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    New England Biolabs t4 polynucleotide kinase pnk buffer buffer new england biolabs
    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 Pnk Buffer Buffer New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    New England Biolabs pnk buffer
    ( A ) Effect of enzymes on 5′ monophosporylated (5′Pi) or capped RNA (CAP). <t>T4</t> RNAP synthesized RNA (264 nt) was kinased using T4 <t>PNK</t> and [α- 32 P]ATP or capped with the Vaccinia Capping System (M2080) and [α- 32 P]GTP, as described by the manufacturer. ( B ) Comparison of RppH activity on 32 P-capped RNA in buffer NEB II vs. NEB T4 RNA ligase buffer. ( C ) 32 P-capped RNA (10 pmol) (264 nt) incubated with 0.5 U of RppH at 37 °C and 20 °C. ( D ) [ 32 P]5′-adenylated oligo (20 pmol) (55 nt) incubated with 2 U of RppH at 20 °C and 37 °C in T4 RNA ligase buffer. ( E ) Assessment of run-on length: nuclei were run on using the described run-on conditions (20 nM CTP-limiting) for the indicated time in the presence and absence of 4 ng/µL α-amanitin, a concentration efficiently inhibiting RNAP II transcription. For visualization of actual run-on length, nuclei were incubated in Freezing Buffer + RNase A (0.25 mg/mL) for 20 min at 4 °C followed by 5 min at RT and consecutively washed three times before run-on.
    Pnk Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 645 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 polynucleotide kinase
    ( A ) Effect of enzymes on 5′ monophosporylated (5′Pi) or capped RNA (CAP). <t>T4</t> RNAP synthesized RNA (264 nt) was kinased using T4 <t>PNK</t> and [α- 32 P]ATP or capped with the Vaccinia Capping System (M2080) and [α- 32 P]GTP, as described by the manufacturer. ( B ) Comparison of RppH activity on 32 P-capped RNA in buffer NEB II vs. NEB T4 RNA ligase buffer. ( C ) 32 P-capped RNA (10 pmol) (264 nt) incubated with 0.5 U of RppH at 37 °C and 20 °C. ( D ) [ 32 P]5′-adenylated oligo (20 pmol) (55 nt) incubated with 2 U of RppH at 20 °C and 37 °C in T4 RNA ligase buffer. ( E ) Assessment of run-on length: nuclei were run on using the described run-on conditions (20 nM CTP-limiting) for the indicated time in the presence and absence of 4 ng/µL α-amanitin, a concentration efficiently inhibiting RNAP II transcription. For visualization of actual run-on length, nuclei were incubated in Freezing Buffer + RNase A (0.25 mg/mL) for 20 min at 4 °C followed by 5 min at RT and consecutively washed three times before run-on.
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 29387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    New England Biolabs 10x pnk buffer
    ( A ) Effect of enzymes on 5′ monophosporylated (5′Pi) or capped RNA (CAP). <t>T4</t> RNAP synthesized RNA (264 nt) was kinased using T4 <t>PNK</t> and [α- 32 P]ATP or capped with the Vaccinia Capping System (M2080) and [α- 32 P]GTP, as described by the manufacturer. ( B ) Comparison of RppH activity on 32 P-capped RNA in buffer NEB II vs. NEB T4 RNA ligase buffer. ( C ) 32 P-capped RNA (10 pmol) (264 nt) incubated with 0.5 U of RppH at 37 °C and 20 °C. ( D ) [ 32 P]5′-adenylated oligo (20 pmol) (55 nt) incubated with 2 U of RppH at 20 °C and 37 °C in T4 RNA ligase buffer. ( E ) Assessment of run-on length: nuclei were run on using the described run-on conditions (20 nM CTP-limiting) for the indicated time in the presence and absence of 4 ng/µL α-amanitin, a concentration efficiently inhibiting RNAP II transcription. For visualization of actual run-on length, nuclei were incubated in Freezing Buffer + RNase A (0.25 mg/mL) for 20 min at 4 °C followed by 5 min at RT and consecutively washed three times before run-on.
    10x Pnk Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    New England Biolabs pnk buffer 1x
    ( A ) Effect of enzymes on 5′ monophosporylated (5′Pi) or capped RNA (CAP). <t>T4</t> RNAP synthesized RNA (264 nt) was kinased using T4 <t>PNK</t> and [α- 32 P]ATP or capped with the Vaccinia Capping System (M2080) and [α- 32 P]GTP, as described by the manufacturer. ( B ) Comparison of RppH activity on 32 P-capped RNA in buffer NEB II vs. NEB T4 RNA ligase buffer. ( C ) 32 P-capped RNA (10 pmol) (264 nt) incubated with 0.5 U of RppH at 37 °C and 20 °C. ( D ) [ 32 P]5′-adenylated oligo (20 pmol) (55 nt) incubated with 2 U of RppH at 20 °C and 37 °C in T4 RNA ligase buffer. ( E ) Assessment of run-on length: nuclei were run on using the described run-on conditions (20 nM CTP-limiting) for the indicated time in the presence and absence of 4 ng/µL α-amanitin, a concentration efficiently inhibiting RNAP II transcription. For visualization of actual run-on length, nuclei were incubated in Freezing Buffer + RNase A (0.25 mg/mL) for 20 min at 4 °C followed by 5 min at RT and consecutively washed three times before run-on.
    Pnk Buffer 1x, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    New England Biolabs 1x t4 pnk buffer
    ( A ) Effect of enzymes on 5′ monophosporylated (5′Pi) or capped RNA (CAP). <t>T4</t> RNAP synthesized RNA (264 nt) was kinased using T4 <t>PNK</t> and [α- 32 P]ATP or capped with the Vaccinia Capping System (M2080) and [α- 32 P]GTP, as described by the manufacturer. ( B ) Comparison of RppH activity on 32 P-capped RNA in buffer NEB II vs. NEB T4 RNA ligase buffer. ( C ) 32 P-capped RNA (10 pmol) (264 nt) incubated with 0.5 U of RppH at 37 °C and 20 °C. ( D ) [ 32 P]5′-adenylated oligo (20 pmol) (55 nt) incubated with 2 U of RppH at 20 °C and 37 °C in T4 RNA ligase buffer. ( E ) Assessment of run-on length: nuclei were run on using the described run-on conditions (20 nM CTP-limiting) for the indicated time in the presence and absence of 4 ng/µL α-amanitin, a concentration efficiently inhibiting RNAP II transcription. For visualization of actual run-on length, nuclei were incubated in Freezing Buffer + RNase A (0.25 mg/mL) for 20 min at 4 °C followed by 5 min at RT and consecutively washed three times before run-on.
    1x T4 Pnk Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    ( A ) Effect of enzymes on 5′ monophosporylated (5′Pi) or capped RNA (CAP). T4 RNAP synthesized RNA (264 nt) was kinased using T4 PNK and [α- 32 P]ATP or capped with the Vaccinia Capping System (M2080) and [α- 32 P]GTP, as described by the manufacturer. ( B ) Comparison of RppH activity on 32 P-capped RNA in buffer NEB II vs. NEB T4 RNA ligase buffer. ( C ) 32 P-capped RNA (10 pmol) (264 nt) incubated with 0.5 U of RppH at 37 °C and 20 °C. ( D ) [ 32 P]5′-adenylated oligo (20 pmol) (55 nt) incubated with 2 U of RppH at 20 °C and 37 °C in T4 RNA ligase buffer. ( E ) Assessment of run-on length: nuclei were run on using the described run-on conditions (20 nM CTP-limiting) for the indicated time in the presence and absence of 4 ng/µL α-amanitin, a concentration efficiently inhibiting RNAP II transcription. For visualization of actual run-on length, nuclei were incubated in Freezing Buffer + RNase A (0.25 mg/mL) for 20 min at 4 °C followed by 5 min at RT and consecutively washed three times before run-on.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Nascent RNA sequencing reveals distinct features in plant transcription

