t4 polynucleotide kinase pnk buffer  (New England Biolabs)


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    Name:
    T4 Polynucleotide Kinase - 2
    Description:

    Catalog Number:
    M0201L
    Price:
    None
    Score:
    85
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    New England Biolabs t4 polynucleotide kinase pnk buffer

    https://www.bioz.com/result/t4 polynucleotide kinase pnk buffer/product/New England Biolabs
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    t4 polynucleotide kinase pnk buffer - by Bioz Stars, 2020-01
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    Related Articles

    Transduction:

    Article Title: G-quadruplex recognition activities of E. Coli MutS
    Article Snippet: End labeling used T4 PNK (New England Biolabs (NEB), Ipswich, MA) and 32 PγATP (from either MP biomedicals or Perkin Elmer). .. End labeling used T4 PNK (New England Biolabs (NEB), Ipswich, MA) and 32 PγATP (from either MP biomedicals or Perkin Elmer).

    Centrifugation:

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: After precipitation, RNAs were pelleted by centrifugation and resuspended in the 5' end labeling reaction consisting of 10 units of T4 PNK, 70 mM Tris-HCl pH 7.6, 10 mM MgCl2 , 5 mM DTT and 0.03 μCi of [γ-32 P] ATP (3000 Ci/mmol). .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Amplification:

    Article Title: Dissecting neural differentiation regulatory networks through epigenetic footprinting
    Article Snippet: Fragments smaller than 200 bps were eliminated (Zymo, R1016) and the remaining fraction was treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L). .. Fragments smaller than 200 bps were eliminated (Zymo, R1016) and the remaining fraction was treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L).

    Filtration:

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element
    Article Snippet: Transcripts were purified using organic extraction followed by gel filtration using Micro Bio-Spin 30 Columns (BioRad). .. Dephosphorylated transcripts were end-labeled in the presence of [γ32 P] ATP (3000 Ci/mmole; Perkin Elmer Easy Tides) and T4 PNK (NEB) at 20 units/pmole RNA.

    Binding Assay:

    Article Title: Genetic and Molecular Functional Characterization of Variants within TNFSF13B, a Positional Candidate Preeclampsia Susceptibility Gene on 13q
    Article Snippet: All oligonucleotides were 5′ end-labeled using T4 polynucleotide kinase (PNK) (New England Biolabs) and [γ33P] ATP (3000 Ci/mmol) (PerkinElmer) and annealed to their complementary unlabeled oligonucleotides as previously described . .. The samples were purified according to manufacturers' instructions through G25 Microspin™ columns (GE Healthcare).

    Stable Transfection:

    Article Title: The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity
    Article Snippet: Genomic DNA was isolated from confluent 10 cm culture dishes of the original HeLa-EM2-11ht cells and FLAG-TPP1 expressing stable cell lines using the GenElute kit (Sigma). .. A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel.

    Synthesized:

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element
    Article Snippet: For UV cross-linking and REMSA analyses, RNAs were synthesized in the presence of [α32 P] UTP (800 Ci/mmole; Perkin Elmer Easy Tides) using the RiboMAX™ Large Scale RNA Production Systems (T7) (Promega). .. Dephosphorylated transcripts were end-labeled in the presence of [γ32 P] ATP (3000 Ci/mmole; Perkin Elmer Easy Tides) and T4 PNK (NEB) at 20 units/pmole RNA.

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: Fluorescently-labeled U4 and U6 were made via ligation of an in vitro transcribed RNA to a synthesized fluorescently labeled RNA (IDT) using T4 RNA ligase 2. .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Autoradiography:

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. The reaction volume of 10 μl containing the 32 P-labeled helicase substrate (1000 cpm/10 μl) in appropriate buffer (20 mMTris-HCl, pH 8.0, 8 mM DTT, 1.0 mM MgCl2 , 20 mMKCl and 16 μg/ml BSA) and PfUDN was incubated at 37°C for 60 min.

    Construct:

    Article Title: A highly conserved Tyrosine residue of family B DNA polymerases contributes to dictate translesion synthesis past 8-oxo-7,8-dihydro-2?-deoxyguanosine
    Article Snippet: Hybridizations were performed in the presence of 0.2 M NaCl and 50 mM Tris-HCl (pH 7.5), resulting in primer/template structures. .. Phage T4 polynucleotide kinase was obtained from New England Biolabs. ϕ29 DNA polymerase variants at Tyr390 residue Y390F and Y390S were constructed by J. Saturno in an exonuclease deficient background (D12A/D66A) (hereafter PolY390F Exo− and PolY390S Exo− , respectively (unpublished data) and further purified from Escherichia coli BL21(DE3) cells harbouring the corresponding recombinant plasmid ( ). .. The hybrid molecules Pber-2/T4 and Pber-2/8oxodG, containing the unmodified dG and an 8oxodG lesion at the +3 position of the template, respectively, can be used both as substrate for the 3′–5′ exonuclease activity and for DNA-dependent DNA polymerization.

    Electrophoresis:

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. The reaction volume of 10 μl containing the 32 P-labeled helicase substrate (1000 cpm/10 μl) in appropriate buffer (20 mMTris-HCl, pH 8.0, 8 mM DTT, 1.0 mM MgCl2 , 20 mMKCl and 16 μg/ml BSA) and PfUDN was incubated at 37°C for 60 min.

