Structured Review

Roche t4 polynucleotide kinase
Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The <t>T4</t> polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).
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Images

1) Product Images from "Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi"

Article Title: Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr478

Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The T4 polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).
Figure Legend Snippet: Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The T4 polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).

Techniques Used: Clone Assay, Construct, Negative Control, Labeling

2) Product Images from "Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay"

Article Title: Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay

Journal: RNA

doi: 10.1261/rna.064501.117

cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).
Figure Legend Snippet: cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).

Techniques Used: Labeling, Marker

3) Product Images from "Human Cytomegalovirus Reactivation during Lactation and Mother-to-Child Transmission in Preterm Infants"

Article Title: Human Cytomegalovirus Reactivation during Lactation and Mother-to-Child Transmission in Preterm Infants

Journal:

doi: 10.1128/JCM.43.3.1318-1324.2005

) end labeled by T4 polynucleotide kinase and [γ-
Figure Legend Snippet: ) end labeled by T4 polynucleotide kinase and [γ-

Techniques Used: Labeling

Related Articles

Clone Assay:

Article Title: Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials
Article Snippet: Fragments between 1000 and 2000 bp were excised from the gel (Qiagen, Venlo, The Netherlands), end repaired using Klenow - T4 polymerase mixture (Fermentas, St.-Leon-Rot, Germany) and phosphorylated using T4 polynucleotide kinase (Roche, Vilvoorde, Belgium). .. This resulted in over 10 000 positive clones after blue white screening for each bacteriophage, of which more than 90% contained inserts between 1 and 2 kb.

Amplification:

Article Title: BcsKC Is an Essential Protein for the Type VI Secretion System Activity in Burkholderia cenocepacia That Forms an Outer Membrane Complex with BcsLB *
Article Snippet: T4 polynucleotide kinase and T4 DNA ligase (Roche Applied Science) were used as recommended by the manufacturers. .. DNA amplification by PCR was performed using a PTC-221 DNA engine (MJ Research, Incline Village, Nevada) with Taq , Proof Start, or HotStar HiFidelity DNA polymerases (Qiagen).

Article Title: Human Cytomegalovirus Reactivation during Lactation and Mother-to-Child Transmission in Preterm Infants
Article Snippet: .. The specificity of the amplified DNA fragments was confirmed by Southern hybridization with the internal oligonucleotide probe P2 terminally labeled with [γ-32 P]ATP (Hartmann Analytik, Braunschweig, Germany) and T4 polynucleotide kinase (Roche, Mannheim, Germany) (21-23). .. DNA samples negative for HCMV were probed for successful DNA preparation by beta-globin PCR according to Bauer et al. ( ).

Filtration:

Article Title: On the mechanism of strand assimilation by the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8)
Article Snippet: PB11 was 5′ 32 P-labeled with T4 polynucleotide kinase and purified using Sephadex G-25 (fine) Quick spin columns (Roche Molecular Biochemicals). .. Alternatively, pUC18 form I was isolated from Brij58-lysed cells followed by anion exchange and gel filtration chromatography [Q high (BioRad) and Chroma Spin + TE-1000 columns (BD Biosciences)].

Synthesized:

Article Title: RNA-binding Site of Escherichia coli Peptidyl-tRNA Hydrolase
Article Snippet: To prepare duplexHis , oligoribonucleotides 5′-CGC ACU AGC CAG CCC A-3′ and 5′-GCU GGC UAG UGC G-3′ were chemically synthesized as described above for the tetraloopHis . .. To aminoacylate the above duplex with histidyl-tRNA synthetase, phosphorylation at the two 5′-ends was ensured by a 30-min incubation at 37 °C in the presence of 50 m m Tris-HCl (pH 8.0), 10 m m MgCl2 , 5 m m dithiothreitol, 2 m m ATP and 0.5 units/μl of T4 polynucleotide kinase (Roche Applied Science).

Article Title: On the mechanism of strand assimilation by the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8)
Article Snippet: Oligodeoxyribonucleotide PB11 (100mer) , complementary to residues 379–478 of the minus strand of pUC18, was synthesized and gel purified by Sigma Genosys. .. PB11 was 5′ 32 P-labeled with T4 polynucleotide kinase and purified using Sephadex G-25 (fine) Quick spin columns (Roche Molecular Biochemicals).

