10×t4 pnk buffer  (New England Biolabs)


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    Structured Review

    New England Biolabs 10×t4 pnk buffer
    10×T4 Pnk Buffer, supplied by New England Biolabs, 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/10×t4 pnk buffer/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    10×t4 pnk buffer - by Bioz Stars, 2020-04
    93/100 stars

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    Synthesized:

    Article Title: m6A Facilitates eIF4F-Independent mRNA Translation
    Article Snippet: The capped or non-capped mRNA (Hsp70-5'UTR) was synthesized using the mMessage mMachine T7 Ultra kit (Ambion). .. The probe was labeled in a 50ul reaction mixture containing 2 µl RNA probe (1 µM), 5 µl 10×T4 PNK buffer (NEB), 1 µl T4 PNK (NEB), 40 U ml−1 RNaseOUT (Thermo Scientific), 1 µl [32 P]ATP and 40 µl RNase-free water at 37°C for 1 h. The labeled probe was then purified by RNase-free micro bio-spin columns with bio-gel P30 (Bio-Rad 732-6250) according to manufacturer's protocols.

    Labeling:

    Article Title: m6A Facilitates eIF4F-Independent mRNA Translation
    Article Snippet: .. The probe was labeled in a 50ul reaction mixture containing 2 µl RNA probe (1 µM), 5 µl 10×T4 PNK buffer (NEB), 1 µl T4 PNK (NEB), 40 U ml−1 RNaseOUT (Thermo Scientific), 1 µl [32 P]ATP and 40 µl RNase-free water at 37°C for 1 h. The labeled probe was then purified by RNase-free micro bio-spin columns with bio-gel P30 (Bio-Rad 732-6250) according to manufacturer's protocols. .. After adding 2.5 µl 20×SSC (Promega) buffer, the RNA was denatured at 65 °C for 10 min and slowly cooled down.

    Purification:

    Article Title: m6A Facilitates eIF4F-Independent mRNA Translation
    Article Snippet: .. The probe was labeled in a 50ul reaction mixture containing 2 µl RNA probe (1 µM), 5 µl 10×T4 PNK buffer (NEB), 1 µl T4 PNK (NEB), 40 U ml−1 RNaseOUT (Thermo Scientific), 1 µl [32 P]ATP and 40 µl RNase-free water at 37°C for 1 h. The labeled probe was then purified by RNase-free micro bio-spin columns with bio-gel P30 (Bio-Rad 732-6250) according to manufacturer's protocols. .. After adding 2.5 µl 20×SSC (Promega) buffer, the RNA was denatured at 65 °C for 10 min and slowly cooled down.

    Incubation:

    Article Title: m6A Facilitates eIF4F-Independent mRNA Translation
    Article Snippet: The probe was labeled in a 50ul reaction mixture containing 2 µl RNA probe (1 µM), 5 µl 10×T4 PNK buffer (NEB), 1 µl T4 PNK (NEB), 40 U ml−1 RNaseOUT (Thermo Scientific), 1 µl [32 P]ATP and 40 µl RNase-free water at 37°C for 1 h. The labeled probe was then purified by RNase-free micro bio-spin columns with bio-gel P30 (Bio-Rad 732-6250) according to manufacturer's protocols. .. The purified probe (20 fmol) was incubated with increasing amount of GST-METTL3 at 4 °C for 1 h in binding buffer containing 10 mM HEPES, pH 8.0, 50 mM KCl, 1 mM EDTA, 0.05% Triton-X-100, 5% glycerol, 10 µg ml−1 salmon DNA, 1 mM DTT and 40 U ml-1 RNaseOUT (Thermo Scientific).

    Electrophoretic Mobility Shift Assay:

    Article Title: m6A Facilitates eIF4F-Independent mRNA Translation
    Article Snippet: Paragraph title: Electrophoretic mobility shift assay ... The probe was labeled in a 50ul reaction mixture containing 2 µl RNA probe (1 µM), 5 µl 10×T4 PNK buffer (NEB), 1 µl T4 PNK (NEB), 40 U ml−1 RNaseOUT (Thermo Scientific), 1 µl [32 P]ATP and 40 µl RNase-free water at 37°C for 1 h. The labeled probe was then purified by RNase-free micro bio-spin columns with bio-gel P30 (Bio-Rad 732-6250) according to manufacturer's protocols.

