monophosphate pre mirnas  (New England Biolabs)


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    Name:
    T7 RNA Polymerase
    Description:
    T7 RNA Polymerase 25 000 units
    Catalog Number:
    m0251l
    Price:
    282
    Size:
    25 000 units
    Category:
    RNA Polymerases
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    Structured Review

    New England Biolabs monophosphate pre mirnas
    T7 RNA Polymerase
    T7 RNA Polymerase 25 000 units
    https://www.bioz.com/result/monophosphate pre mirnas/product/New England Biolabs
    Average 92 stars, based on 1470 article reviews
    Price from $9.99 to $1999.99
    monophosphate pre mirnas - by Bioz Stars, 2020-09
    92/100 stars

    Images

    1) Product Images from "Dicer cleaves 5′-extended microRNA precursors originating from RNA polymerase II transcription start sites"

    Article Title: Dicer cleaves 5′-extended microRNA precursors originating from RNA polymerase II transcription start sites

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky306

    Processing of 5′-extended pre-miRNAs is independent of Dicer 5′ pocket. ( A ) In vitro transcribed 5′-extended pre-miR-HSUR4s were incubated with four different human Dicers purified by Flag IP. [WT: wild type Dicer; TN: transdominant negative mutation Dicer; 3′ mut: 3′ pocket mutation Dicer (Y926A); 5′ mut: 5′ pocket mutation Dicer (R778A/R780A/H982A)]. Northern blots were performed using probes targeting miR-HSUR4-3p. ( B ) Quantitations of relative mature miRNA levels (mature miRNA/pre-miRNA) compared to WT Dicer processing in (A) (mean ± standard deviation) were derived from three independent experiments. FC: fold change. ( C ) In vitro transcribed 5′-monophosphate pre-miR-HSUR4s without 5′ extension (–2 nt) but with 1 nt or 2 nt 3′ overhang were incubated with four different human Dicers purified by Flag IP. Schematics of pre-miR-HSUR4s with 1 nt or 2 nt 3′ overhang are given. ( D ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (C) were derived from three independent experiments. ( E ) In vitro transcribed 5′-monophosphate pre-let-7a-1 and m 7 G-capped +15 nt extended pre-let-7a-1 were incubated with four different human Dicers purified by Flag IP. Northern blots were performed using probes targeting let-7a-1 (5p). Schematic of +15 nt extended pre-let-7a-1 is shown, with extended sequence underlined. ( F ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (E) were derived from three independent experiments.
    Figure Legend Snippet: Processing of 5′-extended pre-miRNAs is independent of Dicer 5′ pocket. ( A ) In vitro transcribed 5′-extended pre-miR-HSUR4s were incubated with four different human Dicers purified by Flag IP. [WT: wild type Dicer; TN: transdominant negative mutation Dicer; 3′ mut: 3′ pocket mutation Dicer (Y926A); 5′ mut: 5′ pocket mutation Dicer (R778A/R780A/H982A)]. Northern blots were performed using probes targeting miR-HSUR4-3p. ( B ) Quantitations of relative mature miRNA levels (mature miRNA/pre-miRNA) compared to WT Dicer processing in (A) (mean ± standard deviation) were derived from three independent experiments. FC: fold change. ( C ) In vitro transcribed 5′-monophosphate pre-miR-HSUR4s without 5′ extension (–2 nt) but with 1 nt or 2 nt 3′ overhang were incubated with four different human Dicers purified by Flag IP. Schematics of pre-miR-HSUR4s with 1 nt or 2 nt 3′ overhang are given. ( D ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (C) were derived from three independent experiments. ( E ) In vitro transcribed 5′-monophosphate pre-let-7a-1 and m 7 G-capped +15 nt extended pre-let-7a-1 were incubated with four different human Dicers purified by Flag IP. Northern blots were performed using probes targeting let-7a-1 (5p). Schematic of +15 nt extended pre-let-7a-1 is shown, with extended sequence underlined. ( F ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (E) were derived from three independent experiments.

    Techniques Used: In Vitro, Incubation, Purification, Mutagenesis, Northern Blot, Standard Deviation, Derivative Assay, Sequencing

    2) Product Images from "Dicer cleaves 5′-extended microRNA precursors originating from RNA polymerase II transcription start sites"

    Article Title: Dicer cleaves 5′-extended microRNA precursors originating from RNA polymerase II transcription start sites