Construct:

Article Title: Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi
Article Snippet: For the cleavage 3′-product of the pGEM-T easy construct, one-half of the product was dephosphorylated at 37°C for 30 min with 1 U of calf intestinal alkaline phosphatase (Roche) and subsequently gel purified (3cGEMT*). .. Aliquots of each sample RNA were split into two tubes to insure the same radiolabeling level for samples with and without T4 polynucleotide kinase (T4 PNK, Roche).

Electrophoresis:

Article Title: Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi
Article Snippet: Aliquots of each sample RNA were split into two tubes to insure the same radiolabeling level for samples with and without T4 polynucleotide kinase (T4 PNK, Roche). .. Reactions were stopped by addition of 10 µl of 2× formamide buffer and resolved by electrophoresis in 8% polyacrylamide, 7 M urea, 1× TBE gel.

Nuclear Magnetic Resonance:

Article Title: RNA-binding Site of Escherichia coli Peptidyl-tRNA Hydrolase
Article Snippet: Before use, oligonucleotides were dissolved in NMR buffer (50 m m sodium acetate (pH 6.0), 200 m m NaCl) at a final concentration of 1040 μ m each, incubated for 1 h at 60 °C, and slowly cooled down to room temperature. .. To aminoacylate the above duplex with histidyl-tRNA synthetase, phosphorylation at the two 5′-ends was ensured by a 30-min incubation at 37 °C in the presence of 50 m m Tris-HCl (pH 8.0), 10 m m MgCl2 , 5 m m dithiothreitol, 2 m m ATP and 0.5 units/μl of T4 polynucleotide kinase (Roche Applied Science).

Activity Assay:

Article Title: Heterogeneity in Expression of DNA Polymerase β and DNA Repair Activity in Human Tumor Cell Lines
Article Snippet: The gap-filling synthesis activity in nuclear extracts of all cell lines (50 μg protein) was determined using a 51-bp DNA template with a G:U mismatch at the 22 bp position ( ). .. The 51-bp template was labeled at the 5′ end by [γ-32 P]ATP (Amersham Pharmacia Biotech, Piscataway, NJ) and T4 polynucleotide kinase (Roche Molecular Biochemicals, Indianapolis, IN).

Modification:

Article Title: Quantitative Deep Sequencing Reveals Dynamic HIV-1 Escape and Large Population Shifts during CCR5 Antagonist Therapy In Vivo
Article Snippet: Deep Sequencing, Data Filtering and Alignment The V3 loop amplicons were first end-repaired with T4 DNA polymerase and 5′-phosphorylated with T4 Polynucleotide Kinase (454/Roche). .. The modified amplicons including adapters were column purified (MinElute, Qiagen), immobilized onto 28 µm streptavidin beads, and blunt ended (454 adapters have a 5′ overhang) using a Fill-in polymerase (454/Roche).

Transformation Assay:

Article Title: BcsKC Is an Essential Protein for the Type VI Secretion System Activity in Burkholderia cenocepacia That Forms an Outer Membrane Complex with BcsLB *
Article Snippet: T4 polynucleotide kinase and T4 DNA ligase (Roche Applied Science) were used as recommended by the manufacturers. .. Transformation of E. coli DH5α, E. coli SY327, and E. coli KEM5 was done by the calcium chloride protocol ( ).

Article Title: Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials
Article Snippet: Fragments between 1000 and 2000 bp were excised from the gel (Qiagen, Venlo, The Netherlands), end repaired using Klenow - T4 polymerase mixture (Fermentas, St.-Leon-Rot, Germany) and phosphorylated using T4 polynucleotide kinase (Roche, Vilvoorde, Belgium). .. Subsequently, fragments were ligated using T4 ligase (Fermentas) into Sma I-linearised pUC19 plasmids and transformed to Escherichia coli XL1 blue MRF.

Gel Purification:

Article Title: The requirement for the highly conserved G−1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase
Article Snippet: .. Purified tRNAHis (20 pmol) was treated with calf intestinal phosphatase (Roche) to remove the 5′ phosphate, phenol-extracted, and ethanol-precipitated, and resuspended tRNA was labeled with 25 pmol of [γ-32 P]ATP (7000 Ci/mmol MP Biologicals) using T4 polynucleotide kinase (Roche), followed by removal of excess label using a Micro Bio-spin 6 chromatography column (Bio-Rad), and gel purification. .. His6-Hts1 was purified by immobilized metal ion affinity chromatography , and 5′ end-labeled tRNAHis species from wild-type or thg1 -Δ [ 2μ LEU2 tRNAHis A73, 2μ HIS3 HTS1 ] strains were incubated in a buffer containing 50 mM HEPES (pH 7.5), 4 mM DTT, 20 mM KCl, 10 mM MgCl2 , 2.5 mM ATP, 1 mM L-Histidine, and 2.5 μM yeast HisRS (His6-Hts1) for 30 min at 30°C, essentially as described ( ).