    doi: 10.1073/pnas.1603217113

    Figure Lengend Snippet: ( A ) Effect of enzymes on 5′ monophosporylated (5′Pi) or capped RNA (CAP). T4 RNAP synthesized RNA (264 nt) was kinased using T4 PNK and [α- 32 P]ATP or capped with the Vaccinia Capping System (M2080) and [α- 32 P]GTP, as described by the manufacturer. ( B ) Comparison of RppH activity on 32 P-capped RNA in buffer NEB II vs. NEB T4 RNA ligase buffer. ( C ) 32 P-capped RNA (10 pmol) (264 nt) incubated with 0.5 U of RppH at 37 °C and 20 °C. ( D ) [ 32 P]5′-adenylated oligo (20 pmol) (55 nt) incubated with 2 U of RppH at 20 °C and 37 °C in T4 RNA ligase buffer. ( E ) Assessment of run-on length: nuclei were run on using the described run-on conditions (20 nM CTP-limiting) for the indicated time in the presence and absence of 4 ng/µL α-amanitin, a concentration efficiently inhibiting RNAP II transcription. For visualization of actual run-on length, nuclei were incubated in Freezing Buffer + RNase A (0.25 mg/mL) for 20 min at 4 °C followed by 5 min at RT and consecutively washed three times before run-on.

    Article Snippet: Add kinasing master mix containing 10 µL of PNK buffer (NEB), 1 µL of T4 Polynucleotide Kinase, 5 µL of 10 mM ATP, and dH2O+T for a 100-µL final volume and incubate another 1 h at 37 °C.

    Techniques: Synthesized, Activity Assay, Incubation, Concentration Assay