    Microarray:

    Article Title: Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template
    Article Snippet: The surface of the microarray slide, containing surface attached primers, was conditioned by incubation with 10 μl of 1 × T4 ligase buffer (50 mM Tris–HCl, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 25 ug/ml BSA, pH 7.5, New England Biolabs, Beverly, MA, USA) at 37°C for 1 h with shaking at 300 rpm on a slide incubator (Thermomixer Comfort, Eppendorf, Hamburg, Germany). .. A 10 μl ligation mix containing the following was prepared: 1 × T4 ligase buffer, 20 pmol β-globin template, 1 μl of 50% PEG 4000 (Fermentas, Vilnius, Lithuania), 1 μl (400 U = 6 Weiss U) of T4 DNA ligase (NEB), 1 μl (10 U) of T4 PNK kinase (NEB) and 50 pmol of 10mer degenerate ON library for each of the four colors (Cy3, Cy5, Fluorescein, Alexa594) ( Supplementary Table 1 ).

    Incubation:

    Article Title: Unconventional miR-122 binding stabilizes the HCV genome by forming a trimolecular RNA structure
    Article Snippet: MiR-122 RNAs (IDT) were 5′32 P-labelled using [γ-32 P]ATP (PerkinElmer) and T4 PNK (NEB) [10 µL of reaction; 1× T4 PNK buffer, 50 pmol miR-122 RNA, 50 µCi [γ-32 P]ATP, 10 U T4 PNK] at 37°C for 1 h and subsequently diluted with nuclease-free water (40 µL). .. MiR-122 RNAs (IDT) were 5′32 P-labelled using [γ-32 P]ATP (PerkinElmer) and T4 PNK (NEB) [10 µL of reaction; 1× T4 PNK buffer, 50 pmol miR-122 RNA, 50 µCi [γ-32 P]ATP, 10 U T4 PNK] at 37°C for 1 h and subsequently diluted with nuclease-free water (40 µL).

    Article Title: RNAi triggered by specialized machinery silences developmental genes and retrotransposons
    Article Snippet: Adapters were in molar excess of RNA, and reactions contained 200 units of T4 Rnl2 truncated enzyme and 20 units of murine RNase inhibitor in a total volume of 20 μl. .. 10μl of the 3′-ligated RNA was phosphorylated by adding 3 μl of water, 0.5 μl of T4 Rnl1 buffer and 0.5 μl (20 U) of murine RNase inhibitor, and 1 μl (10 units) of T4 PNK (NEB) followed by incubation at 37°C for 30 min. .. Final buffer composition for this reaction was 50 mM Tris pH 7.5, 10 mM MgCl2 , 1mM DTT, 8.25% PEG, 0.33 mM ATP in a 15 μl reaction volume.

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: RNAse I (200 U/110 OD, Ambion) was added and the mixture was incubated at 23°C for 1 h. The remaining extract was used for total RNA extraction for subsequent total RNA sequencing. .. RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB).

    Article Title: Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions
    Article Snippet: RNA was fragmented by chemical hydrolysis; heating to 95°C, 10 min in 1× RNase III buffer (AM2290, Life Technologies), and snap cooled on ice. .. ATP (0.83 mM, 11140965001, Roche) and 10 U of T4 PNK (M0201L, NEB) were added and incubated at 37°C for 30 min. RNA was purified using Purelink RNA Micro Kit (12183-016, Life Technologies). .. Equimolar pools of RNA-seq libraries were made following quantitative PCR quantification using a Kapa Library Quantification kit (KK4823, Kapa Biosystems).

    Article Title: Genetic and Molecular Functional Characterization of Variants within TNFSF13B, a Positional Candidate Preeclampsia Susceptibility Gene on 13q
    Article Snippet: All oligonucleotides were 5′ end-labeled using T4 polynucleotide kinase (PNK) (New England Biolabs) and [γ33P] ATP (3000 Ci/mmol) (PerkinElmer) and annealed to their complementary unlabeled oligonucleotides as previously described . .. The EMSA reactions were carried out in binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris–HCl (pH 7.5), 0.25 mg/ml poly(dI-dC)) in a final volume of 10 µl.

    Article Title: Dissecting domains necessary for activation and repression of splicing by muscleblind-like protein 1
    Article Snippet: For the PCR reaction, the 3’ primer was 5’ end labeled with [32 P]-ATP,. .. Oligo primer (4 pmoles per PCR reaction) was incubated at 37°C for 60 minutes in 50 mM Tris 10 mM MgCl2 T4 polynucleotide kinase enzyme (NEB) and 0.1 μl [α-32 P]-UTP per PCR reaction. .. After incubation the solution was phenol extracted, and purified on a G-50 spin column (GE Healthcare).

    Article Title: Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template
    Article Snippet: The surface of the microarray slide, containing surface attached primers, was conditioned by incubation with 10 μl of 1 × T4 ligase buffer (50 mM Tris–HCl, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 25 ug/ml BSA, pH 7.5, New England Biolabs, Beverly, MA, USA) at 37°C for 1 h with shaking at 300 rpm on a slide incubator (Thermomixer Comfort, Eppendorf, Hamburg, Germany). .. A 10 μl ligation mix containing the following was prepared: 1 × T4 ligase buffer, 20 pmol β-globin template, 1 μl of 50% PEG 4000 (Fermentas, Vilnius, Lithuania), 1 μl (400 U = 6 Weiss U) of T4 DNA ligase (NEB), 1 μl (10 U) of T4 PNK kinase (NEB) and 50 pmol of 10mer degenerate ON library for each of the four colors (Cy3, Cy5, Fluorescein, Alexa594) ( Supplementary Table 1 ).

