pnk  (New England Biolabs)


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    Name:
    T4 Polynucleotide Kinase
    Description:
    T4 Polynucleotide Kinase 2 500 units
    Catalog Number:
    M0201L
    Price:
    228
    Category:
    Polynucleotide Kinases
    Size:
    2 500 units
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    Structured Review

    New England Biolabs pnk
    T4 Polynucleotide Kinase
    T4 Polynucleotide Kinase 2 500 units
    https://www.bioz.com/result/pnk/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pnk - by Bioz Stars, 2021-05
    99/100 stars

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    1) Product Images from "Nucleobase modification by an RNA enzyme"

    Article Title: Nucleobase modification by an RNA enzyme

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1199

    Identification of nucleotidyl adduct. ( A ) MALDI-TOF mass spectrometry of input substrate d3.1 (top left), 5΄-thiophosphorylated d3.1 that was generated by treatment with ATPγS and PNK (bottom left), ribozyme-catalyzed product that was formed by treatment of d3.1 with K28min and GTPγS (top right), and acid-decomposition of the ribozyme-catalyzed product (bottom right). The M–H peaks produced by each treatment are indicated: blue triangles, 6784.28 m/z (expected 6782.17 a.m.u for input d3.1 substrate); magenta triangles, 6880.38 m/z (expected 6878.11 a.m.u for mono-thiophosphorylated d3.1); green triangles, 7322.00 m/z (expected 7319.10 a.m.u for adduct shown in panel F top); red triangles, 7225.3 m/z (expected 7223.16 a.m.u for adduct shown in panel F bottom). ( B ) Incorporation of α- 32 P label into unlabeled acceptor strand. Controls in first two lanes show 5΄- 32 P-labeled d3.1 substrate alone (NR) or incubated with ribozyme K28min and GTPγS (sP). For remaining lanes, non-radiolabeled d3.1 substrate was incubated with ribozyme K28min and [α- 32 P]GTP for 5 min before adding the indicated amount of unlabeled GTP. Reactions were performed at 10°C for 18 h. ( C ) Thiophosphorylation reactions containing the indicated combinations of ribozyme, substrate and donor were treated with sodium periodate, followed by amination with Cy3 hydrazide dye. For the sample in Lane 4, the product of the ribozyme-catalyzed reaction was purified from an APM gel prior to the dye labeling reaction. Gel was scanned for Cy3 fluorescence (Ex. 532 nm Em. 570 nm). Phosphorimages are of 20% denaturing PAGE.
    Figure Legend Snippet: Identification of nucleotidyl adduct. ( A ) MALDI-TOF mass spectrometry of input substrate d3.1 (top left), 5΄-thiophosphorylated d3.1 that was generated by treatment with ATPγS and PNK (bottom left), ribozyme-catalyzed product that was formed by treatment of d3.1 with K28min and GTPγS (top right), and acid-decomposition of the ribozyme-catalyzed product (bottom right). The M–H peaks produced by each treatment are indicated: blue triangles, 6784.28 m/z (expected 6782.17 a.m.u for input d3.1 substrate); magenta triangles, 6880.38 m/z (expected 6878.11 a.m.u for mono-thiophosphorylated d3.1); green triangles, 7322.00 m/z (expected 7319.10 a.m.u for adduct shown in panel F top); red triangles, 7225.3 m/z (expected 7223.16 a.m.u for adduct shown in panel F bottom). ( B ) Incorporation of α- 32 P label into unlabeled acceptor strand. Controls in first two lanes show 5΄- 32 P-labeled d3.1 substrate alone (NR) or incubated with ribozyme K28min and GTPγS (sP). For remaining lanes, non-radiolabeled d3.1 substrate was incubated with ribozyme K28min and [α- 32 P]GTP for 5 min before adding the indicated amount of unlabeled GTP. Reactions were performed at 10°C for 18 h. ( C ) Thiophosphorylation reactions containing the indicated combinations of ribozyme, substrate and donor were treated with sodium periodate, followed by amination with Cy3 hydrazide dye. For the sample in Lane 4, the product of the ribozyme-catalyzed reaction was purified from an APM gel prior to the dye labeling reaction. Gel was scanned for Cy3 fluorescence (Ex. 532 nm Em. 570 nm). Phosphorimages are of 20% denaturing PAGE.

    Techniques Used: Mass Spectrometry, Generated, Produced, Labeling, Incubation, Purification, Fluorescence, Polyacrylamide Gel Electrophoresis

    Related Articles

    Labeling:

    Article Title: Axl-Gas6 Interaction Counteracts E1A-Mediated Cell Growth Suppression and Proapoptotic Activity
    Article Snippet: Single-stranded cDNA was synthesized with a kit provided by GIBCO-BRL. .. Primer 1 was labeled with [γ-32 P]ATP (Amersham Life Science) and T4 polynucleotide kinase (New England Biolabs) before PCR ( Taq DNA polymerase; Fisher Biotech). .. The RT-PCR products were analyzed by gel electrophoresis with an 8% polyacrylamide gel.