Countercurrent Chromatography:

Article Title: RNA-binding Site of Escherichia coli Peptidyl-tRNA Hydrolase
Article Snippet: To prepare duplexHis , oligoribonucleotides 5′-CGC ACU AGC CAG CCC A-3′ and 5′-GCU GGC UAG UGC G-3′ were chemically synthesized as described above for the tetraloopHis . .. To aminoacylate the above duplex with histidyl-tRNA synthetase, phosphorylation at the two 5′-ends was ensured by a 30-min incubation at 37 °C in the presence of 50 m m Tris-HCl (pH 8.0), 10 m m MgCl2 , 5 m m dithiothreitol, 2 m m ATP and 0.5 units/μl of T4 polynucleotide kinase (Roche Applied Science).

Conjugation Assay:

Article Title: Genetic Characterization of a Multicomponent Signal Transduction System Controlling the Expression of Cable Pili in Burkholderia cenocepacia
Article Snippet: DNA-modifying enzymes, including restriction endonucleases, T4 polynucleotide kinase, T4 DNA ligase, T4 polymerase, and Taq polymerase, were obtained from Roche, New England Biolabs, Promega, and Invitrogen. .. Recombinant plasmids were introduced into E. coli and/or B. cenocepacia by either electroporation or conjugation, as previously described ( ).

Chromatography:

Article Title: The requirement for the highly conserved G−1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase
Article Snippet: .. Purified tRNAHis (20 pmol) was treated with calf intestinal phosphatase (Roche) to remove the 5′ phosphate, phenol-extracted, and ethanol-precipitated, and resuspended tRNA was labeled with 25 pmol of [γ-32 P]ATP (7000 Ci/mmol MP Biologicals) using T4 polynucleotide kinase (Roche), followed by removal of excess label using a Micro Bio-spin 6 chromatography column (Bio-Rad), and gel purification. .. His6-Hts1 was purified by immobilized metal ion affinity chromatography , and 5′ end-labeled tRNAHis species from wild-type or thg1 -Δ [ 2μ LEU2 tRNAHis A73, 2μ HIS3 HTS1 ] strains were incubated in a buffer containing 50 mM HEPES (pH 7.5), 4 mM DTT, 20 mM KCl, 10 mM MgCl2 , 2.5 mM ATP, 1 mM L-Histidine, and 2.5 μM yeast HisRS (His6-Hts1) for 30 min at 30°C, essentially as described ( ).

Article Title: On the mechanism of strand assimilation by the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8)
Article Snippet: PB11 was 5′ 32 P-labeled with T4 polynucleotide kinase and purified using Sephadex G-25 (fine) Quick spin columns (Roche Molecular Biochemicals). .. Alternatively, pUC18 form I was isolated from Brij58-lysed cells followed by anion exchange and gel filtration chromatography [Q high (BioRad) and Chroma Spin + TE-1000 columns (BD Biosciences)].

Generated:

Article Title: Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi
Article Snippet: Checking for 5′-hydroxyl ends The 77 nt length 3′-fragments generated by co-transcriptional cleavage reactions of pGEM-T-61/L1Tc+77, 7134-171/L1Tc+77 and 55-171/L1Tc+77 constructs were gel purified as described above for uncleaved RNA. .. Aliquots of each sample RNA were split into two tubes to insure the same radiolabeling level for samples with and without T4 polynucleotide kinase (T4 PNK, Roche).

other:

Article Title: Functional analysis of the promoter of the mitochondrial phosphate carrier human gene: identification of activator and repressor elements and their transcription factors
Article Snippet: The double-stranded oligonucleotide probes were 5′-end labelled using T4 polynucleotide kinase and [γ-32 P]ATP at 37 °C for 30 min. Where indicated, the unlabelled DNA probe, the mutated probe or an unrelated oligonucleotide was added.