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. Using gel filtration through a Sepharose 4B column (Pharmacia, Sweden) the non-hybridized oligodeoxynucleotide was removed [ ].

    Article Title: A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome
    Article Snippet: The RNA was eluted from the beads in 100 μl of low TE. .. 3′ phosphates were removed from the RNA by addition 8.2 μl of 10× T4 polynucleotide buffer to 75 μl of the RNA solution and 4 μl of ATP-free T4 polynucleotide kinase (NEB) was added and incubated for 15 min. Hydrophilic streptavidin magnetic beads (NEB) were prepared by washing 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 50 mM NaCl, 1 mM EDTA and 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 500 mM NaCl, 1 mM EDTA and suspended in their original suspension concentration of 4 mg/ml in wash buffer A. .. 50 μl of the kinase treated RNA was added to 30 μl of the prewashed streptavidin beads at room temperature with occasional resuspension for 20 min.

    Diffusion-based Assay:

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: RNAs were extracted from the gel by passive diffusion into 300 mM sodium acetate pH 5.2 and ethanol precipitated. .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Activity Assay:

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: This oligodeoxynucleotide of the nucleotide sequence 5'- (T)15 GTTTTCCCAGTCACGAC(T)15 -3' contains 15 base-pairs of non-complementary region (T)15 at both the 5’ and 3’ ends. .. Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. Using gel filtration through a Sepharose 4B column (Pharmacia, Sweden) the non-hybridized oligodeoxynucleotide was removed [ ].

    Expressing:

    Article Title: The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity
    Article Snippet: Genomic DNA was isolated from confluent 10 cm culture dishes of the original HeLa-EM2-11ht cells and FLAG-TPP1 expressing stable cell lines using the GenElute kit (Sigma). .. A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel.

    Modification:

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: Fluorescent U4 was created by ligation of a 5′-Cy5 labeled RNA consisting of U4 nucleotides 1–13 to an in vitro transcribed U4 containing nucleotides 14–160 (modified from the pUC118 plasmid described previously). .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB). .. U6(13–112) was modified using only T4 PNK due to the presence of a 5'-OH group in the original transcript.

    Gel Purification:

    Article Title: RNAi triggered by specialized machinery silences developmental genes and retrotransposons
    Article Snippet: Small RNA fractions < 40 nt were prepared by gel purification from denaturing acrylamide gels, or by using flashPAGE™ fractionation (Life Technologies) according to the manufacturer’s recommendation . .. 10μl of the 3′-ligated RNA was phosphorylated by adding 3 μl of water, 0.5 μl of T4 Rnl1 buffer and 0.5 μl (20 U) of murine RNase inhibitor, and 1 μl (10 units) of T4 PNK (NEB) followed by incubation at 37°C for 30 min.

    Transfection:

    Article Title: Dissecting domains necessary for activation and repression of splicing by muscleblind-like protein 1
    Article Snippet: Paragraph title: RNA and protein analysis of transfections ... Oligo primer (4 pmoles per PCR reaction) was incubated at 37°C for 60 minutes in 50 mM Tris 10 mM MgCl2 T4 polynucleotide kinase enzyme (NEB) and 0.1 μl [α-32 P]-UTP per PCR reaction.

    Ligation:

    Article Title: RNAi triggered by specialized machinery silences developmental genes and retrotransposons
    Article Snippet: 10μl of the 3′-ligated RNA was phosphorylated by adding 3 μl of water, 0.5 μl of T4 Rnl1 buffer and 0.5 μl (20 U) of murine RNase inhibitor, and 1 μl (10 units) of T4 PNK (NEB) followed by incubation at 37°C for 30 min. .. 10μl of the 3′-ligated RNA was phosphorylated by adding 3 μl of water, 0.5 μl of T4 Rnl1 buffer and 0.5 μl (20 U) of murine RNase inhibitor, and 1 μl (10 units) of T4 PNK (NEB) followed by incubation at 37°C for 30 min.

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: The library was then prepared using the TruSeq Small RNA kit (Illumina), whereby the RNA was precipitated for at least 4 h in between each of the following steps. .. RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB). .. The 5′ end was then re-phosphorylated using T4 PNK (NEB) supplied with ATP, followed by 5′ adapter ligation, cDNA synthesis and PCR.

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: Fluorescent U4 was created by ligation of a 5′-Cy5 labeled RNA consisting of U4 nucleotides 1–13 to an in vitro transcribed U4 containing nucleotides 14–160 (modified from the pUC118 plasmid described previously). .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Article Title: Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template
    Article Snippet: The surface of the microarray slide, containing surface attached primers, was conditioned by incubation with 10 μl of 1 × T4 ligase buffer (50 mM Tris–HCl, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 25 ug/ml BSA, pH 7.5, New England Biolabs, Beverly, MA, USA) at 37°C for 1 h with shaking at 300 rpm on a slide incubator (Thermomixer Comfort, Eppendorf, Hamburg, Germany). .. A 10 μl ligation mix containing the following was prepared: 1 × T4 ligase buffer, 20 pmol β-globin template, 1 μl of 50% PEG 4000 (Fermentas, Vilnius, Lithuania), 1 μl (400 U = 6 Weiss U) of T4 DNA ligase (NEB), 1 μl (10 U) of T4 PNK kinase (NEB) and 50 pmol of 10mer degenerate ON library for each of the four colors (Cy3, Cy5, Fluorescein, Alexa594) ( Supplementary Table 1 ). .. The mix was placed on the slide under a coverslip and incubated at 46°C for 1 h with acoustic wave mixing (ArrayBooster, Implen, Munich, Germany).

