t4 polynucleotide kinase  (Thermo Fisher)


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  • 98
    Name:
    Anza T4 PNK Kit
    Description:
    The Invitrogen Anza T4 PNK (polynucleotide kinase) Kit is used to perform 5’-phosphorylation of DNA and oligonucleotides. T4 PNK exhibits 5’ polynucleotide kinase and 3’ phosphatase activity, catalyzing the transfer of the terminal phosphate of ATP to a 5’ hydroxyl group of a nucleic acid. When preparing DNA for the ligation step in cloning, either the insert DNA or the vector DNA should contain a 5’ phosphate. Often PCR-amplified DNA is treated with T4 PNK to phosphorylate the 5’ end for subsequent cloning ligation. Benefits: • Fast 15-minute incubation at room temperature • Phosphorylaton can be performed with DNA or oligonucleotides in water, TE, or 1X Anza buffers The Anza T4 PNK Kit is part of the Anza Restriction Enzyme Cloning System, unifying the traditional cloning processes. Other Anza DNA Modifying Enzymes include the Anza T4 DNA Ligase Master Mix, Anza DNA Blunt End Kit, and Anza DNA End Repair Kit.
    Catalog Number:
    IVGN2304
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Size:
    50 reactions
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, DNA⁄RNA Modifying Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher t4 polynucleotide kinase
    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).
    The Invitrogen Anza T4 PNK (polynucleotide kinase) Kit is used to perform 5’-phosphorylation of DNA and oligonucleotides. T4 PNK exhibits 5’ polynucleotide kinase and 3’ phosphatase activity, catalyzing the transfer of the terminal phosphate of ATP to a 5’ hydroxyl group of a nucleic acid. When preparing DNA for the ligation step in cloning, either the insert DNA or the vector DNA should contain a 5’ phosphate. Often PCR-amplified DNA is treated with T4 PNK to phosphorylate the 5’ end for subsequent cloning ligation. Benefits: • Fast 15-minute incubation at room temperature • Phosphorylaton can be performed with DNA or oligonucleotides in water, TE, or 1X Anza buffers The Anza T4 PNK Kit is part of the Anza Restriction Enzyme Cloning System, unifying the traditional cloning processes. Other Anza DNA Modifying Enzymes include the Anza T4 DNA Ligase Master Mix, Anza DNA Blunt End Kit, and Anza DNA End Repair Kit.
    https://www.bioz.com/result/t4 polynucleotide kinase/product/Thermo Fisher
    Average 98 stars, based on 514 article reviews
    Price from $9.99 to $1999.99
    t4 polynucleotide kinase - by Bioz Stars, 2019-10
    98/100 stars

    Images

    1) Product Images from "Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA"

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq572

    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).
    Figure Legend Snippet: Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).

    Techniques Used: Ligation, Purification, Sequencing, Blocking Assay

    2) Product Images from "Reconstitution of a Staphylococcal Plasmid-Protein Relaxation Complex In Vitro"

    Article Title: Reconstitution of a Staphylococcal Plasmid-Protein Relaxation Complex In Vitro

    Journal:

    doi: 10.1128/JB.186.11.3374-3383.2004

    The pC221 cop903 nic 5′ end is resistant to phosphorylation. (A) Overview of expected BstXI/BstUI fragment of pC221 cop903 after cleavage with BsrFI or nicking by MobA subsequently 5′ end labeled with T4 polynucleotide kinase; (B) autoradiograph
    Figure Legend Snippet: The pC221 cop903 nic 5′ end is resistant to phosphorylation. (A) Overview of expected BstXI/BstUI fragment of pC221 cop903 after cleavage with BsrFI or nicking by MobA subsequently 5′ end labeled with T4 polynucleotide kinase; (B) autoradiograph

    Techniques Used: Labeling, Autoradiography

    3) Product Images from "Conversion of Topoisomerase I Cleavage Complexes on the Leading Strand of Ribosomal DNA into 5?-Phosphorylated DNA Double-Strand Breaks by Replication Runoff"

    Article Title: Conversion of Topoisomerase I Cleavage Complexes on the Leading Strand of Ribosomal DNA into 5?-Phosphorylated DNA Double-Strand Breaks by Replication Runoff

    Journal:

    doi:

    Diagram of the LM-PCR protocol to detect top1-induced DNA single-strand breaks and replication-mediated DNA double-strand breaks. Top1 is shown as a shaded oval with covalent linkage to the 3′ end of a DNA single-strand break. In the assay for top1-induced DNA single-strand breaks, top1-induced DNA single-strand breaks (i.e., top1 cleavage complexes) were detected (upper left) as described previously by annealing primer 1 (P1) to denatured genomic DNA. After primer extension and in vitro phosphorylation of the 5′-OH termini with T4 polynucleotide kinase, ligation to the double-stranded linker was performed. Thereafter, rRNA gene-specific DNA fragments were amplified with Taq DNA polymerase using the linker-primer and a nested, gene-specific PCR primer. After 26 cycles of PCR, a third primer (5′ end labeled with 32 P; star) was used for two primer extension cycles before the samples were separated in 7% denaturing polyacrylamide gels. In the assay for replication-mediated DNA double-strand breaks, collision between a replication fork and a top1 cleavage complex is proposed to lead to replication runoff, with generation of a DNA double-strand break (upper right). Because of in vivo 5′-end phosphorylation of replication-mediated DNA double-strand breaks, ligation to the linker could be performed without prior T4 polynucleotide kinase reaction. The following reaction steps were the same as for the detection of top1-induced DNA single-strand breaks. Note that the single-strand break assay detects both single- and double-strand breaks.
    Figure Legend Snippet: Diagram of the LM-PCR protocol to detect top1-induced DNA single-strand breaks and replication-mediated DNA double-strand breaks. Top1 is shown as a shaded oval with covalent linkage to the 3′ end of a DNA single-strand break. In the assay for top1-induced DNA single-strand breaks, top1-induced DNA single-strand breaks (i.e., top1 cleavage complexes) were detected (upper left) as described previously by annealing primer 1 (P1) to denatured genomic DNA. After primer extension and in vitro phosphorylation of the 5′-OH termini with T4 polynucleotide kinase, ligation to the double-stranded linker was performed. Thereafter, rRNA gene-specific DNA fragments were amplified with Taq DNA polymerase using the linker-primer and a nested, gene-specific PCR primer. After 26 cycles of PCR, a third primer (5′ end labeled with 32 P; star) was used for two primer extension cycles before the samples were separated in 7% denaturing polyacrylamide gels. In the assay for replication-mediated DNA double-strand breaks, collision between a replication fork and a top1 cleavage complex is proposed to lead to replication runoff, with generation of a DNA double-strand break (upper right). Because of in vivo 5′-end phosphorylation of replication-mediated DNA double-strand breaks, ligation to the linker could be performed without prior T4 polynucleotide kinase reaction. The following reaction steps were the same as for the detection of top1-induced DNA single-strand breaks. Note that the single-strand break assay detects both single- and double-strand breaks.