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky306

    Processing of 5′-extended pre-miRNAs is independent of Dicer 5′ pocket. ( A ) In vitro transcribed 5′-extended pre-miR-HSUR4s were incubated with four different human Dicers purified by Flag IP. [WT: wild type Dicer; TN: transdominant negative mutation Dicer; 3′ mut: 3′ pocket mutation Dicer (Y926A); 5′ mut: 5′ pocket mutation Dicer (R778A/R780A/H982A)]. Northern blots were performed using probes targeting miR-HSUR4-3p. ( B ) Quantitations of relative mature miRNA levels (mature miRNA/pre-miRNA) compared to WT Dicer processing in (A) (mean ± standard deviation) were derived from three independent experiments. FC: fold change. ( C ) In vitro transcribed 5′-monophosphate pre-miR-HSUR4s without 5′ extension (–2 nt) but with 1 nt or 2 nt 3′ overhang were incubated with four different human Dicers purified by Flag IP. Schematics of pre-miR-HSUR4s with 1 nt or 2 nt 3′ overhang are given. ( D ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (C) were derived from three independent experiments. ( E ) In vitro transcribed 5′-monophosphate pre-let-7a-1 and m 7 G-capped +15 nt extended pre-let-7a-1 were incubated with four different human Dicers purified by Flag IP. Northern blots were performed using probes targeting let-7a-1 (5p). Schematic of +15 nt extended pre-let-7a-1 is shown, with extended sequence underlined. ( F ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (E) were derived from three independent experiments.
    Figure Legend Snippet: Processing of 5′-extended pre-miRNAs is independent of Dicer 5′ pocket. ( A ) In vitro transcribed 5′-extended pre-miR-HSUR4s were incubated with four different human Dicers purified by Flag IP. [WT: wild type Dicer; TN: transdominant negative mutation Dicer; 3′ mut: 3′ pocket mutation Dicer (Y926A); 5′ mut: 5′ pocket mutation Dicer (R778A/R780A/H982A)]. Northern blots were performed using probes targeting miR-HSUR4-3p. ( B ) Quantitations of relative mature miRNA levels (mature miRNA/pre-miRNA) compared to WT Dicer processing in (A) (mean ± standard deviation) were derived from three independent experiments. FC: fold change. ( C ) In vitro transcribed 5′-monophosphate pre-miR-HSUR4s without 5′ extension (–2 nt) but with 1 nt or 2 nt 3′ overhang were incubated with four different human Dicers purified by Flag IP. Schematics of pre-miR-HSUR4s with 1 nt or 2 nt 3′ overhang are given. ( D ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (C) were derived from three independent experiments. ( E ) In vitro transcribed 5′-monophosphate pre-let-7a-1 and m 7 G-capped +15 nt extended pre-let-7a-1 were incubated with four different human Dicers purified by Flag IP. Northern blots were performed using probes targeting let-7a-1 (5p). Schematic of +15 nt extended pre-let-7a-1 is shown, with extended sequence underlined. ( F ) Quantitations of relative mature miRNA levels compared to WT Dicer processing in (E) were derived from three independent experiments.

    Techniques Used: In Vitro, Incubation, Purification, Mutagenesis, Northern Blot, Standard Deviation, Derivative Assay, Sequencing

    Related Articles

    Amplification:

    Article Title: The Hepatitis C Virus RNA 3?-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site ▿
    Article Snippet: .. The amplified DNA fragments were gel purified, extracted with phenol-chloroform, ethanol precipitated, dissolved in water, and used for transcription using T7 RNA polymerase (New England Biolabs). pRL-FMDV-wt-FL was linearized with XbaI downstream of the RLuc sequence and transcribed with T7 RNA polymerase to obtain RLuc control RNA for cotransfections. .. Reactions were treated with RNase-free DNase I to digest DNA templates, and RNA was purified with RNeasy kits (QIAGEN).

    In Vitro:

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy
    Article Snippet: .. The in vitro transcription reaction was carried out with 10 μg of linearized plasmid DNA in a total volume of 100 μL containing 20 μL 5× RRL buffer (400 mM HEPES (pH 7.5), 60 mM MgCl2 , 10 mM spermidine and 200 mM DTT), NTP-Mix (3.125 mM ATP, CTP and UTP and 1.56 mM GTP), 1 U/μL RNasin (Promega, Madison, WI, USA), 2 U/μL T7 RNA polymerase (New England Biolabs) and 1 mM anti-reverse cap analogue (ARCA; 3′-O-Me- m7G(5′)ppp(5′)G; New England Biolabs). .. After incubation at 37 °C for 2.5 h, 1 U/μL T7 RNA polymerase was added followed by additional 2.5 h incubation at 37 °C.

    Article Title: Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach
    Article Snippet: .. Anti-sgRNA and sgRNA where transcribed in vitro by T7 RNA Polymerase (NEB) from linear DNA (IDT) overnight and then extracted with Phenol-Chloroform. .. The concentration of the RNA was determined by comparing a SYBR Green II stained band in a denaturing PAGE (8M Urea at 45°C) to the RNA Ladder (NEB, N0364S).