    Footprinting:

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element
    Article Snippet: For RNase footprinting experiments, cold synthetic transcripts were dephosphorylated with SuperSAP (Affymetrix), purified, and resuspended in nuclease-free water. .. Dephosphorylated transcripts were end-labeled in the presence of [γ32 P] ATP (3000 Ci/mmole; Perkin Elmer Easy Tides) and T4 PNK (NEB) at 20 units/pmole RNA.

    Hemagglutination Assay:

    Article Title: Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3'-Terminal Fragment of 16S rRNA in E. coli
    Article Snippet: It can be hypothesized that the structure of the binary rRNA–protein complex that is formed during the initial stages of the small ribosomal subunit assembly must undergo rearrangement during the formation of an intact subunit. .. T4 polynucleotide kinase (PNK) and PNK buffer (New England Biolabs, USA), reverse transcriptase of the avian myeloblastosis virus (RT-AMV), Taq DNA polymerase, RNase inhibitor, proteinase K, nucleoside triphosphate and its dideoxy derivatives (Roche, Germany), [γ- 32 Р]АТР (Amersham, Germany), bovine serum albumin (BSА, MBI, Fermentas, Lithuania), 0.45 µm nitrocellulose filters (Millipore HA, USA;Schleicher & Schuell BA85, Germany), Ni-NTA-agarose (QIAGEN, Germany), phenylmethylsulfonyl fluoride (PMSF, Merk, Germany) were used. .. The рFD3LH plasmid was kindly provided by L. Brakier-Gingras (University of Montreal, Canada).

    other:

    Article Title: Involvement of the TPR2 subdomain movement in the activities of ?29 DNA polymerase
    Article Snippet: Phage T4 polynucleotide kinase was obtained from New England Biolabs.

    DNA Sequencing:

    Article Title: G-quadruplex recognition activities of E. Coli MutS
    Article Snippet: End labeling used T4 PNK (New England Biolabs (NEB), Ipswich, MA) and 32 PγATP (from either MP biomedicals or Perkin Elmer). .. The mutS:Tn10 allele from FC1124 (provided by Dr. Pat Foster, Indiana University, Bloomington, IN) was transferred to NM522 (NEB) by P1 transduction to create JW1 then tested for mutator phenotype by nalidixic acid screening.

    Polymerase Chain Reaction:

    Article Title: G-quadruplex recognition activities of E. Coli MutS
    Article Snippet: End labeling used T4 PNK (New England Biolabs (NEB), Ipswich, MA) and 32 PγATP (from either MP biomedicals or Perkin Elmer). .. The mutS:Tn10 allele from FC1124 (provided by Dr. Pat Foster, Indiana University, Bloomington, IN) was transferred to NM522 (NEB) by P1 transduction to create JW1 then tested for mutator phenotype by nalidixic acid screening.

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB). .. The 5′ end was then re-phosphorylated using T4 PNK (NEB) supplied with ATP, followed by 5′ adapter ligation, cDNA synthesis and PCR.

    Article Title: Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions
    Article Snippet: ATP (0.83 mM, 11140965001, Roche) and 10 U of T4 PNK (M0201L, NEB) were added and incubated at 37°C for 30 min. RNA was purified using Purelink RNA Micro Kit (12183-016, Life Technologies). .. Equimolar pools of RNA-seq libraries were made following quantitative PCR quantification using a Kapa Library Quantification kit (KK4823, Kapa Biosystems).

    Article Title: Dissecting neural differentiation regulatory networks through epigenetic footprinting
    Article Snippet: Fragments smaller than 200 bps were eliminated (Zymo, R1016) and the remaining fraction was treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L). .. Fragments smaller than 200 bps were eliminated (Zymo, R1016) and the remaining fraction was treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L).

    Article Title: Dissecting domains necessary for activation and repression of splicing by muscleblind-like protein 1
    Article Snippet: For the PCR reaction, the 3’ primer was 5’ end labeled with [32 P]-ATP,. .. Oligo primer (4 pmoles per PCR reaction) was incubated at 37°C for 60 minutes in 50 mM Tris 10 mM MgCl2 T4 polynucleotide kinase enzyme (NEB) and 0.1 μl [α-32 P]-UTP per PCR reaction. .. After incubation the solution was phenol extracted, and purified on a G-50 spin column (GE Healthcare).

    Hybridization:

    Article Title: The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity
    Article Snippet: A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel. .. A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel.

    Nucleic Acid Electrophoresis:

    Article Title: Unconventional miR-122 binding stabilizes the HCV genome by forming a trimolecular RNA structure
    Article Snippet: MiR-122 RNAs (IDT) were 5′32 P-labelled using [γ-32 P]ATP (PerkinElmer) and T4 PNK (NEB) [10 µL of reaction; 1× T4 PNK buffer, 50 pmol miR-122 RNA, 50 µCi [γ-32 P]ATP, 10 U T4 PNK] at 37°C for 1 h and subsequently diluted with nuclease-free water (40 µL). .. The pre-folded s1 and s2 RNAs were serial diluted by 2-fold in folding buffer to obtain desired concentration range before incubation with ∼3000 cpm/µL (∼0.5–1 pmol) in a 5 µL of reaction for 20–30 min at 37°C.