    Sequencing:

    Article Title: m6A Facilitates eIF4F-Independent mRNA Translation
    Article Snippet: The m6 A-modified or -unmodified RNA probe was synthesized by Thermo Scientific with the sequence of 5'-CGAUCCUCGGCCAGGXCCAGCCUUCCCCA-3' (X=A or m6 A). .. The probe was labeled in a 50ul reaction mixture containing 2 µl RNA probe (1 µM), 5 µl 10×T4 PNK buffer (NEB), 1 µl T4 PNK (NEB), 40 U ml−1 RNaseOUT (Thermo Scientific), 1 µl [32 P]ATP and 40 µl RNase-free water at 37°C for 1 h. The labeled probe was then purified by RNase-free micro bio-spin columns with bio-gel P30 (Bio-Rad 732-6250) according to manufacturer's protocols.

    Binding Assay:

    Article Title: m6A Facilitates eIF4F-Independent mRNA Translation
    Article Snippet: The probe was labeled in a 50ul reaction mixture containing 2 µl RNA probe (1 µM), 5 µl 10×T4 PNK buffer (NEB), 1 µl T4 PNK (NEB), 40 U ml−1 RNaseOUT (Thermo Scientific), 1 µl [32 P]ATP and 40 µl RNase-free water at 37°C for 1 h. The labeled probe was then purified by RNase-free micro bio-spin columns with bio-gel P30 (Bio-Rad 732-6250) according to manufacturer's protocols. .. The purified probe (20 fmol) was incubated with increasing amount of GST-METTL3 at 4 °C for 1 h in binding buffer containing 10 mM HEPES, pH 8.0, 50 mM KCl, 1 mM EDTA, 0.05% Triton-X-100, 5% glycerol, 10 µg ml−1 salmon DNA, 1 mM DTT and 40 U ml-1 RNaseOUT (Thermo Scientific).

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    New England Biolabs t4 pnk buffer
    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and <t>T4</t> PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.
    T4 Pnk Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 pnk buffer/product/New England Biolabs
    Average 90 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
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    99
    New England Biolabs t4 polynucleotide kinase
    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and <t>T4</t> PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 polynucleotide kinase/product/New England Biolabs
    Average 99 stars, based on 148 article reviews
    Price from $9.99 to $1999.99
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    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi

    doi: 10.1016/j.omtn.2017.07.008

    Figure Lengend Snippet: Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.

    Article Snippet: The protocol was modified as follows when CIP or T4 PNK treatment is necessary: (1) for CIP treatment, in 20 μL of products from one in vitro transcription reaction before DNase treatment, we added 1 μL of DNase (supplied with T7 Transcription Kit), 1 μL of CIP, 4 μL of 10× CutSmart buffer (NEB), and water to total volume of 40 μL, and incubated at 37°C for 15 min; and (2) for T4 PNK treatment, in 20 μL of products from one in vitro transcription reaction before DNase treatment, we added 1 μL of DNase (supplied with T7 Transcription Kit), 1 μL of T4 PNK, 4 μL of 10× T4 PNK buffer (NEB), and water to total volume of 40 μL, and incubated at 37°C for 15 min. All T7 in vitro transcription products were purified by Micro Bio-Spin P-30 Gel Columns, Tris Buffer, from Bio-Rad.

    Techniques: Transfection, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing

    Read distribution of ex‑mRNA reads across the full-length mRNA transcripts. ( A and B ) Read coverage for the hemoglobin A2 transcript ( A ) and the albumin transcript ( B ) by sample type for untreated and T4 PNK end-treated samples. Exon boundaries (HBA2: 3 exons, ALB: 15 exons) are indicated by alternating intensities of gray, and UTRs are distinguished from CDS by thinner bars. ( C ) Metagene analysis with relative read coverage (percentage) across 5′ UTRs, CDSs, and 3′ UTRs for untreated and PNK-treated samples as well as corresponding data obtained after 100 random simulations (across an average of 2342–3500 captured transcripts for untreated samples and an average of 12,789–16,487 captured transcripts for PNK-treated samples, depending on sample type). Shown are results from n = 6 individual samples per condition.