    Techniques Used: Polymerase Chain Reaction, In Vitro, Ligation, Amplification, Labeling, In Vivo

    4) Product Images from "Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate"

    Article Title: Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate

    Journal: Nature

    doi: 10.1038/s41586-018-0557-5

    Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using T4 polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).
    Figure Legend Snippet: Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using T4 polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).

    Techniques Used: Thin Layer Chromatography, Generated, Incubation

    5) Product Images from "A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing"

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing

    Journal: Molecular Microbiology

    doi: 10.1111/mmi.12810

    Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the  rrnE  operon, which is representative of all seven rRNA operons in  E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF  strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).
    Figure Legend Snippet: Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the rrnE operon, which is representative of all seven rRNA operons in E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).

    Techniques Used: RNA Sequencing Assay, Sequencing, Amplification, Produced, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Isolation, Staining, Incubation, Nucleic Acid Electrophoresis

    6) Product Images from "An In Vitro DNA Double-Strand Break Repair Assay Based on End-Joining of Defined Duplex Oligonucleotides"

    Article Title: An In Vitro DNA Double-Strand Break Repair Assay Based on End-Joining of Defined Duplex Oligonucleotides

    Journal:

    doi: 10.1007/978-1-61779-998-3_33

    The sequences and duplex formation scheme for a typical undamaged control duplex oligonucleotide end-joining substrate. T4-PNK T4-polynucleotide kinase.
    Figure Legend Snippet: The sequences and duplex formation scheme for a typical undamaged control duplex oligonucleotide end-joining substrate. T4-PNK T4-polynucleotide kinase.

    Techniques Used:

    7) Product Images from "Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideu"

    Article Title: Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideu

    Journal:

    doi: 10.1074/jbc.M110.208306

    Principle of the cRT-PCR method used to determine the ends of RNA molecules. Total RNA is dephosphorylated by shrimp alkaline phosphatase ( SAP ) and then 5′-monophosphorylated by T4 polynucleotide kinase ( T4 PNK ). The resulting 5′-phosphate
    Figure Legend Snippet: Principle of the cRT-PCR method used to determine the ends of RNA molecules. Total RNA is dephosphorylated by shrimp alkaline phosphatase ( SAP ) and then 5′-monophosphorylated by T4 polynucleotide kinase ( T4 PNK ). The resulting 5′-phosphate

    Techniques Used: Polymerase Chain Reaction

    8) Product Images from "A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA"

    Article Title: A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA

    Journal: Scientific Reports

    doi: 10.1038/srep00126

    T4 polynucleotide kinase does not preferentially label m 6 Am or Am containing ends. The oligonucleotides SK-526 (m 6 Am) and SK-524 (Am) were mixed in ratios of 5:1, 2:1, 1:1. 1:2 and 1:5, end labelled using T4 polynucleotide kinase, digested to 5' phosphate mononucleotides and separated by TLC. The relative intensities of the resulting spots were quantified using phosphorimaging. The calculated percentage of m 6 Am closely matched the actual m 6 Am percentage.
    Figure Legend Snippet: T4 polynucleotide kinase does not preferentially label m 6 Am or Am containing ends. The oligonucleotides SK-526 (m 6 Am) and SK-524 (Am) were mixed in ratios of 5:1, 2:1, 1:1. 1:2 and 1:5, end labelled using T4 polynucleotide kinase, digested to 5' phosphate mononucleotides and separated by TLC. The relative intensities of the resulting spots were quantified using phosphorimaging. The calculated percentage of m 6 Am closely matched the actual m 6 Am percentage.

    Techniques Used: Thin Layer Chromatography

    9) Product Images from "Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideum"

    Article Title: Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideum

    Journal:

    doi: 10.1074/jbc.M110.208306

    Principle of the cRT-PCR method used to determine the ends of RNA molecules. Total RNA is dephosphorylated by shrimp alkaline phosphatase ( SAP ) and then 5′-monophosphorylated by T4 polynucleotide kinase ( T4 PNK ). The resulting 5′-phosphate
    Figure Legend Snippet: Principle of the cRT-PCR method used to determine the ends of RNA molecules. Total RNA is dephosphorylated by shrimp alkaline phosphatase ( SAP ) and then 5′-monophosphorylated by T4 polynucleotide kinase ( T4 PNK ). The resulting 5′-phosphate

    Techniques Used: Polymerase Chain Reaction

    10) Product Images from "The 5?-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex"

    Article Title: The 5?-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp764

    The phosphorylation status of mivaRNAIs derived from VA RNAI(A) and VA RNAI(G). The RISC associated RNAs from pVA RNAI(A) or pVA RNAI(G) transfected cells were treated with combinations of calf intestinal alkaline phosphatase (CIAP), T4 polynucleotide kinase (PNK) and Terminator 5′-exonuclease (TE). After enzyme treatment, the 5′- ( A ) or 3′-strands ( B ) of mivaRNAI were analyzed by northern blotting.
    Figure Legend Snippet: The phosphorylation status of mivaRNAIs derived from VA RNAI(A) and VA RNAI(G). The RISC associated RNAs from pVA RNAI(A) or pVA RNAI(G) transfected cells were treated with combinations of calf intestinal alkaline phosphatase (CIAP), T4 polynucleotide kinase (PNK) and Terminator 5′-exonuclease (TE). After enzyme treatment, the 5′- ( A ) or 3′-strands ( B ) of mivaRNAI were analyzed by northern blotting.

    Techniques Used: Derivative Assay, Transfection, Northern Blot

    Related Articles

    Amplification:

    Article Title: Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideu
    Article Snippet: After precipitation, the RNA was incubated with 20 units of T4 polynucleotide kinase (Fermentas) and 20 units of RiboLock RNase Inhibitor (Fermentas) in 30 μl of 50 m m Tris-HCl (pH 7.6), 10 m m MgCl2 , 5 m m DTT, 100 μ m spermidine, 1 m m ATP for 30 min at 37 °C. .. After precipitation, the RNA was incubated with 20 units of T4 polynucleotide kinase (Fermentas) and 20 units of RiboLock RNase Inhibitor (Fermentas) in 30 μl of 50 m m Tris-HCl (pH 7.6), 10 m m MgCl2 , 5 m m DTT, 100 μ m spermidine, 1 m m ATP for 30 min at 37 °C.