    Synthesized:

    Article Title: Protein Expression Redirects Vesicular Stomatitis Virus RNA Synthesis to Cytoplasmic Inclusions
    Article Snippet: .. As a control, transcripts were also synthesized by T7 RNA polymerase (New England Biolabs, Beverly MA) using the previously described VSV expression plasmid pN . .. Incorporation of BrUTP into RNA in cells Approximately 30,000 BSR-T7 cells grown on cover slips in 24 well plates were infected with VSV at the specified MOI (3–500).

    Purification:

    Article Title: Native purification and labeling of RNA for single molecule fluorescence studies
    Article Snippet: .. 3 Plasmids encoding for the T7 RNA polymerase gene for protein overexpression and purification are available ( ) or the enzyme is available for purchase through New England Biolabs. ..

    Article Title: The Hepatitis C Virus RNA 3?-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site ▿
    Article Snippet: .. The amplified DNA fragments were gel purified, extracted with phenol-chloroform, ethanol precipitated, dissolved in water, and used for transcription using T7 RNA polymerase (New England Biolabs). pRL-FMDV-wt-FL was linearized with XbaI downstream of the RLuc sequence and transcribed with T7 RNA polymerase to obtain RLuc control RNA for cotransfections. .. Reactions were treated with RNase-free DNase I to digest DNA templates, and RNA was purified with RNeasy kits (QIAGEN).

    Incubation:

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy
    Article Snippet: .. After incubation at 37 °C for 2.5 h, 1 U/μL T7 RNA polymerase was added followed by additional 2.5 h incubation at 37 °C. .. DNA was digested with DNaseI for one hour and RNA was purified by acidic phenol-chloroform extraction and isopropanol precipitation.

    Expressing:

    Article Title: Protein Expression Redirects Vesicular Stomatitis Virus RNA Synthesis to Cytoplasmic Inclusions
    Article Snippet: .. As a control, transcripts were also synthesized by T7 RNA polymerase (New England Biolabs, Beverly MA) using the previously described VSV expression plasmid pN . .. Incorporation of BrUTP into RNA in cells Approximately 30,000 BSR-T7 cells grown on cover slips in 24 well plates were infected with VSV at the specified MOI (3–500).

    Sequencing:

    Article Title: The Hepatitis C Virus RNA 3?-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site ▿
    Article Snippet: .. The amplified DNA fragments were gel purified, extracted with phenol-chloroform, ethanol precipitated, dissolved in water, and used for transcription using T7 RNA polymerase (New England Biolabs). pRL-FMDV-wt-FL was linearized with XbaI downstream of the RLuc sequence and transcribed with T7 RNA polymerase to obtain RLuc control RNA for cotransfections. .. Reactions were treated with RNase-free DNase I to digest DNA templates, and RNA was purified with RNeasy kits (QIAGEN).

    Recombinant:

    Article Title: RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication
    Article Snippet: .. The T7 RNA transcription was performed overnight at 37 °C in a reaction mixture containing 3.3 μM dsDNA template, 7.5 mM of each rNTP, 34 mM MgCl2 , 1 U/μL Recombinant RNasin Ribonuclease Inhibitor (Promega), 10 mM DTT, 0.1 U/μL inorganic pyrophosphatase, and 10 U/μL T7 RNA polymerase (New England Biolabs) in 1× RNAPol Reaction Buffer (40 mM Tris⋅HCl, pH 7.9, 6 mM MgCl2 , 2 mM spermidine, 10 mM DTT). ..

    Over Expression:

    Article Title: Native purification and labeling of RNA for single molecule fluorescence studies
    Article Snippet: .. 3 Plasmids encoding for the T7 RNA polymerase gene for protein overexpression and purification are available ( ) or the enzyme is available for purchase through New England Biolabs. ..

    Plasmid Preparation:

    Article Title: Protein Expression Redirects Vesicular Stomatitis Virus RNA Synthesis to Cytoplasmic Inclusions
    Article Snippet: .. As a control, transcripts were also synthesized by T7 RNA polymerase (New England Biolabs, Beverly MA) using the previously described VSV expression plasmid pN . .. Incorporation of BrUTP into RNA in cells Approximately 30,000 BSR-T7 cells grown on cover slips in 24 well plates were infected with VSV at the specified MOI (3–500).

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy
    Article Snippet: .. The in vitro transcription reaction was carried out with 10 μg of linearized plasmid DNA in a total volume of 100 μL containing 20 μL 5× RRL buffer (400 mM HEPES (pH 7.5), 60 mM MgCl2 , 10 mM spermidine and 200 mM DTT), NTP-Mix (3.125 mM ATP, CTP and UTP and 1.56 mM GTP), 1 U/μL RNasin (Promega, Madison, WI, USA), 2 U/μL T7 RNA polymerase (New England Biolabs) and 1 mM anti-reverse cap analogue (ARCA; 3′-O-Me- m7G(5′)ppp(5′)G; New England Biolabs). .. After incubation at 37 °C for 2.5 h, 1 U/μL T7 RNA polymerase was added followed by additional 2.5 h incubation at 37 °C.

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