starved for 30 min in methionine/cysteine-free medium and concomitantly treated with DMSO, 30 μg/ml CHX, 2 μ m QL47, or 2 μ m compound 14. The medium was next supplemented with a mixture of 35 S-Met and 35 S-Cys for 30 min. Total cell lysates were analyzed by SDS-PAGE, followed by autoradiography to measure bulk protein synthesis. Neosynthesized protein abundance is presented as a percentage of the DMSO-treated control samples. One representative experiment is shown from three independent experiments. E , Huh7 cells were treated with DMSO or 2 μ m QL47, and metabolic labeling ( red boxes ) was performed as indicated in D 1 h and 24 h post-treatment. Analysis of the neosynthesized proteins was performed as in D , and their abundance is presented as a percentage of the DMSO-treated control samples at each time point. One representative experiment is shown from two independent experiments.
    Figure Legend Snippet: QL47 inhibits protein synthesis in live cells. A , chemical structures of QL47 and the derivatives YKL-04-085, QL47R, and YKL-03-109 (compound 14) ( 6 , 9 ). QL47R is an inactive analog due to replacement of the cysteine-reactive acrylamide moiety with a nonreactive propyl amide. Compound 14 is a negative control compound with significantly diminished antiviral activity. B , HEK293T cells were transfected with an in vitro transcribed reporter DV subgenomic RNA bearing the virus's seven nonstructural genes ( NS1–NS5 ) as well as a NanoLuc®-proline, glutamate, serine, threonine ( NlucP ) luciferase reporter gene. The intracellular reporter activity was immediately measured for 1 h at 37 °C. Cells were treated with DMSO or 2 μ m QL47 at 1 h post-transfection, and the intracellular luciferase signal was then measured continuously for 3 h at 37 °C. The luciferase signal obtained from mock-transfected cells was subtracted, and data are presented as means ± S.D. of three experimental replicates. One representative experiment is shown from two independent experiments. RLU , relative light units. C , measurement of puromycin incorporation in nascent cellular proteins. Huh7 cells were treated with DMSO, 50 μg/ml CHX, or 2 μ m QL47 for 1 h. The cells were next pulse-labeled with 1 μ m puromycin for 30 min, and their cellular contents were analyzed by Western blotting. Prematurely terminated polypeptides were detected using a puromycin-specific antibody. Their abundance was normalized to the loading control (tubulin) and is presented as a percentage of the DMSO-treated control samples. One representative experiment is shown from two independent experiments. D , metabolic labeling of cellular proteins with radiolabeled amino acids. Huh7 cells were starved for 30 min in methionine/cysteine-free medium and concomitantly treated with DMSO, 30 μg/ml CHX, 2 μ m QL47, or 2 μ m compound 14. The medium was next supplemented with a mixture of 35 S-Met and 35 S-Cys for 30 min. Total cell lysates were analyzed by SDS-PAGE, followed by autoradiography to measure bulk protein synthesis. Neosynthesized protein abundance is presented as a percentage of the DMSO-treated control samples. One representative experiment is shown from three independent experiments. E , Huh7 cells were treated with DMSO or 2 μ m QL47, and metabolic labeling ( red boxes ) was performed as indicated in D 1 h and 24 h post-treatment. Analysis of the neosynthesized proteins was performed as in D , and their abundance is presented as a percentage of the DMSO-treated control samples at each time point. One representative experiment is shown from two independent experiments.

    Techniques Used: Negative Control, Activity Assay, Transfection, In Vitro, Luciferase, Labeling, Western Blot, SDS Page, Autoradiography