    Article Title: A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA
    Article Snippet: Prior to amplification, the forward oligonucleotides were 5' phosphorylated, the amplification products were subsequently digested with lambda nuclease (New England BioLabs) to leave the single stranded antisense DNA strand. .. After phenol-chloroform extraction and ethanol precipitation, RNA samples were resuspended in 20 µl of sterile distilled water and 5' ends were labelled using 30 units T4 polynucleotide kinase (PNK, Fermentas) and 7.4 MBq [γ-32 P] ATP at 37 °C for 30 minutes.

    Cycling Probe Technology:

    Article Title: Conversion of Topoisomerase I Cleavage Complexes on the Leading Strand of Ribosomal DNA into 5?-Phosphorylated DNA Double-Strand Breaks by Replication Runoff
    Article Snippet: 20-( S )-Camptothecin lactone (CPT) was obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute. .. T4 polynucleotide kinase (10 U/μl) was purchased from Gibco-BRL, Life Technologies (Gaithersburg, Md.), and aphidicolin was obtained from Sigma Chemical Co. (St. Louis, Mo.).

    Positive Control:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: The positive control was oligo 11 phosphorylated by T4 PNK. .. 25 µl of the positive control mixture contained 1 x PNK buffer A (50 mM Tris-HCl, pH 7.6 at 25°C, 10 mM MgCl2 , 5 mM DTT, 0.1 mM spermidine and 0.1 mM EDTA), 2 µM of oligo 11, 0.6 µCi/µl of [γ-32P] ATP, and 0.6 U/µl of T4 PNK (Fermentas). .. The positive control was then diluted 10-fold with 1 x TE.

    Synthesized:

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response
    Article Snippet: For site-specific m6 A detection, DNA primers were firstly 5′ labeled with 32 P using T4 polynucleotide kinase (Invitrogen) and purified by ethanol precipitation. .. For site-specific m6 A detection, DNA primers were firstly 5′ labeled with 32 P using T4 polynucleotide kinase (Invitrogen) and purified by ethanol precipitation.

    Article Title: m6A-dependent regulation of messenger RNA stability
    Article Snippet: The RNA probe was synthesized by a previously reported method with the sequence of 5’-AUGGGCCGUUCAUCUGCUAAAAGGXCUGCUUUUGGGGCUUGU-3’(X = A or m6 A). .. After the synthesis, the RNA probe was labeled in a reaction mixture of 2 µL RNA probe (1 µM), 5 µL 5×T4 PNK buffer A (Fermentas), 1 µL T4 PNK (Fermentas), 1 µL P-ATP and 41 µL RNase-free water (final RNA concentration 40 nM) at 37°C for 1 hour.

    Electrophoresis:

    Article Title: Reconstitution of a Staphylococcal Plasmid-Protein Relaxation Complex In Vitro
    Article Snippet: Then, 75 fmol of DNA was treated with 12.5 U of T4 polynucleotide kinase (Invitrogen) in a phosphate exchange reaction for 14 min at 37°C in the presence of [γ-33 P]ATP (15 μCi) in a final volume of 7 μl. .. Then, 75 fmol of DNA was treated with 12.5 U of T4 polynucleotide kinase (Invitrogen) in a phosphate exchange reaction for 14 min at 37°C in the presence of [γ-33 P]ATP (15 μCi) in a final volume of 7 μl.

    Incubation:

    Article Title: Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate
    Article Snippet: For generating linear oligoadenylates A2 > P (5’ hydroxyl-Ap-Ap with a 2’,3’-cyclic phosphate) and A4 > P, 30 μM A2 (AAACAUCAG) or A4 (AAAAACAUCAG) RNA was incubated with MazF in FXa buffer for 1 h at 37 °C. .. For use as standards, A2 > P and A4 > P linear oligoadenylates were 5’-end labelled using 32 P-γ-ATP and T4 Polynucleotide Kinase (PNK; Thermo Fisher Scientific) via its forward reaction.

    Article Title: DNA-guided DNA interference by a prokaryotic Argonaute
    Article Snippet: Proteinase K (Ambion) and CaCl2 (final concentration, 5 mM) were added to purified proteins and samples were incubated for 1 h at 37 °C. .. Purified nucleic acids were [γ-32 P]ATP labelled with T4 PNK (Fermentas) in exchange- or forward-labelling reactions and thereafter separated from free [γ-32 P] ATP using a Sephadex G-25 column (GE).

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA
    Article Snippet: See Supplementary Table S2 for sample information. .. For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP. .. Reactions were purified using the MinElute PCR Purification kit.

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing
    Article Snippet: For each sample, an aliquot of 5–10 μg was mixed with an equal volume of 2× RNA-loading dye (New England BioLabs), denatured by incubation at 90°C for 90 s, chilled on ice, and analysed along with other samples by denaturing electrophoreses using a 6% sequencing-type gel [acrylamide : bis -acrylamide (29:1), 1× TBE, 7 M urea]. .. Specific E. coli transcripts were probed using complementary oligonucleotides (see ) labelled at their 5′ ends with 32 P using T4 polynucleotide kinase (Thermo Scientific) and γ-32 P-ATP (3000 Ci mmol−1 , 10 mCi ml−1 , 250 μCi, Perkin Elmer).

    Article Title: Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideu
    Article Snippet: To generate RNA ends suitable for circularization, 15 μg of DNase I-treated total RNA was dephosphorylated by incubation with 1 unit of SAP (Fermentas) in 30 μl of 10 m m Tris-HCl (pH 7.5), 10 m m MgCl2 , 100 μg/ml BSA for 30 min at 37 °C. .. After precipitation, the RNA was incubated with 20 units of T4 polynucleotide kinase (Fermentas) and 20 units of RiboLock RNase Inhibitor (Fermentas) in 30 μl of 50 m m Tris-HCl (pH 7.6), 10 m m MgCl2 , 5 m m DTT, 100 μ m spermidine, 1 m m ATP for 30 min at 37 °C. .. After protein extraction with phenol and chloroform, RNA was precipitated with ethanol.

    Article Title: A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA
    Article Snippet: The 5' phosphate of the exposed cap adjacent nucleotide was removed by the addition of 10 units of Alkaline Phosphatase (Fermentas) and incubation for a further 15 minutes at 37 °C. .. After phenol-chloroform extraction and ethanol precipitation, RNA samples were resuspended in 20 µl of sterile distilled water and 5' ends were labelled using 30 units T4 polynucleotide kinase (PNK, Fermentas) and 7.4 MBq [γ-32 P] ATP at 37 °C for 30 minutes.

    Article Title: N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions
    Article Snippet: The synthetic RNA oligos were 5′ end-labeled with γ-32 P-ATP by T4 PNK (70031, Affymetrix), gel purified, and re-folded. .. The synthetic RNA oligos were 5′ end-labeled with γ-32 P-ATP by T4 PNK (70031, Affymetrix), gel purified, and re-folded.

