t4 pnk buffer  (New England Biolabs)


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    Name:
    T4 Polynucleotide Kinase Reaction Buffer
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    T4 Polynucleotide Kinase Reaction Buffer 4 0 ml
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    B0201S
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    New England Biolabs t4 pnk buffer
    T4 Polynucleotide Kinase Reaction Buffer
    T4 Polynucleotide Kinase Reaction Buffer 4 0 ml
    https://www.bioz.com/result/t4 pnk buffer/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
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    Images

    1) Product Images from "No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5’-OH ends phosphorylated by Trl1"

    Article Title: No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5’-OH ends phosphorylated by Trl1

    Journal: bioRxiv

    doi: 10.1101/465633

    Endonucleolytically cleaved 5’-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3’-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in ( a ). c 12% PAGE followed by northern blot analysis using probe prA. Treatment using T4 PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro . A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d) calculated from three independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: Endonucleolytically cleaved 5’-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3’-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in ( a ). c 12% PAGE followed by northern blot analysis using probe prA. Treatment using T4 PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro . A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d) calculated from three independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Northern Blot, RNA Detection, Mutagenesis, In Vitro, Standard Deviation

    2) Product Images from "The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit, Vps25"

    Article Title: The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit, Vps25

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003718

    Analysis of ESCRT-II/RNA binding in vitro . A , Coomassie Blue-stained gel of the recombinant Xenopus ( Xen ) and human ( Hu ) ESCRT-II complexes used in the in vitro RNA-binding assays. ΔMBD lacks the membrane-binding domains of human ESCRT-II. B–D , autoradiographs of UV–cross-linked in vitro binding reactions with: B , Xenopus ESCRT-II and 5′-end-labeled total egg RNA; C , Xenopus ESCRT-II and individual 5′-end-labeled in vitro transcribed RNAs that are under-represented in ESCRT-II immunoprecipitations; and D , full-length HuESCRT-II ( FL ) or HuESCRT-IIΔMBD (ΔMBD) and a body-labeled, in vitro transcribed GA-rich CLIP tag (a region of the ctr9 mRNA). B and C , a covalent intermediate of PNK and [γ- 32 P]ATP (used to radiolabel the RNA fragments) is indicated. D , Folch fraction liposomes were included in the binding reactions at the indicated concentrations. A fluorescent Western blotting ( WB ) of the same nitrocellulose membrane shown in the autoradiograph is shown as a loading control. The asterisk represents a nonspecific band. A–D , the expected migrations of the ESCRT-II subunits are indicated. E , quantification of the autoradiograph shown in D and two additional, independent replicates depicting the fraction of RNA bound by each ESCRT-II subunit at the indicated concentrations of Folch fraction liposomes relative to binding with no liposomes present. Error bars are S.E.
    Figure Legend Snippet: Analysis of ESCRT-II/RNA binding in vitro . A , Coomassie Blue-stained gel of the recombinant Xenopus ( Xen ) and human ( Hu ) ESCRT-II complexes used in the in vitro RNA-binding assays. ΔMBD lacks the membrane-binding domains of human ESCRT-II. B–D , autoradiographs of UV–cross-linked in vitro binding reactions with: B , Xenopus ESCRT-II and 5′-end-labeled total egg RNA; C , Xenopus ESCRT-II and individual 5′-end-labeled in vitro transcribed RNAs that are under-represented in ESCRT-II immunoprecipitations; and D , full-length HuESCRT-II ( FL ) or HuESCRT-IIΔMBD (ΔMBD) and a body-labeled, in vitro transcribed GA-rich CLIP tag (a region of the ctr9 mRNA). B and C , a covalent intermediate of PNK and [γ- 32 P]ATP (used to radiolabel the RNA fragments) is indicated. D , Folch fraction liposomes were included in the binding reactions at the indicated concentrations. A fluorescent Western blotting ( WB ) of the same nitrocellulose membrane shown in the autoradiograph is shown as a loading control. The asterisk represents a nonspecific band. A–D , the expected migrations of the ESCRT-II subunits are indicated. E , quantification of the autoradiograph shown in D and two additional, independent replicates depicting the fraction of RNA bound by each ESCRT-II subunit at the indicated concentrations of Folch fraction liposomes relative to binding with no liposomes present. Error bars are S.E.