    QL47 inhibits eukaryotic but not prokaryotic protein synthesis. A , E. coli cells carrying the pUA66- rrnB plasmid that constitutively expresses GFP ( 24 ) were treated with DMSO, 250 μg/ml G418, or 50 μ m QL47. The intracellular GFP fluorescence signal was then measured continuously for 14 h at 37 °C. The signal obtained from growth medium was subtracted, and data are presented as means ± S.D. of 12 experimental replicates. One representative experiment is shown from two independent experiments. B , analysis of in vitro translation assays performed in rabbit reticulocyte lysates, yeast cell lysates, or a reconstituted E. coli cell-free synthesis system (PURExpress®). Translation in rabbit reticulocyte lysates was performed in the presence of DMSO, 30 μg/ml CHX, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed reporter DV subgenomic RNA was used as a template, and the luciferase signal was measured after 90-min incubation at 30 °C. Data are presented as means normalized to DMSO ± S.D. of four experimental replicates. Translation in yeast cell lysates was performed in the presence of DMSO, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed vesicular stomatitis virus (VSV) RNA bearing a luciferase reporter gene ( 44 ) was used as a template, and the luciferase signal was measured after 2-h incubation at 25 °C. Data are presented as means normalized to DMSO ± S.D. of three experimental replicates. Translation in a reconstituted E. coli cell-free synthesis system (PURExpress®) was performed in the presence of DMSO, 250 μg/ml G418, 100 μ m QL47, or 100 μ m compound 14. A plasmid expressing GFP under control of a T7 promoter was used as a template. After 1-h incubation at 37 °C, the total protein content was analyzed by Western blotting. The reporter protein was detected using a GFP antibody, and its abundance was normalized to the loading control (histidine tag). Data are presented as means normalized to DMSO ± S.D. of two technical replicates. One representative experiment is shown from four (rabbit reticulocyte lysates) or two (yeast cell lysates and E. coli cell-free synthesis system) independent experiments. Asterisks indicate that the differences between experimental samples and the DMSO-treated control samples are statistically significant when compared using unpaired t test: ***, p
    Figure Legend Snippet: QL47 inhibits eukaryotic but not prokaryotic protein synthesis. A , E. coli cells carrying the pUA66- rrnB plasmid that constitutively expresses GFP ( 24 ) were treated with DMSO, 250 μg/ml G418, or 50 μ m QL47. The intracellular GFP fluorescence signal was then measured continuously for 14 h at 37 °C. The signal obtained from growth medium was subtracted, and data are presented as means ± S.D. of 12 experimental replicates. One representative experiment is shown from two independent experiments. B , analysis of in vitro translation assays performed in rabbit reticulocyte lysates, yeast cell lysates, or a reconstituted E. coli cell-free synthesis system (PURExpress®). Translation in rabbit reticulocyte lysates was performed in the presence of DMSO, 30 μg/ml CHX, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed reporter DV subgenomic RNA was used as a template, and the luciferase signal was measured after 90-min incubation at 30 °C. Data are presented as means normalized to DMSO ± S.D. of four experimental replicates. Translation in yeast cell lysates was performed in the presence of DMSO, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed vesicular stomatitis virus (VSV) RNA bearing a luciferase reporter gene ( 44 ) was used as a template, and the luciferase signal was measured after 2-h incubation at 25 °C. Data are presented as means normalized to DMSO ± S.D. of three experimental replicates. Translation in a reconstituted E. coli cell-free synthesis system (PURExpress®) was performed in the presence of DMSO, 250 μg/ml G418, 100 μ m QL47, or 100 μ m compound 14. A plasmid expressing GFP under control of a T7 promoter was used as a template. After 1-h incubation at 37 °C, the total protein content was analyzed by Western blotting. The reporter protein was detected using a GFP antibody, and its abundance was normalized to the loading control (histidine tag). Data are presented as means normalized to DMSO ± S.D. of two technical replicates. One representative experiment is shown from four (rabbit reticulocyte lysates) or two (yeast cell lysates and E. coli cell-free synthesis system) independent experiments. Asterisks indicate that the differences between experimental samples and the DMSO-treated control samples are statistically significant when compared using unpaired t test: ***, p

    Techniques Used: Plasmid Preparation, Fluorescence, In Vitro, Luciferase, Incubation, Expressing, Western Blot

    Related Articles

    In Vitro:

    Article Title: A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation
    Article Snippet: .. For pcDNA3-RLUC-POLIRES-FLUC and pcDNA3-RLUC-CrPV-IRES-FLUC, in vitro transcripts were synthesized from Xho I-linearized plasmids using the mMessage mMachine T7 transcription kit (Thermo Fisher Scientific, AM1344) and then polyadenylated using E. coli poly(A) polymerase (New England Biolabs, M0276). .. In vitro transcripts were synthesized from Xho I-linearized pBS-EMCV-Fluci and Xba I-linearized mung bean nuclease–treated pSGR-JFH1/Luc using the AmpliScribe T7-Flash transcription kit (Lucigen, ASF3257).