    RNA Sequencing Assay:

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: RNAse I (200 U/110 OD, Ambion) was added and the mixture was incubated at 23°C for 1 h. The remaining extract was used for total RNA extraction for subsequent total RNA sequencing. .. RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB).

    Article Title: Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions
    Article Snippet: Paragraph title: RNA-Seq ... ATP (0.83 mM, 11140965001, Roche) and 10 U of T4 PNK (M0201L, NEB) were added and incubated at 37°C for 30 min. RNA was purified using Purelink RNA Micro Kit (12183-016, Life Technologies).

    Article Title: Dissecting neural differentiation regulatory networks through epigenetic footprinting
    Article Snippet: Paragraph title: Strand Specific RNA-Sequencing Library Construction ... Fragments smaller than 200 bps were eliminated (Zymo, R1016) and the remaining fraction was treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L).

    Magnetic Beads:

    Article Title: A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome
    Article Snippet: The RNA was eluted from the beads in 100 μl of low TE. .. 3′ phosphates were removed from the RNA by addition 8.2 μl of 10× T4 polynucleotide buffer to 75 μl of the RNA solution and 4 μl of ATP-free T4 polynucleotide kinase (NEB) was added and incubated for 15 min. Hydrophilic streptavidin magnetic beads (NEB) were prepared by washing 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 50 mM NaCl, 1 mM EDTA and 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 500 mM NaCl, 1 mM EDTA and suspended in their original suspension concentration of 4 mg/ml in wash buffer A. .. 50 μl of the kinase treated RNA was added to 30 μl of the prewashed streptavidin beads at room temperature with occasional resuspension for 20 min.

    Isolation:

    Article Title: The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity
    Article Snippet: Genomic DNA was isolated from confluent 10 cm culture dishes of the original HeLa-EM2-11ht cells and FLAG-TPP1 expressing stable cell lines using the GenElute kit (Sigma). .. A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel.

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: Ribosome-protected fragments (RPFs) were then isolated as described above for total RNA extraction. .. RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB).

    Article Title: Dissecting neural differentiation regulatory networks through epigenetic footprinting
    Article Snippet: Poly(A) RNA was isolated using Oligo d (T25 ) beads (NEB, E7490L). .. Fragments smaller than 200 bps were eliminated (Zymo, R1016) and the remaining fraction was treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L).

    Transferring:

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: This oligodeoxynucleotide of the nucleotide sequence 5'- (T)15 GTTTTCCCAGTCACGAC(T)15 -3' contains 15 base-pairs of non-complementary region (T)15 at both the 5’ and 3’ ends. .. Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. Using gel filtration through a Sepharose 4B column (Pharmacia, Sweden) the non-hybridized oligodeoxynucleotide was removed [ ].

    Electrophoretic Mobility Shift Assay:

    Article Title: Unconventional miR-122 binding stabilizes the HCV genome by forming a trimolecular RNA structure
    Article Snippet: Paragraph title: Electrophoretic mobility shift assays with HCV s1 and s2 RNAs ... MiR-122 RNAs (IDT) were 5′32 P-labelled using [γ-32 P]ATP (PerkinElmer) and T4 PNK (NEB) [10 µL of reaction; 1× T4 PNK buffer, 50 pmol miR-122 RNA, 50 µCi [γ-32 P]ATP, 10 U T4 PNK] at 37°C for 1 h and subsequently diluted with nuclease-free water (40 µL).

    Article Title: Genetic and Molecular Functional Characterization of Variants within TNFSF13B, a Positional Candidate Preeclampsia Susceptibility Gene on 13q
    Article Snippet: Paragraph title: Electrophoretic Mobility Shift Assays (EMSA) ... All oligonucleotides were 5′ end-labeled using T4 polynucleotide kinase (PNK) (New England Biolabs) and [γ33P] ATP (3000 Ci/mmol) (PerkinElmer) and annealed to their complementary unlabeled oligonucleotides as previously described .

    Purification:

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element
    Article Snippet: For RNase footprinting experiments, cold synthetic transcripts were dephosphorylated with SuperSAP (Affymetrix), purified, and resuspended in nuclease-free water. .. Dephosphorylated transcripts were end-labeled in the presence of [γ32 P] ATP (3000 Ci/mmole; Perkin Elmer Easy Tides) and T4 PNK (NEB) at 20 units/pmole RNA.

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB). .. U6(13–112) was modified using only T4 PNK due to the presence of a 5'-OH group in the original transcript.

    Article Title: Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions
    Article Snippet: RNA was fragmented by chemical hydrolysis; heating to 95°C, 10 min in 1× RNase III buffer (AM2290, Life Technologies), and snap cooled on ice. .. ATP (0.83 mM, 11140965001, Roche) and 10 U of T4 PNK (M0201L, NEB) were added and incubated at 37°C for 30 min. RNA was purified using Purelink RNA Micro Kit (12183-016, Life Technologies). .. Equimolar pools of RNA-seq libraries were made following quantitative PCR quantification using a Kapa Library Quantification kit (KK4823, Kapa Biosystems).

    Article Title: The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach
    Article Snippet: The ChIP DNA and the Input DNA ends were repaired using T4 DNA polymerase, Klenow enzyme, and T4 polynucleotide kinase (PNK) (New England Biolabs), followed by treatment with Klenow exo- to add an A base to the 3' end. .. The ChIP DNA and the Input DNA ends were repaired using T4 DNA polymerase, Klenow enzyme, and T4 polynucleotide kinase (PNK) (New England Biolabs), followed by treatment with Klenow exo- to add an A base to the 3' end.