    Techniques Used: RNA Binding Assay, In Vitro, Staining, Recombinant, Binding Assay, Labeling, Cross-linking Immunoprecipitation, Western Blot, Autoradiography

    3) Product Images from "CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini"

    Article Title: CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini

    Journal: Cell Discovery

    doi: 10.1038/s41421-021-00265-2

    CPA-seq. a The workflow of sRNA library preparation for CPA-seq. Purified small RNAs are incubated in deacylation buffer to remove 3′-aminoacyl (3′-aa), treated with Cap-Clip to remove 5′ m 7 G and m 3 G caps, then treated with T4 PNK to convert 5′-OH to 5′-P, and to convert 3′-P and 3′-cP to 3′-OH, followed by treatment with a mix of AlkB and AlkB(D135S) to remove methylations in m 1 G, m 3 C, and m 1 A. The pretreated small RNAs were ligated with 3′ and 5′ adapters, reverse transcribed by TGIRT-III, and then PCR amplified for sequencing. b Northern blotting of RNA samples from HEK293T with/without treatment of deacylation buffer. c Cap-Clip treated synthetic 5′-m 7 G-RNA (31 nt) was ligated with a 5′-adapter (26 nt). d T4 PNK-treated synthetic 5′-OH RNA (27 nt) was ligated with a 5′-adapter (26 nt). e T4 PNK-treated synthetic 3′-P RNA (27 nt) was ligated with 3′-adapter (29 nt). f LC-MS/MS analysis showed that sequential treatments with deacylation buffer, Cap-Clip, T4 PNK, and AlkB mix (CPA) efficiently removed methylations in m 1 G, m 3 C, and m 1 A of small RNAs extracted from HEK293T cells ( n = 3).
    Figure Legend Snippet: CPA-seq. a The workflow of sRNA library preparation for CPA-seq. Purified small RNAs are incubated in deacylation buffer to remove 3′-aminoacyl (3′-aa), treated with Cap-Clip to remove 5′ m 7 G and m 3 G caps, then treated with T4 PNK to convert 5′-OH to 5′-P, and to convert 3′-P and 3′-cP to 3′-OH, followed by treatment with a mix of AlkB and AlkB(D135S) to remove methylations in m 1 G, m 3 C, and m 1 A. The pretreated small RNAs were ligated with 3′ and 5′ adapters, reverse transcribed by TGIRT-III, and then PCR amplified for sequencing. b Northern blotting of RNA samples from HEK293T with/without treatment of deacylation buffer. c Cap-Clip treated synthetic 5′-m 7 G-RNA (31 nt) was ligated with a 5′-adapter (26 nt). d T4 PNK-treated synthetic 5′-OH RNA (27 nt) was ligated with a 5′-adapter (26 nt). e T4 PNK-treated synthetic 3′-P RNA (27 nt) was ligated with 3′-adapter (29 nt). f LC-MS/MS analysis showed that sequential treatments with deacylation buffer, Cap-Clip, T4 PNK, and AlkB mix (CPA) efficiently removed methylations in m 1 G, m 3 C, and m 1 A of small RNAs extracted from HEK293T cells ( n = 3).

    Techniques Used: Purification, Incubation, Cross-linking Immunoprecipitation, Polymerase Chain Reaction, Amplification, Sequencing, Northern Blot, Liquid Chromatography with Mass Spectroscopy