    Journal: JCI Insight

    Article Title: Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis

    doi: 10.1172/jci.insight.127317

    Figure Lengend Snippet: Read distribution of ex‑mRNA reads across the full-length mRNA transcripts. ( A and B ) Read coverage for the hemoglobin A2 transcript ( A ) and the albumin transcript ( B ) by sample type for untreated and T4 PNK end-treated samples. Exon boundaries (HBA2: 3 exons, ALB: 15 exons) are indicated by alternating intensities of gray, and UTRs are distinguished from CDS by thinner bars. ( C ) Metagene analysis with relative read coverage (percentage) across 5′ UTRs, CDSs, and 3′ UTRs for untreated and PNK-treated samples as well as corresponding data obtained after 100 random simulations (across an average of 2342–3500 captured transcripts for untreated samples and an average of 12,789–16,487 captured transcripts for PNK-treated samples, depending on sample type). Shown are results from n = 6 individual samples per condition.

    Article Snippet: To half of the eluted exRNA, i.e., 14 μl, we added 6 μl of a master mix corresponding to the equivalent of 2 μl ×10 T4 PNK buffer, 2 μl 10 mM ATP, 1 μl RNase-free water, and 1 μl T4 PNK (NEB, catalog M0201S) for a final reaction volume of 20 μl in a 1.5 ml siliconized microcentrifuge tube.

    Techniques:

    Treatment of total extracellular RNA with T4 polynucleotide kinase followed by small-RNA-sequencing. ( A ) Total RNA was isolated from 450 μl serum or platelet-depleted EDTA, acid citrate dextrose (ACD), and heparin plasma from 6 healthy individuals and purified using silica-based spin columns. Half of the RNA was treated with T4 polynucleotide kinase (T4 PNK) and repurified (PNK treated), and multiplexed small-RNA-sequencing (sRNA-seq) libraries were prepared separately for the untreated (libraries 1 and 3) and PNK-treated RNA (libraries 2 and 4). ( B ) Differences in read annotation in the 4 sample types for untreated RNA and PNK-treated RNA using initial annotation settings (reads 12–42 nt, up to 2 mismatches, multimapping). ( C ) Differences in ex‑mRNA capture between untreated and PNK-treated RNA using final annotation criteria (reads  > 15 nt, no mismatch and up to 2 mapping locations). Box plots show the median and first and third quartiles (bottom and top hinges). Whiskers extend at most ×1.5 interquartile range from the hinges; any data outside this are shown as individual outlier points. Shown are results from  n  = 6 individual samples per condition.

    Journal: JCI Insight

    Article Title: Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis

    doi: 10.1172/jci.insight.127317

    Figure Lengend Snippet: Treatment of total extracellular RNA with T4 polynucleotide kinase followed by small-RNA-sequencing. ( A ) Total RNA was isolated from 450 μl serum or platelet-depleted EDTA, acid citrate dextrose (ACD), and heparin plasma from 6 healthy individuals and purified using silica-based spin columns. Half of the RNA was treated with T4 polynucleotide kinase (T4 PNK) and repurified (PNK treated), and multiplexed small-RNA-sequencing (sRNA-seq) libraries were prepared separately for the untreated (libraries 1 and 3) and PNK-treated RNA (libraries 2 and 4). ( B ) Differences in read annotation in the 4 sample types for untreated RNA and PNK-treated RNA using initial annotation settings (reads 12–42 nt, up to 2 mismatches, multimapping). ( C ) Differences in ex‑mRNA capture between untreated and PNK-treated RNA using final annotation criteria (reads > 15 nt, no mismatch and up to 2 mapping locations). Box plots show the median and first and third quartiles (bottom and top hinges). Whiskers extend at most ×1.5 interquartile range from the hinges; any data outside this are shown as individual outlier points. Shown are results from n = 6 individual samples per condition.

    Article Snippet: To half of the eluted exRNA, i.e., 14 μl, we added 6 μl of a master mix corresponding to the equivalent of 2 μl ×10 T4 PNK buffer, 2 μl 10 mM ATP, 1 μl RNase-free water, and 1 μl T4 PNK (NEB, catalog M0201S) for a final reaction volume of 20 μl in a 1.5 ml siliconized microcentrifuge tube.

    Techniques: RNA Sequencing Assay, Isolation, Purification