    Article Title: Pancreatic β-cell tRNA hypomethylation and fragmentation link TRMT10A deficiency with diabetes
    Article Snippet: Oligonucleotides used for primer extension assays and DNA probes used in northern blots were 5′-end-labeled using ATP [γ-32 P] and T4 polynucleotide kinase (T4 PNK) (ThermoFisher). .. The labeling reaction was performed by mixing 20 pmol oligonucleotide primers or 200 ng DNA probes with 150 μCi ATP [γ-32 P], 2 μl 10× T4 PNK reaction buffer, 10U T4 PNK and DEPC-treated water up to 20 μl.

    Modification:

    Article Title: The 5?-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex
    Article Snippet: To analyze the 5′-end modification on mivaRNAI, small RNAs extracted from immuno-purified RISC complexes were treated with different enzyme combinations in a total reaction volume of 23 µl under the reaction conditions recommended by the manufacturer. .. RNAs were treated with 20 U of calf intestine alkaline phosphatase (CIAP, Fermentas) for 30 min at 37°C, 10 U of T4 polynucleotide kinase (PNK, Fermentas) for 20 min at 37°C and 1 U of Terminator 5′-exonuclease (TE, Epicentre) for 1 h at 30°C.

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: Paragraph title: Internal modification of DNA ... To later remove the template/splint (DNA5/6) after either reaction, the 5′-terminus was phosphorylated by T4 PNK [100 μM DNA, 1x T4 DNA Ligase buffer (Thermo Scientific; 40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP, pH 7.8 at 25°C), 0.5 U/μl T4 PNK], so that 5′-monophosphate-specific λ-exonuclease could be used for digestion of DNA5/6.

    Kinase Assay:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: Paragraph title: Kinase Assay for T4 DNA Ligase ... 25 µl of the positive control mixture contained 1 x PNK buffer A (50 mM Tris-HCl, pH 7.6 at 25°C, 10 mM MgCl2 , 5 mM DTT, 0.1 mM spermidine and 0.1 mM EDTA), 2 µM of oligo 11, 0.6 µCi/µl of [γ-32P] ATP, and 0.6 U/µl of T4 PNK (Fermentas).

    Hybridization:

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing
    Article Snippet: Specific E. coli transcripts were probed using complementary oligonucleotides (see ) labelled at their 5′ ends with 32 P using T4 polynucleotide kinase (Thermo Scientific) and γ-32 P-ATP (3000 Ci mmol−1 , 10 mCi ml−1 , 250 μCi, Perkin Elmer). .. Specific E. coli transcripts were probed using complementary oligonucleotides (see ) labelled at their 5′ ends with 32 P using T4 polynucleotide kinase (Thermo Scientific) and γ-32 P-ATP (3000 Ci mmol−1 , 10 mCi ml−1 , 250 μCi, Perkin Elmer).

    Ligation:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: To explore the ligation mechanism of DNA linkers with 5′-OH ends, oligo 11 of linker F were phosphorylated by using [γ-32P] ATP and T4 DNA ligase. .. 25 µl of the positive control mixture contained 1 x PNK buffer A (50 mM Tris-HCl, pH 7.6 at 25°C, 10 mM MgCl2 , 5 mM DTT, 0.1 mM spermidine and 0.1 mM EDTA), 2 µM of oligo 11, 0.6 µCi/µl of [γ-32P] ATP, and 0.6 U/µl of T4 PNK (Fermentas).

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: To prepare internally alkyne-modified DNA2, alkyne-tailed DNA2 was either used as a primer in primer extension or as a ligation fragment in a splinted ligation. .. To later remove the template/splint (DNA5/6) after either reaction, the 5′-terminus was phosphorylated by T4 PNK [100 μM DNA, 1x T4 DNA Ligase buffer (Thermo Scientific; 40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP, pH 7.8 at 25°C), 0.5 U/μl T4 PNK], so that 5′-monophosphate-specific λ-exonuclease could be used for digestion of DNA5/6.

    Northern Blot:

    Article Title: Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideu
    Article Snippet: Paragraph title: Northern Analysis ... As probes, DNA oligonucleotides were 5′ end-labeled by incubating 10 pmol of primer with 10 units of T4 polynucleotide kinase (Fermentas) in 50 m m Tris-HCl (pH 7.6), 10 m m MgCl2 , 5 m m DTT, 100 μ m spermidine, and 0.37 MBq of [γ-32 P]ATP.

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing
    Article Snippet: Paragraph title: RLM-RT-PCR analysis and northern blotting ... Specific E. coli transcripts were probed using complementary oligonucleotides (see ) labelled at their 5′ ends with 32 P using T4 polynucleotide kinase (Thermo Scientific) and γ-32 P-ATP (3000 Ci mmol−1 , 10 mCi ml−1 , 250 μCi, Perkin Elmer).

    Article Title: CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III
    Article Snippet: Paragraph title: Northern blot analysis ... For primer labeling, 40 pmol of oligonucleotide were denaturated for 5 min at 95°C and then mixed with 20 μCi of 32 P-γ-ATP and 10 Units of T4 polynucleotide kinase (Fermentas).

    Article Title: The human Obg protein GTPBP10 is involved in mitoribosomal biogenesis
    Article Snippet: Paragraph title: RNA isolation and northern blot ... [32 P]-radiolabeled probes were generated utilizing T4 Polynucleotide Kinase (Thermo Scientific) according to the manufacturer's instructions.

    Article Title: Pancreatic β-cell tRNA hypomethylation and fragmentation link TRMT10A deficiency with diabetes
    Article Snippet: Guo was eluted at 13.2 min, whereas retention time of m1 G was 15.7 min. For both m1 G and Guo, quantification was performed by external calibration, and the results are given as the number of m1 G per 1000 Guo. .. Oligonucleotides used for primer extension assays and DNA probes used in northern blots were 5′-end-labeled using ATP [γ-32 P] and T4 polynucleotide kinase (T4 PNK) (ThermoFisher). .. The labeling reaction was performed by mixing 20 pmol oligonucleotide primers or 200 ng DNA probes with 150 μCi ATP [γ-32 P], 2 μl 10× T4 PNK reaction buffer, 10U T4 PNK and DEPC-treated water up to 20 μl.

    Oligonucleotide Labeling:

    Article Title: An In Vitro DNA Double-Strand Break Repair Assay Based on End-Joining of Defined Duplex Oligonucleotides
    Article Snippet: Paragraph title: 2.3. Oligonucleotide Labeling and Duplex Formation ... T4 polynucleotide kinase and 10× buffer (Fermentas; Hanover, MD).