    CPA-seq reveals sRNAs with diverse termini. a Distribution of different types of sRNAs extracted from HEK293T cells that we process with the full CPA-seq process or with various combinations of the Cap-Clip, T4 PNK, and AlkB mix enzymes ( n = 2). b The number of sRNA species revealed with different treatments ( n = 2). c Distribution of different types of sRNAs responsive to T4 PNK treatment (unique reads that were highly detected in CPA group, but lowly detected in CA group with the fold change > 30, n = 2). d Reads of sRNA responsive to T4 PNK treatment mapping to 5S, 18S, and 28S ribosomal RNAs have been combined to show detection of rsRNAs containing diverse termini (rsRNAs with RPM > 300 are shown in the structural map). e Reads of sRNA responsive to T4 PNK treatment mapping to cytosolic tsRNAs have been combined to show detection of tsRNAs containing diverse termini. f Reads of tsRNAs responsive to T4 PNK treatment. g Northern blotting of GluCTC 5′tsRNAs that are responsive to T4 PNK treatment.
    Figure Legend Snippet: CPA-seq reveals sRNAs with diverse termini. a Distribution of different types of sRNAs extracted from HEK293T cells that we process with the full CPA-seq process or with various combinations of the Cap-Clip, T4 PNK, and AlkB mix enzymes ( n = 2). b The number of sRNA species revealed with different treatments ( n = 2). c Distribution of different types of sRNAs responsive to T4 PNK treatment (unique reads that were highly detected in CPA group, but lowly detected in CA group with the fold change > 30, n = 2). d Reads of sRNA responsive to T4 PNK treatment mapping to 5S, 18S, and 28S ribosomal RNAs have been combined to show detection of rsRNAs containing diverse termini (rsRNAs with RPM > 300 are shown in the structural map). e Reads of sRNA responsive to T4 PNK treatment mapping to cytosolic tsRNAs have been combined to show detection of tsRNAs containing diverse termini. f Reads of tsRNAs responsive to T4 PNK treatment. g Northern blotting of GluCTC 5′tsRNAs that are responsive to T4 PNK treatment.

    Techniques Used: Cross-linking Immunoprecipitation, Northern Blot

    4) Product Images from "Nascent RNA sequencing reveals distinct features in plant transcription"

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

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

    doi: 10.1073/pnas.1603217113

    ( 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.
    Figure Legend 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.

    Techniques Used: Synthesized, Activity Assay, Incubation, Concentration Assay

    5) Product Images from "A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi"

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

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2017.07.008

    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.
    Figure Legend 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.

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

    6) Product Images from "No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5′-OH ends phosphorylated by Trl1"

    Article Title: No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5′-OH ends phosphorylated by Trl1

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13991-9

    Endonucleolytically cleaved 5′-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3′-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in a . c 12% PAGE followed by northern blot analysis using probe prA. Treatment using T4 PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro. A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d.) calculated from three independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: Endonucleolytically cleaved 5′-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3′-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in a . c 12% PAGE followed by northern blot analysis using probe prA. Treatment using T4 PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro. A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d.) calculated from three independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Northern Blot, RNA Detection, Mutagenesis, In Vitro, Standard Deviation

    Related Articles

    De-Phosphorylation Assay:

    Article Title: The landscape of transcriptional and translational changes over 22 years of bacterial adaptation
    Article Snippet: .. 3’ dephosphorylation was performed by incubating fragments with 10 U/uL T4 Polynucleotide Kinase (New England Biolabs M0201S) in the supplied buffer (NEB B0201S) along with SUPERase-In for 1 hour at 37 C in a reaction volume of 5 uL. .. Linker ligation took place by adding the following reagents to the above reaction to the indicated final concentrations: 17% w/v PEG-8000, 200 U/uL of T4 RNA Ligase 2 (NEB M0351S), 1X T4 RNA Ligase Reaction Buffer (NEB B0216L), and 20 uM pre-adenylated linkers.

    Modification:

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

    In Vitro:

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

    Incubation:

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

    Article Title: CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini
    Article Snippet: The small RNA was purified from the reaction by ethanol precipitation. .. Then the recovered small RNA was incubated with 1 U Cap-Clip Acid Pyrophosphatase (Cellscript) in 1× Cap-Clip Acid Pyrophosphatase reaction buffer (Cellscript) at 37 °C for 30 min. Then, the reaction was added with 20 U T4 PNK (NEB) in 1× T4 PNK reaction buffer (NEB) and 1 mM ATP (NEB) and incubated at 37 °C for 30 min. .. The small RNA was purified from the reaction by phenol-chloroform extraction and ethanol precipitation.