    Article Title: Dissecting domains necessary for activation and repression of splicing by muscleblind-like protein 1
    Article Snippet: For RNA analysis, cells in 6 well plates were washed using twice with 2 ml PBS, then to each well 1 ml tri-reagent (Sigma-Aldrich) was added, and purified according to the manufacturers’ protocols. .. Oligo primer (4 pmoles per PCR reaction) was incubated at 37°C for 60 minutes in 50 mM Tris 10 mM MgCl2 T4 polynucleotide kinase enzyme (NEB) and 0.1 μl [α-32 P]-UTP per PCR reaction.

    Article Title: A highly conserved Tyrosine residue of family B DNA polymerases contributes to dictate translesion synthesis past 8-oxo-7,8-dihydro-2?-deoxyguanosine
    Article Snippet: Hybridizations were performed in the presence of 0.2 M NaCl and 50 mM Tris-HCl (pH 7.5), resulting in primer/template structures. .. Phage T4 polynucleotide kinase was obtained from New England Biolabs. ϕ29 DNA polymerase variants at Tyr390 residue Y390F and Y390S were constructed by J. Saturno in an exonuclease deficient background (D12A/D66A) (hereafter PolY390F Exo− and PolY390S Exo− , respectively (unpublished data) and further purified from Escherichia coli BL21(DE3) cells harbouring the corresponding recombinant plasmid ( ). .. The hybrid molecules Pber-2/T4 and Pber-2/8oxodG, containing the unmodified dG and an 8oxodG lesion at the +3 position of the template, respectively, can be used both as substrate for the 3′–5′ exonuclease activity and for DNA-dependent DNA polymerization.

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: Helicase assay was demonstrated using the purified fraction of PfUDN. .. Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature.

    Article Title: Purification and Characterization of the RecA Protein from Neisseria gonorrhoeae
    Article Snippet: The E. coli RecA, SSB , and LexA proteins were purified as described. .. Restriction enzymes, Klenow, polynucleotide kinase (PNK), T4 DNA ligase, and M13K07 Helper Phage were purchased from New England Biolabs.

    Sequencing:

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB). .. The PCR product was then loaded on a Novex 6% TBE-Urea gel (Life Technologies) and the band around 150 bp (5′ adapter + 30 nt RPF + 3′ adapter) was excised from the gel.

    Article Title: The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach
    Article Snippet: The ChIP DNA and the Input DNA ends were repaired using T4 DNA polymerase, Klenow enzyme, and T4 polynucleotide kinase (PNK) (New England Biolabs), followed by treatment with Klenow exo- to add an A base to the 3' end. .. The samples were purified using the QIAquick MinElute kit (Qiagen) at each preparation step.

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: This oligodeoxynucleotide of the nucleotide sequence 5'- (T)15 GTTTTCCCAGTCACGAC(T)15 -3' contains 15 base-pairs of non-complementary region (T)15 at both the 5’ and 3’ ends. .. Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature.

    Recombinant:

    Article Title: A highly conserved Tyrosine residue of family B DNA polymerases contributes to dictate translesion synthesis past 8-oxo-7,8-dihydro-2?-deoxyguanosine
    Article Snippet: Hybridizations were performed in the presence of 0.2 M NaCl and 50 mM Tris-HCl (pH 7.5), resulting in primer/template structures. .. Phage T4 polynucleotide kinase was obtained from New England Biolabs. ϕ29 DNA polymerase variants at Tyr390 residue Y390F and Y390S were constructed by J. Saturno in an exonuclease deficient background (D12A/D66A) (hereafter PolY390F Exo− and PolY390S Exo− , respectively (unpublished data) and further purified from Escherichia coli BL21(DE3) cells harbouring the corresponding recombinant plasmid ( ). .. The hybrid molecules Pber-2/T4 and Pber-2/8oxodG, containing the unmodified dG and an 8oxodG lesion at the +3 position of the template, respectively, can be used both as substrate for the 3′–5′ exonuclease activity and for DNA-dependent DNA polymerization.

    Labeling:

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: Paragraph title: RNA labeling ... After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Article Title: Genetic and Molecular Functional Characterization of Variants within TNFSF13B, a Positional Candidate Preeclampsia Susceptibility Gene on 13q
    Article Snippet: All oligonucleotides were 5′ end-labeled using T4 polynucleotide kinase (PNK) (New England Biolabs) and [γ33P] ATP (3000 Ci/mmol) (PerkinElmer) and annealed to their complementary unlabeled oligonucleotides as previously described . .. The EMSA reactions were carried out in binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris–HCl (pH 7.5), 0.25 mg/ml poly(dI-dC)) in a final volume of 10 µl.

    Article Title: Dissecting domains necessary for activation and repression of splicing by muscleblind-like protein 1
    Article Snippet: For the PCR reaction, the 3’ primer was 5’ end labeled with [32 P]-ATP,. .. Oligo primer (4 pmoles per PCR reaction) was incubated at 37°C for 60 minutes in 50 mM Tris 10 mM MgCl2 T4 polynucleotide kinase enzyme (NEB) and 0.1 μl [α-32 P]-UTP per PCR reaction.

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: This oligodeoxynucleotide of the nucleotide sequence 5'- (T)15 GTTTTCCCAGTCACGAC(T)15 -3' contains 15 base-pairs of non-complementary region (T)15 at both the 5’ and 3’ ends. .. Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. Using gel filtration through a Sepharose 4B column (Pharmacia, Sweden) the non-hybridized oligodeoxynucleotide was removed [ ].