    Generated:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: 25 µl of the positive control mixture contained 1 x PNK buffer A (50 mM Tris-HCl, pH 7.6 at 25°C, 10 mM MgCl2 , 5 mM DTT, 0.1 mM spermidine and 0.1 mM EDTA), 2 µM of oligo 11, 0.6 µCi/µl of [γ-32P] ATP, and 0.6 U/µl of T4 PNK (Fermentas). .. 25 µl of the positive control mixture contained 1 x PNK buffer A (50 mM Tris-HCl, pH 7.6 at 25°C, 10 mM MgCl2 , 5 mM DTT, 0.1 mM spermidine and 0.1 mM EDTA), 2 µM of oligo 11, 0.6 µCi/µl of [γ-32P] ATP, and 0.6 U/µl of T4 PNK (Fermentas).

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA
    Article Snippet: For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP. .. For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP.

    Article Title: The human Obg protein GTPBP10 is involved in mitoribosomal biogenesis
    Article Snippet: RNA separation on a denaturing formaldehyde/formamide 1.2% agarose gel was performed as previously described ( ). .. [32 P]-radiolabeled probes were generated utilizing T4 Polynucleotide Kinase (Thermo Scientific) according to the manufacturer's instructions. .. Imaging was performed with Typhoon imaging system (GE Healthcare).

    Imaging:

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing
    Article Snippet: Specific E. coli transcripts were probed using complementary oligonucleotides (see ) labelled at their 5′ ends with 32 P using T4 polynucleotide kinase (Thermo Scientific) and γ-32 P-ATP (3000 Ci mmol−1 , 10 mCi ml−1 , 250 μCi, Perkin Elmer). .. The membrane was pre-hybridized with 3 ml of ULTRAhyb-Oligo Hybridization Buffer (Ambion) at 42o C for 30 min. Radiolabelled probe, which had been denatured by incubation at 90°C for 90 s and chilled on ice, was added to the hybridization tube.

    Polymerase Chain Reaction:

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA
    Article Snippet: See Supplementary Table S2 for sample information. .. For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP. .. Reactions were purified using the MinElute PCR Purification kit.

    Article Title: Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideu
    Article Snippet: After precipitation, the RNA was incubated with 20 units of T4 polynucleotide kinase (Fermentas) and 20 units of RiboLock RNase Inhibitor (Fermentas) in 30 μl of 50 m m Tris-HCl (pH 7.6), 10 m m MgCl2 , 5 m m DTT, 100 μ m spermidine, 1 m m ATP for 30 min at 37 °C. .. After precipitation, the RNA was incubated with 20 units of T4 polynucleotide kinase (Fermentas) and 20 units of RiboLock RNase Inhibitor (Fermentas) in 30 μl of 50 m m Tris-HCl (pH 7.6), 10 m m MgCl2 , 5 m m DTT, 100 μ m spermidine, 1 m m ATP for 30 min at 37 °C.

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: To later remove the template/splint (DNA5/6) after either reaction, the 5′-terminus was phosphorylated by T4 PNK [100 μM DNA, 1x T4 DNA Ligase buffer (Thermo Scientific; 40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP, pH 7.8 at 25°C), 0.5 U/μl T4 PNK], so that 5′-monophosphate-specific λ-exonuclease could be used for digestion of DNA5/6. .. To avoid misligation of DNA5/6 and ligation fragment DNA7, DNA was 3′-blocked by TdT reaction with ddGTP (100 μM DNA, 1 mM ddGTP, 90 min) and precipitated prior to phosphorylation.

    Sonication:

    Article Title: A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA
    Article Snippet: Membranes were prehybridised at 42 °C in 40 % formamide with 5 × Denhardt's, 3 % SDS, 0.3 M NaCl, 50 mM sodium phosphate buffer (pH 7.0) and 0.1 mg ml−1 sonicated salmon sperm DNA. .. After phenol-chloroform extraction and ethanol precipitation, RNA samples were resuspended in 20 µl of sterile distilled water and 5' ends were labelled using 30 units T4 polynucleotide kinase (PNK, Fermentas) and 7.4 MBq [γ-32 P] ATP at 37 °C for 30 minutes.

    Copurification:

    Article Title: DNA-guided DNA interference by a prokaryotic Argonaute
    Article Snippet: Paragraph title: Guide co-purification and sequencing ... Purified nucleic acids were [γ-32 P]ATP labelled with T4 PNK (Fermentas) in exchange- or forward-labelling reactions and thereafter separated from free [γ-32 P] ATP using a Sephadex G-25 column (GE).

    Radioactivity:

    Article Title: DNA-guided DNA interference by a prokaryotic Argonaute
    Article Snippet: Purified nucleic acids were [γ-32 P]ATP labelled with T4 PNK (Fermentas) in exchange- or forward-labelling reactions and thereafter separated from free [γ-32 P] ATP using a Sephadex G-25 column (GE). .. After nuclease treatment, samples were mixed with Loading Buffer (95% (deionized) formamide, 5 mM EDTA, 0.025% SDS, 0.025% bromophenol blue and 0.025% xylene cyanol), heated for 5 min at 95 °C and resolved on 15% or 20% denaturing polyacrylamide gels.

    RNA Sequencing Assay:

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing
    Article Snippet: RNA for northern blotting was isolated as described for the RNA-seq analysis, with the exception that the mRNA was not enriched, from a second batch of cultures. .. Specific E. coli transcripts were probed using complementary oligonucleotides (see ) labelled at their 5′ ends with 32 P using T4 polynucleotide kinase (Thermo Scientific) and γ-32 P-ATP (3000 Ci mmol−1 , 10 mCi ml−1 , 250 μCi, Perkin Elmer).

    Isolation:

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing
    Article Snippet: E. coli K-12 MG1655 (seq) was grown in Luria–Bertani (Amresco) as well as M9 minimal media (Sigma) supplemented with glucose (0.4%, w/v) at 37°C with shaking (100 r.p.m.) until an OD600 of ∼ 0.5 at which point RNA was isolated as described previously (Kime et al ., ). .. Specific E. coli transcripts were probed using complementary oligonucleotides (see ) labelled at their 5′ ends with 32 P using T4 polynucleotide kinase (Thermo Scientific) and γ-32 P-ATP (3000 Ci mmol−1 , 10 mCi ml−1 , 250 μCi, Perkin Elmer).

    Article Title: RNA modification landscape of the human mitochondrial tRNALys regulates protein synthesis
    Article Snippet: Deacylation of total RNA (at < 0.5 μg/μL) was performed in 100 mM Tris-HCl pH = 9.0 at 37 °C for 30 min. Small RNA was isolated from total RNA by using the small RNA isolation protocol for the RNA Clean and Concentrator Kit (Zymo R1015). .. Small RNA was treated with T4 PNK (Affymetrix, final concentration 0.1U/μL) in 1× T4 PNK buffer at 37 °C for 30 min.