    Purification:

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

    other:

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

    Isolation:

    Article Title: The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit, Vps25
    Article Snippet: The samples were then UV–cross-linked for 5 min on ice in a Stratalinker 2400 (Stratagene) equipped with UVC bulbs and then treated with RNase A (Sigma type XIIA) at 0.1 mg/ml for 30 min at room temperature. .. ESCRT-II was then isolated by immunoprecipitation with anti-ESCRT-II coupled to protein A–Dynabeads, and after washing, the beads were resuspended in PNK buffer and treated with PNK (NEB) and [γ-32 P]ATP (PerkinElmer) for 45 min at 37 °C. .. The beads were washed with PBS, resuspended in protein loading buffer, and analyzed by SDS-PAGE and autoradiography.

    Immunoprecipitation:

    Article Title: The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit, Vps25
    Article Snippet: The samples were then UV–cross-linked for 5 min on ice in a Stratalinker 2400 (Stratagene) equipped with UVC bulbs and then treated with RNase A (Sigma type XIIA) at 0.1 mg/ml for 30 min at room temperature. .. ESCRT-II was then isolated by immunoprecipitation with anti-ESCRT-II coupled to protein A–Dynabeads, and after washing, the beads were resuspended in PNK buffer and treated with PNK (NEB) and [γ-32 P]ATP (PerkinElmer) for 45 min at 37 °C. .. The beads were washed with PBS, resuspended in protein loading buffer, and analyzed by SDS-PAGE and autoradiography.

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    New England Biolabs t4 pnk buffer
    Endonucleolytically cleaved 5’-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3’-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in ( a ). c 12% PAGE followed by northern blot analysis using probe prA. Treatment using <t>T4</t> PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro . A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d) calculated from three independent experiments. Source data are provided as a Source Data file.
    T4 Pnk Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Endonucleolytically cleaved 5’-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3’-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in ( a ). c 12% PAGE followed by northern blot analysis using probe prA. Treatment using T4 PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro . A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d) calculated from three independent experiments. Source data are provided as a Source Data file.

    Journal: bioRxiv

    Article Title: No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5’-OH ends phosphorylated by Trl1

    doi: 10.1101/465633

    Figure Lengend Snippet: Endonucleolytically cleaved 5’-OH RNAs are phosphorylated by Trl1. a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3’-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in ( a ). c 12% PAGE followed by northern blot analysis using probe prA. Treatment using T4 PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a , Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro . A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d) calculated from three independent experiments. Source data are provided as a Source Data file.

    Article Snippet: NEB Buffer 3 was replaced by T4 PNK buffer (NEB) in kinase assays in the presence or absence of Xrn1 (Figs and ).

    Techniques: Polyacrylamide Gel Electrophoresis, Northern Blot, RNA Detection, Mutagenesis, In Vitro, Standard Deviation

    Analysis of ESCRT-II/RNA binding in vitro . A , Coomassie Blue-stained gel of the recombinant Xenopus ( Xen ) and human ( Hu ) ESCRT-II complexes used in the in vitro RNA-binding assays. ΔMBD lacks the membrane-binding domains of human ESCRT-II. B–D , autoradiographs of UV–cross-linked in vitro binding reactions with: B , Xenopus ESCRT-II and 5′-end-labeled total egg RNA; C , Xenopus ESCRT-II and individual 5′-end-labeled in vitro transcribed RNAs that are under-represented in ESCRT-II immunoprecipitations; and D , full-length HuESCRT-II ( FL ) or HuESCRT-IIΔMBD (ΔMBD) and a body-labeled, in vitro transcribed GA-rich CLIP tag (a region of the ctr9 mRNA). B and C , a covalent intermediate of PNK and [γ- 32 P]ATP (used to radiolabel the RNA fragments) is indicated. D , Folch fraction liposomes were included in the binding reactions at the indicated concentrations. A fluorescent Western blotting ( WB ) of the same nitrocellulose membrane shown in the autoradiograph is shown as a loading control. The asterisk represents a nonspecific band. A–D , the expected migrations of the ESCRT-II subunits are indicated. E , quantification of the autoradiograph shown in D and two additional, independent replicates depicting the fraction of RNA bound by each ESCRT-II subunit at the indicated concentrations of Folch fraction liposomes relative to binding with no liposomes present. Error bars are S.E.