    Article Title: A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome
    Article Snippet: The desthiobiotin-GTP labeled RNA was fragmented by adding 2.5 μl of NEB 10× T4 polynucleotide kinase buffer to a 100 μl volume of capped RNA and incubated for 5 min at 94 °C. .. 3′ phosphates were removed from the RNA by addition 8.2 μl of 10× T4 polynucleotide buffer to 75 μl of the RNA solution and 4 μl of ATP-free T4 polynucleotide kinase (NEB) was added and incubated for 15 min. Hydrophilic streptavidin magnetic beads (NEB) were prepared by washing 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 50 mM NaCl, 1 mM EDTA and 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 500 mM NaCl, 1 mM EDTA and suspended in their original suspension concentration of 4 mg/ml in wash buffer A.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. The reaction volume of 10 μl containing the 32 P-labeled helicase substrate (1000 cpm/10 μl) in appropriate buffer (20 mMTris-HCl, pH 8.0, 8 mM DTT, 1.0 mM MgCl2 , 20 mMKCl and 16 μg/ml BSA) and PfUDN was incubated at 37°C for 60 min.

    Chromatin Immunoprecipitation:

    Article Title: The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach
    Article Snippet: For ChIPseq, sample preparation was performed using the ChIP protocol described above. .. The ChIP DNA and the Input DNA ends were repaired using T4 DNA polymerase, Klenow enzyme, and T4 polynucleotide kinase (PNK) (New England Biolabs), followed by treatment with Klenow exo- to add an A base to the 3' end. .. After ligation of the Solexa adaptor using TaKaRa ligation Mix (TaKaRa), the adaptor-ligated DNAs were amplified using Solexa PCR primers for 18 cycles, and the amplified library was isolated from an agarose gel.

    Plasmid Preparation:

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: Fluorescent U4 was created by ligation of a 5′-Cy5 labeled RNA consisting of U4 nucleotides 1–13 to an in vitro transcribed U4 containing nucleotides 14–160 (modified from the pUC118 plasmid described previously). .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Article Title: A highly conserved Tyrosine residue of family B DNA polymerases contributes to dictate translesion synthesis past 8-oxo-7,8-dihydro-2?-deoxyguanosine
    Article Snippet: Hybridizations were performed in the presence of 0.2 M NaCl and 50 mM Tris-HCl (pH 7.5), resulting in primer/template structures. .. Phage T4 polynucleotide kinase was obtained from New England Biolabs. ϕ29 DNA polymerase variants at Tyr390 residue Y390F and Y390S were constructed by J. Saturno in an exonuclease deficient background (D12A/D66A) (hereafter PolY390F Exo− and PolY390S Exo− , respectively (unpublished data) and further purified from Escherichia coli BL21(DE3) cells harbouring the corresponding recombinant plasmid ( ). .. The hybrid molecules Pber-2/T4 and Pber-2/8oxodG, containing the unmodified dG and an 8oxodG lesion at the +3 position of the template, respectively, can be used both as substrate for the 3′–5′ exonuclease activity and for DNA-dependent DNA polymerization.

    Article Title: Purification and Characterization of the RecA Protein from Neisseria gonorrhoeae
    Article Snippet: Restriction enzymes, Klenow, polynucleotide kinase (PNK), T4 DNA ligase, and M13K07 Helper Phage were purchased from New England Biolabs. .. Restriction enzymes, Klenow, polynucleotide kinase (PNK), T4 DNA ligase, and M13K07 Helper Phage were purchased from New England Biolabs.

    Irradiation:

    Article Title: The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity
    Article Snippet: A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel. .. A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel.

    RNA Extraction:

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: Ribosome-protected fragments (RPFs) were then isolated as described above for total RNA extraction. .. RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB).

    Helicase Assay:

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: Paragraph title: Preparation of substrate and DNA helicase assay ... Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature.

    Sample Prep:

    Article Title: The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach
    Article Snippet: For ChIPseq, sample preparation was performed using the ChIP protocol described above. .. The ChIP DNA and the Input DNA ends were repaired using T4 DNA polymerase, Klenow enzyme, and T4 polynucleotide kinase (PNK) (New England Biolabs), followed by treatment with Klenow exo- to add an A base to the 3' end.

    In Vitro:

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: Fluorescent U4 was created by ligation of a 5′-Cy5 labeled RNA consisting of U4 nucleotides 1–13 to an in vitro transcribed U4 containing nucleotides 14–160 (modified from the pUC118 plasmid described previously). .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB). .. U6(13–112) was modified using only T4 PNK due to the presence of a 5'-OH group in the original transcript.

    Ethanol Precipitation:

    Article Title: RNAi triggered by specialized machinery silences developmental genes and retrotransposons
    Article Snippet: After phenol /chloroform/isoamyl alcohol extraction and ethanol precipitation, 3′-adapters (5′-App TCG TAT GCC GTC TTC TGC TTG T NH2 -3′) were ligated overnight at 16°C under conditions that are insensitive to 2′-O-methylation of the terminal nucleotide (50 mM Tris pH 7.5, 10 mM MgCl2, 1mM DTT, 12.5% PEG.) .. 10μl of the 3′-ligated RNA was phosphorylated by adding 3 μl of water, 0.5 μl of T4 Rnl1 buffer and 0.5 μl (20 U) of murine RNase inhibitor, and 1 μl (10 units) of T4 PNK (NEB) followed by incubation at 37°C for 30 min.