    Article Title: The human Obg protein GTPBP10 is involved in mitoribosomal biogenesis
    Article Snippet: Paragraph title: RNA isolation and northern blot ... [32 P]-radiolabeled probes were generated utilizing T4 Polynucleotide Kinase (Thermo Scientific) according to the manufacturer's instructions.

    Labeling:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: The kinase assay for E. coli DNA ligase was not performed because NAD+ labeled with 32P was not available for us. .. 25 µl of the positive control mixture contained 1 x PNK buffer A (50 mM Tris-HCl, pH 7.6 at 25°C, 10 mM MgCl2 , 5 mM DTT, 0.1 mM spermidine and 0.1 mM EDTA), 2 µM of oligo 11, 0.6 µCi/µl of [γ-32P] ATP, and 0.6 U/µl of T4 PNK (Fermentas).

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response
    Article Snippet: Luciferase activity was monitored and recorded using Kronos Dio Luminometer (Atto). .. For site-specific m6 A detection, DNA primers were firstly 5′ labeled with 32 P using T4 polynucleotide kinase (Invitrogen) and purified by ethanol precipitation. .. The primer 5′- AGGGATGCTCTGGGGAAGGCTGG-3′ was used to detect potential m6 A site and the primer 5′-CGCCGCTCGCTCTGCTTCTCTTGTCTTCGCT-3′ was used to detect the non-methylated site.

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: For internal labeling of DNA, DNA2 was TdT-reacted with 5-E -UTP or N6 -P -ATP (100 μM DNA, 100 μM NTP) at 37°C overnight. .. To later remove the template/splint (DNA5/6) after either reaction, the 5′-terminus was phosphorylated by T4 PNK [100 μM DNA, 1x T4 DNA Ligase buffer (Thermo Scientific; 40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP, pH 7.8 at 25°C), 0.5 U/μl T4 PNK], so that 5′-monophosphate-specific λ-exonuclease could be used for digestion of DNA5/6.

    Article Title: CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III
    Article Snippet: In general, 32 P-labeled primers specific to the RNAs of interest were used as probes ( ). .. For primer labeling, 40 pmol of oligonucleotide were denaturated for 5 min at 95°C and then mixed with 20 μCi of 32 P-γ-ATP and 10 Units of T4 polynucleotide kinase (Fermentas). .. Labeled probes were purified prior hybridization using G25 columns (GE Healthcare) in order to remove unincorporated nucleotides.

    Article Title: m6A-dependent regulation of messenger RNA stability
    Article Snippet: The RNA probe was synthesized by a previously reported method with the sequence of 5’-AUGGGCCGUUCAUCUGCUAAAAGGXCUGCUUUUGGGGCUUGU-3’(X = A or m6 A). .. After the synthesis, the RNA probe was labeled in a reaction mixture of 2 µL RNA probe (1 µM), 5 µL 5×T4 PNK buffer A (Fermentas), 1 µL T4 PNK (Fermentas), 1 µL P-ATP and 41 µL RNase-free water (final RNA concentration 40 nM) at 37°C for 1 hour. .. The mixture was then purified by RNase-free micro bio-spin columns with bio-gel P30 in Tris buffer (BioRad 732–6250) to remove hot ATP and other small molecules.

    Article Title: RNA modification landscape of the human mitochondrial tRNALys regulates protein synthesis
    Article Snippet: Small RNA was treated with T4 PNK (Affymetrix, final concentration 0.1U/μL) in 1× T4 PNK buffer at 37 °C for 30 min. .. Small RNA was treated with T4 PNK (Affymetrix, final concentration 0.1U/μL) in 1× T4 PNK buffer at 37 °C for 30 min.

    Article Title: Pancreatic β-cell tRNA hypomethylation and fragmentation link TRMT10A deficiency with diabetes
    Article Snippet: Paragraph title: Radioactive labeling of oligonucleotides ... Oligonucleotides used for primer extension assays and DNA probes used in northern blots were 5′-end-labeled using ATP [γ-32 P] and T4 polynucleotide kinase (T4 PNK) (ThermoFisher).

    Purification:

    Article Title: Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate
    Article Snippet: The E. coli toxin-antitoxin MazEF was purified as previously described . .. For use as standards, A2 > P and A4 > P linear oligoadenylates were 5’-end labelled using 32 P-γ-ATP and T4 Polynucleotide Kinase (PNK; Thermo Fisher Scientific) via its forward reaction.

    Article Title: DNA-guided DNA interference by a prokaryotic Argonaute
    Article Snippet: Precipitation was performed overnight at −20 °C in the presence of linear polymerized acrylamide as carrier. .. Purified nucleic acids were [γ-32 P]ATP labelled with T4 PNK (Fermentas) in exchange- or forward-labelling reactions and thereafter separated from free [γ-32 P] ATP using a Sephadex G-25 column (GE). .. Labelled nucleic acids were incubated with nucleases (DNase-free RNase A (Fermentas),RQ1 RNase-free DNaseI (Promega) or P1 nuclease (Sigma)) for 1 h at 37 °C.

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response
    Article Snippet: Luciferase activity was monitored and recorded using Kronos Dio Luminometer (Atto). .. For site-specific m6 A detection, DNA primers were firstly 5′ labeled with 32 P using T4 polynucleotide kinase (Invitrogen) and purified by ethanol precipitation. .. The primer 5′- AGGGATGCTCTGGGGAAGGCTGG-3′ was used to detect potential m6 A site and the primer 5′-CGCCGCTCGCTCTGCTTCTCTTGTCTTCGCT-3′ was used to detect the non-methylated site.

    Article Title: N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions
    Article Snippet: The eluted RNA was digested with 1 μl (1U/ μl) nuclease P1 at 37 °C for 1 h. Samples were spotted on cellulose TLC plate and 2D TLC was run as described using isobutyric acid: 0.5 M NH4 OH (5:3, v/v) as the first dimension and isopropanol:HCl:water (70:15:15, v/v/v) as the second dimension. .. The synthetic RNA oligos were 5′ end-labeled with γ-32 P-ATP by T4 PNK (70031, Affymetrix), gel purified, and re-folded. .. Structural probing assay with RNase T1, nuclease S1 and RNase V1 was performed as previously described .

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: To later remove the template/splint (DNA5/6) after either reaction, the 5′-terminus was phosphorylated by T4 PNK [100 μM DNA, 1x T4 DNA Ligase buffer (Thermo Scientific; 40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP, pH 7.8 at 25°C), 0.5 U/μl T4 PNK], so that 5′-monophosphate-specific λ-exonuclease could be used for digestion of DNA5/6. .. Primer extension was performed by performing 5 cycles of a polymerase chain reaction (PCR)-like reaction [5 μM tailed DNA2, 5 μM blocked and phosphorylated DNA5/6, 3 mM MgCl2 , 0.5 mM dNTPs and ∼0.25 U/μl lab-prepared Taq Polymerase in 1x Taq polymerase buffer (Rapidozym); denaturation: 94°C, 1 min, annealing: 60°C, 1 min; extension: 72°C, 1 min].