    Journal: The Journal of Biological Chemistry

    Article Title: The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit, Vps25

    doi: 10.1074/jbc.RA118.003718

    Figure Lengend Snippet: Analysis of ESCRT-II/RNA binding in vitro . A , Coomassie Blue-stained gel of the recombinant Xenopus ( Xen ) and human ( Hu ) ESCRT-II complexes used in the in vitro RNA-binding assays. ΔMBD lacks the membrane-binding domains of human ESCRT-II. B–D , autoradiographs of UV–cross-linked in vitro binding reactions with: B , Xenopus ESCRT-II and 5′-end-labeled total egg RNA; C , Xenopus ESCRT-II and individual 5′-end-labeled in vitro transcribed RNAs that are under-represented in ESCRT-II immunoprecipitations; and D , full-length HuESCRT-II ( FL ) or HuESCRT-IIΔMBD (ΔMBD) and a body-labeled, in vitro transcribed GA-rich CLIP tag (a region of the ctr9 mRNA). B and C , a covalent intermediate of PNK and [γ- 32 P]ATP (used to radiolabel the RNA fragments) is indicated. D , Folch fraction liposomes were included in the binding reactions at the indicated concentrations. A fluorescent Western blotting ( WB ) of the same nitrocellulose membrane shown in the autoradiograph is shown as a loading control. The asterisk represents a nonspecific band. A–D , the expected migrations of the ESCRT-II subunits are indicated. E , quantification of the autoradiograph shown in D and two additional, independent replicates depicting the fraction of RNA bound by each ESCRT-II subunit at the indicated concentrations of Folch fraction liposomes relative to binding with no liposomes present. Error bars are S.E.

    Article Snippet: ESCRT-II was then isolated by immunoprecipitation with anti-ESCRT-II coupled to protein A–Dynabeads, and after washing, the beads were resuspended in PNK buffer and treated with PNK (NEB) and [γ-32 P]ATP (PerkinElmer) for 45 min at 37 °C.

    Techniques: RNA Binding Assay, In Vitro, Staining, Recombinant, Binding Assay, Labeling, Cross-linking Immunoprecipitation, Western Blot, Autoradiography

    CPA-seq. a The workflow of sRNA library preparation for CPA-seq. Purified small RNAs are incubated in deacylation buffer to remove 3′-aminoacyl (3′-aa), treated with Cap-Clip to remove 5′ m 7 G and m 3 G caps, then treated with T4 PNK to convert 5′-OH to 5′-P, and to convert 3′-P and 3′-cP to 3′-OH, followed by treatment with a mix of AlkB and AlkB(D135S) to remove methylations in m 1 G, m 3 C, and m 1 A. The pretreated small RNAs were ligated with 3′ and 5′ adapters, reverse transcribed by TGIRT-III, and then PCR amplified for sequencing. b Northern blotting of RNA samples from HEK293T with/without treatment of deacylation buffer. c Cap-Clip treated synthetic 5′-m 7 G-RNA (31 nt) was ligated with a 5′-adapter (26 nt). d T4 PNK-treated synthetic 5′-OH RNA (27 nt) was ligated with a 5′-adapter (26 nt). e T4 PNK-treated synthetic 3′-P RNA (27 nt) was ligated with 3′-adapter (29 nt). f LC-MS/MS analysis showed that sequential treatments with deacylation buffer, Cap-Clip, T4 PNK, and AlkB mix (CPA) efficiently removed methylations in m 1 G, m 3 C, and m 1 A of small RNAs extracted from HEK293T cells ( n = 3).