    Article Title: RISC is a 5? phosphomonoester-producing RNA endonuclease
    Article Snippet: .. 5′ labeling reactions contained 10 pmol oligonucleotide, 5 pmol γ-32 PATP (Amersham, 3000 Ci/mmol), 1 unit T4 polynucleotide kinase (New England Biolabs), and 10 mM MgCl2 /5 mM dithiothreitol/70 mM Tris-HCl (pH 7.6) in a final volume of 10 μL. .. The reaction mixture was incubated for 20 min at 37°C, followed by the addition of 1 μL 100 mM ATP and incubation for another 3 min at 37°C.

    Article Title: Nucleobase modification by an RNA enzyme
    Article Snippet: Radiolabeled nucleotides [γ32 P]-ATP, [α32 P]-CTP and [α32 P]-GTP, were purchased from Perkin-Elmer (Waltham, MA). .. Ribozymes were either transcribed using 33nM [α32 P]-CTP or non-radioactive CTP followed by 5΄ end labeled using [γ32 P]-ATP and PNK (NEB). ..

    Polymerase Chain Reaction:

    Article Title: Axl-Gas6 Interaction Counteracts E1A-Mediated Cell Growth Suppression and Proapoptotic Activity
    Article Snippet: Single-stranded cDNA was synthesized with a kit provided by GIBCO-BRL. .. Primer 1 was labeled with [γ-32 P]ATP (Amersham Life Science) and T4 polynucleotide kinase (New England Biolabs) before PCR ( Taq DNA polymerase; Fisher Biotech). .. The RT-PCR products were analyzed by gel electrophoresis with an 8% polyacrylamide gel.

    Amplification:

    Article Title: Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples
    Article Snippet: All library incubations were performed in 0.5 ml Lobind Eppendorf tubes in an Applied Biosystems 2720 thermal cycler. .. End-repair without T4 DNA Polymerase: for treatments −/+ and −/−, an end-repair mastermix was made by combining 4 μl T4 DNA ligase reaction buffer (New England Biolabs, NEB, Ipswich, Massachusetts, US), 0.5 μl dATP (10mM) (Thermo-Fisher), 2 μl reaction booster mix (consisting of 25 % PEG-4000 (Sigma Aldrich, 50%), 2 mg/ml BSA (Thermo-Fisher) and 400 mM NaCl) , 2 μl T4 PNK (NEB, cat#M0201S, 10 U/μl) and 1.5 μl Klenow Fragment (3’- > 5’ exo-) (NEB, cat#M0212S, 5 U/μl) per amplicon pool reaction. .. Ten μl of this mastermix was then added to each 30 μl amplicon pool, mixed well by pipetting and incubated for 30 minutes at 37°C followed by 30 minutes at 65°C and finally cooled to 4°C.

    Ligation:

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions
    Article Snippet: .. To allow for ligation with the blunt-end of the double-stranded adaptors, 15 µl of purified MNase digested DNA fragments were blunt-ended in a final volume of 20 µl by Klenow fragment of Escherichia coli DNA polymerase (New England Biolabs, CA, USA) and phosphorylated by T4 polynucleotide kinase (New England Biolabs) in a final volume of 30 µl. .. Adaptor ligation The blunt-ended and phosphorylated DNA fragments were ligated to the double-stranded adaptors A and B as described in ( ) with the following modifications.

    Purification:

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions
    Article Snippet: .. To allow for ligation with the blunt-end of the double-stranded adaptors, 15 µl of purified MNase digested DNA fragments were blunt-ended in a final volume of 20 µl by Klenow fragment of Escherichia coli DNA polymerase (New England Biolabs, CA, USA) and phosphorylated by T4 polynucleotide kinase (New England Biolabs) in a final volume of 30 µl. .. Adaptor ligation The blunt-ended and phosphorylated DNA fragments were ligated to the double-stranded adaptors A and B as described in ( ) with the following modifications.

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    New England Biolabs t4 polynucleotide kinase
    Effect of arrest on the transcript arrangement in RNAP. ( A ) RNase A footprinting of the transcript in active EC 26 and in arrested EC 27 . The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. ( B ) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC 20 and EC 27 (lanes 6 and 4) and arrested fraction of EC 27 purified by chase (lane 2) were treated with <t>T4</t> polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.
    T4 Polynucleotide Kinase, 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
    https://www.bioz.com/result/t4 polynucleotide kinase/product/New England Biolabs
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    Effect of arrest on the transcript arrangement in RNAP. ( A ) RNase A footprinting of the transcript in active EC 26 and in arrested EC 27 . The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. ( B ) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC 20 and EC 27 (lanes 6 and 4) and arrested fraction of EC 27 purified by chase (lane 2) were treated with T4 polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.

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

    Article Title: Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3? end of the RNA intact and extruded

    doi:

    Figure Lengend Snippet: Effect of arrest on the transcript arrangement in RNAP. ( A ) RNase A footprinting of the transcript in active EC 26 and in arrested EC 27 . The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. ( B ) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC 20 and EC 27 (lanes 6 and 4) and arrested fraction of EC 27 purified by chase (lane 2) were treated with T4 polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.