    Article Title: RNA modification landscape of the human mitochondrial tRNALys regulates protein synthesis
    Article Snippet: Up to 1.5 μg of small RNA (~60 pmol) was demethylated with 2× molar ratio WT AlkB and 4X molar ratio of D135S AlkB in 25 mM MES pH = 5.0, 30 mM KCl, 2 mM MgCl2 , 2 mM ascorbic acid, 300 μM α-ketoglutarate, 50 μM (NH4 )2 Fe(SO4 )2 in the presence of RNasin at room temperature for 2 h. Demethylated small RNA was purified using an RNA Clean and Concentrator Kit (Zymo). .. Small RNA was treated with T4 PNK (Affymetrix, final concentration 0.1U/μL) in 1× T4 PNK buffer at 37 °C for 30 min.

    Sequencing:

    Article Title: DNA-guided DNA interference by a prokaryotic Argonaute
    Article Snippet: Paragraph title: Guide co-purification and sequencing ... Purified nucleic acids were [γ-32 P]ATP labelled with T4 PNK (Fermentas) in exchange- or forward-labelling reactions and thereafter separated from free [γ-32 P] ATP using a Sephadex G-25 column (GE).

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA
    Article Snippet: Paragraph title: PEP assays and sequencing ... For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP.

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing
    Article Snippet: For each sample, an aliquot of 5–10 μg was mixed with an equal volume of 2× RNA-loading dye (New England BioLabs), denatured by incubation at 90°C for 90 s, chilled on ice, and analysed along with other samples by denaturing electrophoreses using a 6% sequencing-type gel [acrylamide : bis -acrylamide (29:1), 1× TBE, 7 M urea]. .. Specific E. coli transcripts were probed using complementary oligonucleotides (see ) labelled at their 5′ ends with 32 P using T4 polynucleotide kinase (Thermo Scientific) and γ-32 P-ATP (3000 Ci mmol−1 , 10 mCi ml−1 , 250 μCi, Perkin Elmer).

    Article Title: Reconstitution of a Staphylococcal Plasmid-Protein Relaxation Complex In Vitro
    Article Snippet: Then, 75 fmol of DNA was treated with 12.5 U of T4 polynucleotide kinase (Invitrogen) in a phosphate exchange reaction for 14 min at 37°C in the presence of [γ-33 P]ATP (15 μCi) in a final volume of 7 μl. .. Then, 75 fmol of DNA was treated with 12.5 U of T4 polynucleotide kinase (Invitrogen) in a phosphate exchange reaction for 14 min at 37°C in the presence of [γ-33 P]ATP (15 μCi) in a final volume of 7 μl.

    Article Title: m6A-dependent regulation of messenger RNA stability
    Article Snippet: The RNA probe was synthesized by a previously reported method with the sequence of 5’-AUGGGCCGUUCAUCUGCUAAAAGGXCUGCUUUUGGGGCUUGU-3’(X = A or m6 A). .. After the synthesis, the RNA probe was labeled in a reaction mixture of 2 µL RNA probe (1 µM), 5 µL 5×T4 PNK buffer A (Fermentas), 1 µL T4 PNK (Fermentas), 1 µL P-ATP and 41 µL RNase-free water (final RNA concentration 40 nM) at 37°C for 1 hour.

    Electrophoretic Mobility Shift Assay:

    Article Title: m6A-dependent regulation of messenger RNA stability
    Article Snippet: Paragraph title: EMSA (Electrophoretic Mobility Shift Assay / Gel shift assay) ... After the synthesis, the RNA probe was labeled in a reaction mixture of 2 µL RNA probe (1 µM), 5 µL 5×T4 PNK buffer A (Fermentas), 1 µL T4 PNK (Fermentas), 1 µL P-ATP and 41 µL RNase-free water (final RNA concentration 40 nM) at 37°C for 1 hour.

    Negative Control:

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response
    Article Snippet: For site-specific m6 A detection, DNA primers were firstly 5′ labeled with 32 P using T4 polynucleotide kinase (Invitrogen) and purified by ethanol precipitation. .. For site-specific m6 A detection, DNA primers were firstly 5′ labeled with 32 P using T4 polynucleotide kinase (Invitrogen) and purified by ethanol precipitation.

    Binding Assay:

    Article Title: m6A-dependent regulation of messenger RNA stability
    Article Snippet: After the synthesis, the RNA probe was labeled in a reaction mixture of 2 µL RNA probe (1 µM), 5 µL 5×T4 PNK buffer A (Fermentas), 1 µL T4 PNK (Fermentas), 1 µL P-ATP and 41 µL RNase-free water (final RNA concentration 40 nM) at 37°C for 1 hour. .. After the synthesis, the RNA probe was labeled in a reaction mixture of 2 µL RNA probe (1 µM), 5 µL 5×T4 PNK buffer A (Fermentas), 1 µL T4 PNK (Fermentas), 1 µL P-ATP and 41 µL RNase-free water (final RNA concentration 40 nM) at 37°C for 1 hour.

    Agarose Gel Electrophoresis:

    Article Title: The human Obg protein GTPBP10 is involved in mitoribosomal biogenesis
    Article Snippet: RNA separation on a denaturing formaldehyde/formamide 1.2% agarose gel was performed as previously described ( ). .. [32 P]-radiolabeled probes were generated utilizing T4 Polynucleotide Kinase (Thermo Scientific) according to the manufacturer's instructions.

    Ethanol Precipitation:

    Article Title: DNA-guided DNA interference by a prokaryotic Argonaute
    Article Snippet: Nucleic acids were separated from protein content using Roti phenol/chloroform/isoamyl alcohol pH 7.5–8.0 (Carl Roth GmbH) and further purified by ethanol precipitation. .. Purified nucleic acids were [γ-32 P]ATP labelled with T4 PNK (Fermentas) in exchange- or forward-labelling reactions and thereafter separated from free [γ-32 P] ATP using a Sephadex G-25 column (GE).

    Article Title: A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA
    Article Snippet: The 5' phosphate of the exposed cap adjacent nucleotide was removed by the addition of 10 units of Alkaline Phosphatase (Fermentas) and incubation for a further 15 minutes at 37 °C. .. After phenol-chloroform extraction and ethanol precipitation, RNA samples were resuspended in 20 µl of sterile distilled water and 5' ends were labelled using 30 units T4 polynucleotide kinase (PNK, Fermentas) and 7.4 MBq [γ-32 P] ATP at 37 °C for 30 minutes. .. The PNK was heat inactivated (70 °C for 15 min) and the reaction made up to 60 µl with sterile distilled water then passed through a P-30 spin column (Bio-Rad) to remove unincorporated isotope.