    Journal: Cell Discovery

    Article Title: CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini

    doi: 10.1038/s41421-021-00265-2

    Figure Lengend Snippet: CPA-seq. a The workflow of sRNA library preparation for CPA-seq. Purified small RNAs are incubated in deacylation buffer to remove 3′-aminoacyl (3′-aa), treated with Cap-Clip to remove 5′ m 7 G and m 3 G caps, then treated with T4 PNK to convert 5′-OH to 5′-P, and to convert 3′-P and 3′-cP to 3′-OH, followed by treatment with a mix of AlkB and AlkB(D135S) to remove methylations in m 1 G, m 3 C, and m 1 A. The pretreated small RNAs were ligated with 3′ and 5′ adapters, reverse transcribed by TGIRT-III, and then PCR amplified for sequencing. b Northern blotting of RNA samples from HEK293T with/without treatment of deacylation buffer. c Cap-Clip treated synthetic 5′-m 7 G-RNA (31 nt) was ligated with a 5′-adapter (26 nt). d T4 PNK-treated synthetic 5′-OH RNA (27 nt) was ligated with a 5′-adapter (26 nt). e T4 PNK-treated synthetic 3′-P RNA (27 nt) was ligated with 3′-adapter (29 nt). f LC-MS/MS analysis showed that sequential treatments with deacylation buffer, Cap-Clip, T4 PNK, and AlkB mix (CPA) efficiently removed methylations in m 1 G, m 3 C, and m 1 A of small RNAs extracted from HEK293T cells ( n = 3).

    Article Snippet: Then the recovered small RNA was incubated with 1 U Cap-Clip Acid Pyrophosphatase (Cellscript) in 1× Cap-Clip Acid Pyrophosphatase reaction buffer (Cellscript) at 37 °C for 30 min. Then, the reaction was added with 20 U T4 PNK (NEB) in 1× T4 PNK reaction buffer (NEB) and 1 mM ATP (NEB) and incubated at 37 °C for 30 min.

    Techniques: Purification, Incubation, Cross-linking Immunoprecipitation, Polymerase Chain Reaction, Amplification, Sequencing, Northern Blot, Liquid Chromatography with Mass Spectroscopy

    CPA-seq reveals sRNAs with diverse termini. a Distribution of different types of sRNAs extracted from HEK293T cells that we process with the full CPA-seq process or with various combinations of the Cap-Clip, T4 PNK, and AlkB mix enzymes ( n = 2). b The number of sRNA species revealed with different treatments ( n = 2). c Distribution of different types of sRNAs responsive to T4 PNK treatment (unique reads that were highly detected in CPA group, but lowly detected in CA group with the fold change > 30, n = 2). d Reads of sRNA responsive to T4 PNK treatment mapping to 5S, 18S, and 28S ribosomal RNAs have been combined to show detection of rsRNAs containing diverse termini (rsRNAs with RPM > 300 are shown in the structural map). e Reads of sRNA responsive to T4 PNK treatment mapping to cytosolic tsRNAs have been combined to show detection of tsRNAs containing diverse termini. f Reads of tsRNAs responsive to T4 PNK treatment. g Northern blotting of GluCTC 5′tsRNAs that are responsive to T4 PNK treatment.

    Journal: Cell Discovery

    Article Title: CPA-seq reveals small ncRNAs with methylated nucleosides and diverse termini

    doi: 10.1038/s41421-021-00265-2

    Figure Lengend Snippet: CPA-seq reveals sRNAs with diverse termini. a Distribution of different types of sRNAs extracted from HEK293T cells that we process with the full CPA-seq process or with various combinations of the Cap-Clip, T4 PNK, and AlkB mix enzymes ( n = 2). b The number of sRNA species revealed with different treatments ( n = 2). c Distribution of different types of sRNAs responsive to T4 PNK treatment (unique reads that were highly detected in CPA group, but lowly detected in CA group with the fold change > 30, n = 2). d Reads of sRNA responsive to T4 PNK treatment mapping to 5S, 18S, and 28S ribosomal RNAs have been combined to show detection of rsRNAs containing diverse termini (rsRNAs with RPM > 300 are shown in the structural map). e Reads of sRNA responsive to T4 PNK treatment mapping to cytosolic tsRNAs have been combined to show detection of tsRNAs containing diverse termini. f Reads of tsRNAs responsive to T4 PNK treatment. g Northern blotting of GluCTC 5′tsRNAs that are responsive to T4 PNK treatment.

    Article Snippet: Then the recovered small RNA was incubated with 1 U Cap-Clip Acid Pyrophosphatase (Cellscript) in 1× Cap-Clip Acid Pyrophosphatase reaction buffer (Cellscript) at 37 °C for 30 min. Then, the reaction was added with 20 U T4 PNK (NEB) in 1× T4 PNK reaction buffer (NEB) and 1 mM ATP (NEB) and incubated at 37 °C for 30 min.

    Techniques: Cross-linking Immunoprecipitation, Northern Blot

    ( 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