    Article Snippet: EC11 (the numerical index denotes the length of the transcript) was treated with 10 units of T4 polynucleotide kinase (New England Biolabs) and 50 μCi of [α-32 P]ATP (4500 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) for 10 min to label the DNA, washed with TB, and walked to the desired position.

    Techniques: Footprinting, Labeling, Centrifugation, Purification

    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Journal: bioRxiv

    Article Title: Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples

    doi: 10.1101/2020.01.22.915009

    Figure Lengend Snippet: Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Article Snippet: End-repair without T4 DNA Polymerase: for treatments −/+ and −/−, an end-repair mastermix was made by combining 4 μl T4 DNA ligase reaction buffer (New England Biolabs, NEB, Ipswich, Massachusetts, US), 0.5 μl dATP (10mM) (Thermo-Fisher), 2 μl reaction booster mix (consisting of 25 % PEG-4000 (Sigma Aldrich, 50%), 2 mg/ml BSA (Thermo-Fisher) and 400 mM NaCl) , 2 μl T4 PNK (NEB, cat#M0201S, 10 U/μl) and 1.5 μl Klenow Fragment (3’- > 5’ exo-) (NEB, cat#M0212S, 5 U/μl) per amplicon pool reaction.

    Techniques: Polymerase Chain Reaction, Activity Assay, Sequencing, Ligation, Amplification

    Target RNA is cleaved endonucleolytically producing 5′-phosphate and 3′-hydroxyl termini. ( A ) Preparation of site-specifically labeled substrates and cleavage assay. 5′- 32 P-labeled and 3′ aminolinker (L) protected 12-nt oligoribonucleotide was ligated to nonphosphorylated 9-nt oligoribonucleotide using T4 RNA ligase. An aliquot of the ligation product was further 5′- 32 P-labeled using T4 polynucleotide kinase. The purified substrates were incubated with affinity-purified RISC programmed with single-stranded guide siRNA. ( B ) PhosphorImaging of cleavage reactions incubated for 2 h at 30°C, and resolved on a 20% denaturing polyacrylamide gel. 5′- 32 P-labeled 9- and 12-nt oligoribonucleotides were loaded as marker in lanes 1 and 2 , respectively. The cleavage reactions with single- and double-labeled 21-nt substrate are loaded in lanes 4 and 5 , respectively. Lane 3 contains the 12-nt cleavage product isolated from a prior cleavage reaction. ( C ) Two-dimensional thin layer chromatography analysis of the ribonuclease T2-digested RISC-cleavage product. The oval depicts the unlabeled pAp as detected by UV shadowing. The radioactive signal comigrates with the 5′ 32 pAp released by ribonuclease T2 digestion from the gel-purified 12-nt cleavage product.

    Journal: Genes & Development

    Article Title: RISC is a 5? phosphomonoester-producing RNA endonuclease

    doi: 10.1101/gad.1187904

    Figure Lengend Snippet: Target RNA is cleaved endonucleolytically producing 5′-phosphate and 3′-hydroxyl termini. ( A ) Preparation of site-specifically labeled substrates and cleavage assay. 5′- 32 P-labeled and 3′ aminolinker (L) protected 12-nt oligoribonucleotide was ligated to nonphosphorylated 9-nt oligoribonucleotide using T4 RNA ligase. An aliquot of the ligation product was further 5′- 32 P-labeled using T4 polynucleotide kinase. The purified substrates were incubated with affinity-purified RISC programmed with single-stranded guide siRNA. ( B ) PhosphorImaging of cleavage reactions incubated for 2 h at 30°C, and resolved on a 20% denaturing polyacrylamide gel. 5′- 32 P-labeled 9- and 12-nt oligoribonucleotides were loaded as marker in lanes 1 and 2 , respectively. The cleavage reactions with single- and double-labeled 21-nt substrate are loaded in lanes 4 and 5 , respectively. Lane 3 contains the 12-nt cleavage product isolated from a prior cleavage reaction. ( C ) Two-dimensional thin layer chromatography analysis of the ribonuclease T2-digested RISC-cleavage product. The oval depicts the unlabeled pAp as detected by UV shadowing. The radioactive signal comigrates with the 5′ 32 pAp released by ribonuclease T2 digestion from the gel-purified 12-nt cleavage product.

    Article Snippet: 5′ labeling reactions contained 10 pmol oligonucleotide, 5 pmol γ-32 PATP (Amersham, 3000 Ci/mmol), 1 unit T4 polynucleotide kinase (New England Biolabs), and 10 mM MgCl2 /5 mM dithiothreitol/70 mM Tris-HCl (pH 7.6) in a final volume of 10 μL.

    Techniques: Labeling, Cleavage Assay, Ligation, Purification, Incubation, Affinity Purification, Marker, Isolation, Thin Layer Chromatography

    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