    Article Title: Efficient multiplex genome editing using CRISPR-Mb3Cas12a in mice
    Article Snippet: .. Then the in vitro transcribed mRNAs were treated with DNase I (NEB, Cat. M0303S) to remove the plasmid DNA template, followed by poly(A) tailing using E. coli poly(A) polymerase (Cat. M0276S, NEB). .. The poly(A)-tailed Mb3Cas12a mRNAs were purified using the RNA Clean & Concentrator™-5 (Cat. R1016, Zymo Research, Irvine, CA) and eluted in a Tris-EDTA solution (Cat.11-01-02-02, IDT, Coralville, IA). crRNAs were designed using Benchling ( https://benchling.com/ ).

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: .. Next, a polyA tail was added in-vitro by incubating the capped RNA in 50 μ! reaction volume with 20 units E. coli Poly(A) Polymerase (New England Biolabs) and 1mM ATP for 15min at 37°C. .. The capped and tailed RNA was purified using AMPure beads and eluted in 50 μl low TE buffer.

    Synthesized:

    Article Title: A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation
    Article Snippet: .. For pcDNA3-RLUC-POLIRES-FLUC and pcDNA3-RLUC-CrPV-IRES-FLUC, in vitro transcripts were synthesized from Xho I-linearized plasmids using the mMessage mMachine T7 transcription kit (Thermo Fisher Scientific, AM1344) and then polyadenylated using E. coli poly(A) polymerase (New England Biolabs, M0276). .. In vitro transcripts were synthesized from Xho I-linearized pBS-EMCV-Fluci and Xba I-linearized mung bean nuclease–treated pSGR-JFH1/Luc using the AmpliScribe T7-Flash transcription kit (Lucigen, ASF3257).

    Incubation:

    Article Title: Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing
    Article Snippet: .. Briefly, 5 µg RNA, 20 units poly(A) polymerase, 2 µl reaction buffer and 1 mM ATP were incubated for 15 min at 37°C in a total reaction volume of 50 µl. .. To stop the reaction and to remove the enzyme, the poly(A)-tailed RNA was purified with the RNeasy MinElute Cleanup Kit (Qiagen).

    Purification:

    Article Title: RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc
    Article Snippet: .. RNA was then polyadenylated by E. coli poly(A) polymerase (M0276S, NEB) in a 50 μl volume containing 50 mM Tris–HCl (pH 7.9), 250 mM NaCl, 10 mM MgCl2 , 1.5 μg RNA, 2.5 U poly(A) polymerase, and 1 mM ATP. cDNA was generated from the purified tailed RNA using M-MLV Reverse Transcriptase (M368B, Promega). ..

    Generated:

    Article Title: RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc
    Article Snippet: .. RNA was then polyadenylated by E. coli poly(A) polymerase (M0276S, NEB) in a 50 μl volume containing 50 mM Tris–HCl (pH 7.9), 250 mM NaCl, 10 mM MgCl2 , 1.5 μg RNA, 2.5 U poly(A) polymerase, and 1 mM ATP. cDNA was generated from the purified tailed RNA using M-MLV Reverse Transcriptase (M368B, Promega). ..

    Plasmid Preparation:

    Article Title: Efficient multiplex genome editing using CRISPR-Mb3Cas12a in mice
    Article Snippet: .. Then the in vitro transcribed mRNAs were treated with DNase I (NEB, Cat. M0303S) to remove the plasmid DNA template, followed by poly(A) tailing using E. coli poly(A) polymerase (Cat. M0276S, NEB). .. The poly(A)-tailed Mb3Cas12a mRNAs were purified using the RNA Clean & Concentrator™-5 (Cat. R1016, Zymo Research, Irvine, CA) and eluted in a Tris-EDTA solution (Cat.11-01-02-02, IDT, Coralville, IA). crRNAs were designed using Benchling ( https://benchling.com/ ).

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    New England Biolabs m7 gpppa rna cap structure analogue
    M7 Gpppa Rna Cap Structure Analogue, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m7 gpppa rna cap structure analogue/product/New England Biolabs
    Average 90 stars, based on 8 article reviews
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    m7 gpppa rna cap structure analogue - by Bioz Stars, 2020-09
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