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response
    Article Snippet: Luciferase activity was monitored and recorded using Kronos Dio Luminometer (Atto). .. For site-specific m6 A detection, DNA primers were firstly 5′ labeled with 32 P using T4 polynucleotide kinase (Invitrogen) and purified by ethanol precipitation. .. The primer 5′- AGGGATGCTCTGGGGAAGGCTGG-3′ was used to detect potential m6 A site and the primer 5′-CGCCGCTCGCTCTGCTTCTCTTGTCTTCGCT-3′ was used to detect the non-methylated site.

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: To later remove the template/splint (DNA5/6) after either reaction, the 5′-terminus was phosphorylated by T4 PNK [100 μM DNA, 1x T4 DNA Ligase buffer (Thermo Scientific; 40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP, pH 7.8 at 25°C), 0.5 U/μl T4 PNK], so that 5′-monophosphate-specific λ-exonuclease could be used for digestion of DNA5/6. .. Primer extension was performed by performing 5 cycles of a polymerase chain reaction (PCR)-like reaction [5 μM tailed DNA2, 5 μM blocked and phosphorylated DNA5/6, 3 mM MgCl2 , 0.5 mM dNTPs and ∼0.25 U/μl lab-prepared Taq Polymerase in 1x Taq polymerase buffer (Rapidozym); denaturation: 94°C, 1 min, annealing: 60°C, 1 min; extension: 72°C, 1 min].

    Ancient DNA Assay:

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA
    Article Snippet: See Supplementary Table S2 for sample information. .. For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP. .. Reactions were purified using the MinElute PCR Purification kit.

    Concentration Assay:

    Article Title: DNA-guided DNA interference by a prokaryotic Argonaute
    Article Snippet: Proteinase K (Ambion) and CaCl2 (final concentration, 5 mM) were added to purified proteins and samples were incubated for 1 h at 37 °C. .. Purified nucleic acids were [γ-32 P]ATP labelled with T4 PNK (Fermentas) in exchange- or forward-labelling reactions and thereafter separated from free [γ-32 P] ATP using a Sephadex G-25 column (GE).

    Article Title: A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA
    Article Snippet: After phenol-chloroform extraction and ethanol precipitation, RNA samples were resuspended in 20 µl of sterile distilled water and 5' ends were labelled using 30 units T4 polynucleotide kinase (PNK, Fermentas) and 7.4 MBq [γ-32 P] ATP at 37 °C for 30 minutes. .. 1.5 μl of the released 5' monophosphates from this digest was then analysed by 2D TLC as described previously .

    Article Title: Conversion of Topoisomerase I Cleavage Complexes on the Leading Strand of Ribosomal DNA into 5?-Phosphorylated DNA Double-Strand Breaks by Replication Runoff
    Article Snippet: T4 polynucleotide kinase (10 U/μl) was purchased from Gibco-BRL, Life Technologies (Gaithersburg, Md.), and aphidicolin was obtained from Sigma Chemical Co. (St. Louis, Mo.). .. T4 polynucleotide kinase (10 U/μl) was purchased from Gibco-BRL, Life Technologies (Gaithersburg, Md.), and aphidicolin was obtained from Sigma Chemical Co. (St. Louis, Mo.).

    Article Title: m6A-dependent regulation of messenger RNA stability
    Article Snippet: The RNA probe was synthesized by a previously reported method with the sequence of 5’-AUGGGCCGUUCAUCUGCUAAAAGGXCUGCUUUUGGGGCUUGU-3’(X = A or m6 A). .. After the synthesis, the RNA probe was labeled in a reaction mixture of 2 µL RNA probe (1 µM), 5 µL 5×T4 PNK buffer A (Fermentas), 1 µL T4 PNK (Fermentas), 1 µL P-ATP and 41 µL RNase-free water (final RNA concentration 40 nM) at 37°C for 1 hour. .. The mixture was then purified by RNase-free micro bio-spin columns with bio-gel P30 in Tris buffer (BioRad 732–6250) to remove hot ATP and other small molecules.

    Article Title: RNA modification landscape of the human mitochondrial tRNALys regulates protein synthesis
    Article Snippet: Next, 3′-phosphate removal was performed on both demethylase-treated and -untreated small RNA. .. Small RNA was treated with T4 PNK (Affymetrix, final concentration 0.1U/μL) in 1× T4 PNK buffer at 37 °C for 30 min. .. The RNA Clean and Concentrator (Zymo) was again used to clean up the reaction.

    Thin Layer Chromatography:

    Article Title: A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA
    Article Snippet: After phenol-chloroform extraction and ethanol precipitation, RNA samples were resuspended in 20 µl of sterile distilled water and 5' ends were labelled using 30 units T4 polynucleotide kinase (PNK, Fermentas) and 7.4 MBq [γ-32 P] ATP at 37 °C for 30 minutes. .. After phenol-chloroform extraction and ethanol precipitation, RNA samples were resuspended in 20 µl of sterile distilled water and 5' ends were labelled using 30 units T4 polynucleotide kinase (PNK, Fermentas) and 7.4 MBq [γ-32 P] ATP at 37 °C for 30 minutes.

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    Thermo Fisher t4 polynucleotide kinase
    Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using <t>T4</t> polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).
    T4 Polynucleotide Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 515 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using T4 polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).

    Journal: Nature

    Article Title: Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate

    doi: 10.1038/s41586-018-0557-5

    Figure Lengend Snippet: Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using T4 polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).

    Article Snippet: For use as standards, A2 > P and A4 > P linear oligoadenylates were 5’-end labelled using 32 P-γ-ATP and T4 Polynucleotide Kinase (PNK; Thermo Fisher Scientific) via its forward reaction.

    Techniques: Thin Layer Chromatography, Generated, Incubation

    Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the  rrnE  operon, which is representative of all seven rRNA operons in  E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF  strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).

    Journal: Molecular Microbiology

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing

    doi: 10.1111/mmi.12810

    Figure Lengend Snippet: Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the rrnE operon, which is representative of all seven rRNA operons in E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).

    Article Snippet: Specific E. coli transcripts were probed using complementary oligonucleotides (see ) labelled at their 5′ ends with 32 P using T4 polynucleotide kinase (Thermo Scientific) and γ-32 P-ATP (3000 Ci mmol−1 , 10 mCi ml−1 , 250 μCi, Perkin Elmer).

    Techniques: RNA Sequencing Assay, Sequencing, Amplification, Produced, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Isolation, Staining, Incubation, Nucleic Acid Electrophoresis