    Concentration Assay:

    Article Title: Unconventional miR-122 binding stabilizes the HCV genome by forming a trimolecular RNA structure
    Article Snippet: MiR-122 RNAs (IDT) were 5′32 P-labelled using [γ-32 P]ATP (PerkinElmer) and T4 PNK (NEB) [10 µL of reaction; 1× T4 PNK buffer, 50 pmol miR-122 RNA, 50 µCi [γ-32 P]ATP, 10 U T4 PNK] at 37°C for 1 h and subsequently diluted with nuclease-free water (40 µL). .. MiR-122 RNAs (IDT) were 5′32 P-labelled using [γ-32 P]ATP (PerkinElmer) and T4 PNK (NEB) [10 µL of reaction; 1× T4 PNK buffer, 50 pmol miR-122 RNA, 50 µCi [γ-32 P]ATP, 10 U T4 PNK] at 37°C for 1 h and subsequently diluted with nuclease-free water (40 µL).

    Article Title: Dissecting domains necessary for activation and repression of splicing by muscleblind-like protein 1
    Article Snippet: Oligo primer (4 pmoles per PCR reaction) was incubated at 37°C for 60 minutes in 50 mM Tris 10 mM MgCl2 T4 polynucleotide kinase enzyme (NEB) and 0.1 μl [α-32 P]-UTP per PCR reaction. .. After incubation the solution was phenol extracted, and purified on a G-50 spin column (GE Healthcare).

    Article Title: A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome
    Article Snippet: The RNA was eluted from the beads in 100 μl of low TE. .. 3′ phosphates were removed from the RNA by addition 8.2 μl of 10× T4 polynucleotide buffer to 75 μl of the RNA solution and 4 μl of ATP-free T4 polynucleotide kinase (NEB) was added and incubated for 15 min. Hydrophilic streptavidin magnetic beads (NEB) were prepared by washing 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 50 mM NaCl, 1 mM EDTA and 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 500 mM NaCl, 1 mM EDTA and suspended in their original suspension concentration of 4 mg/ml in wash buffer A. .. 50 μl of the kinase treated RNA was added to 30 μl of the prewashed streptavidin beads at room temperature with occasional resuspension for 20 min.

    Fractionation:

    Article Title: RNAi triggered by specialized machinery silences developmental genes and retrotransposons
    Article Snippet: Small RNA fractions < 40 nt were prepared by gel purification from denaturing acrylamide gels, or by using flashPAGE™ fractionation (Life Technologies) according to the manufacturer’s recommendation . .. 10μl of the 3′-ligated RNA was phosphorylated by adding 3 μl of water, 0.5 μl of T4 Rnl1 buffer and 0.5 μl (20 U) of murine RNase inhibitor, and 1 μl (10 units) of T4 PNK (NEB) followed by incubation at 37°C for 30 min.

    DNA Purification:

    Article Title: Purification and Characterization of the RecA Protein from Neisseria gonorrhoeae
    Article Snippet: Restriction enzymes, Klenow, polynucleotide kinase (PNK), T4 DNA ligase, and M13K07 Helper Phage were purchased from New England Biolabs. .. Plasmid pGEM-3Zf(+) was purchased from Promega.

    End Labeling:

    Article Title: G-quadruplex recognition activities of E. Coli MutS
    Article Snippet: All oligonucleotides (detailed in Additional file ) were purchased from Fisher-Operon. .. End labeling used T4 PNK (New England Biolabs (NEB), Ipswich, MA) and 32 PγATP (from either MP biomedicals or Perkin Elmer). .. ATPγS was purchased from MP biomedicals.

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: After precipitation, RNAs were pelleted by centrifugation and resuspended in the 5' end labeling reaction consisting of 10 units of T4 PNK, 70 mM Tris-HCl pH 7.6, 10 mM MgCl2 , 5 mM DTT and 0.03 μCi of [γ-32 P] ATP (3000 Ci/mmol). .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Marker:

    Article Title: The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity
    Article Snippet: The DNA digests were run on a 0.8% Agarose-1X TBE gel at 50 V for a total of 1100 volt-hours. .. A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel. .. Next morning, the gel was shaken in 0.25 M HCl for 15 min followed by two rounds (15 min each) of shaking in solution containing 0.5 M NaOH and 1.5 M NaCl.

    Gel Extraction:

    Article Title: Purification and Characterization of the RecA Protein from Neisseria gonorrhoeae
    Article Snippet: Restriction enzymes, Klenow, polynucleotide kinase (PNK), T4 DNA ligase, and M13K07 Helper Phage were purchased from New England Biolabs. .. Plasmid pGEM-3Zf(+) was purchased from Promega.

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  • 99
    New England Biolabs t4 pnk buffer
    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the   Materials and Methods . Error bars indicate SD.
    T4 Pnk Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 pnk buffer/product/New England Biolabs
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    99
    New England Biolabs t4 polynucleotide kinase
    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the   Materials and Methods . Error bars indicate SD.
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 817 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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    92
    New England Biolabs pnk mix
    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the   Materials and Methods . Error bars indicate SD.
    Pnk Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pnk mix/product/New England Biolabs
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    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the   Materials and Methods . Error bars indicate SD.

    Journal: Molecular Therapy. Nucleic Acids

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

    doi: 10.1016/j.omtn.2017.07.008

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

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

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