Polymerase Chain Reaction:

Article Title: BcsKC Is an Essential Protein for the Type VI Secretion System Activity in Burkholderia cenocepacia That Forms an Outer Membrane Complex with BcsLB *
Article Snippet: T4 polynucleotide kinase and T4 DNA ligase (Roche Applied Science) were used as recommended by the manufacturers. .. DNA amplification by PCR was performed using a PTC-221 DNA engine (MJ Research, Incline Village, Nevada) with Taq , Proof Start, or HotStar HiFidelity DNA polymerases (Qiagen).

Article Title: Human Cytomegalovirus Reactivation during Lactation and Mother-to-Child Transmission in Preterm Infants
Article Snippet: Paragraph title: Nucleic acid preparation and PCR. ... The specificity of the amplified DNA fragments was confirmed by Southern hybridization with the internal oligonucleotide probe P2 terminally labeled with [γ-32 P]ATP (Hartmann Analytik, Braunschweig, Germany) and T4 polynucleotide kinase (Roche, Mannheim, Germany) (21-23).

Sonication:

Article Title: Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials
Article Snippet: Purified DNA from bacteriophages 14/1, PNM and ISP was sonicated for 1 s at 20% intensity using a Sonics Vibracell and separated on a 1% agarose gel (Eurogentec, Seraing, Belgium). .. Fragments between 1000 and 2000 bp were excised from the gel (Qiagen, Venlo, The Netherlands), end repaired using Klenow - T4 polymerase mixture (Fermentas, St.-Leon-Rot, Germany) and phosphorylated using T4 polynucleotide kinase (Roche, Vilvoorde, Belgium).

Recombinant:

Article Title: Genetic Characterization of a Multicomponent Signal Transduction System Controlling the Expression of Cable Pili in Burkholderia cenocepacia
Article Snippet: DNA-modifying enzymes, including restriction endonucleases, T4 polynucleotide kinase, T4 DNA ligase, T4 polymerase, and Taq polymerase, were obtained from Roche, New England Biolabs, Promega, and Invitrogen. .. Recombinant plasmids were introduced into E. coli and/or B. cenocepacia by either electroporation or conjugation, as previously described ( ).

Radioactivity:

Article Title: Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi
Article Snippet: .. Aliquots of each sample RNA were split into two tubes to insure the same radiolabeling level for samples with and without T4 polynucleotide kinase (T4 PNK, Roche). ..

Electroporation:

Article Title: Genetic Characterization of a Multicomponent Signal Transduction System Controlling the Expression of Cable Pili in Burkholderia cenocepacia
Article Snippet: DNA-modifying enzymes, including restriction endonucleases, T4 polynucleotide kinase, T4 DNA ligase, T4 polymerase, and Taq polymerase, were obtained from Roche, New England Biolabs, Promega, and Invitrogen. .. Recombinant plasmids were introduced into E. coli and/or B. cenocepacia by either electroporation or conjugation, as previously described ( ).

Mutagenesis:

Article Title: BcsKC Is an Essential Protein for the Type VI Secretion System Activity in Burkholderia cenocepacia That Forms an Outer Membrane Complex with BcsLB *
Article Snippet: T4 polynucleotide kinase and T4 DNA ligase (Roche Applied Science) were used as recommended by the manufacturers. .. Mobilization of complementing plasmids and mutagenesis plasmids into B. cenocepacia K56-2 was performed by triparental mating using E. coli DH5α carrying the helper plasmid pRK2013 ( , ).

Isolation:

Article Title: On the mechanism of strand assimilation by the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8)
Article Snippet: PB11 was 5′ 32 P-labeled with T4 polynucleotide kinase and purified using Sephadex G-25 (fine) Quick spin columns (Roche Molecular Biochemicals). .. Alternatively, pUC18 form I was isolated from Brij58-lysed cells followed by anion exchange and gel filtration chromatography [Q high (BioRad) and Chroma Spin + TE-1000 columns (BD Biosciences)].

Article Title: Genetic Characterization of a Multicomponent Signal Transduction System Controlling the Expression of Cable Pili in Burkholderia cenocepacia
Article Snippet: DNA-modifying enzymes, including restriction endonucleases, T4 polynucleotide kinase, T4 DNA ligase, T4 polymerase, and Taq polymerase, were obtained from Roche, New England Biolabs, Promega, and Invitrogen. .. Plasmid DNA was isolated by the boiling lysis method ( ) or using the QIAprep Spin Miniprep kit (QIAGEN, Inc.).

Article Title: Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials
Article Snippet: Genome sequencing and proteomic analysis DNA from bacteriophages was isolated using a commercially available kit (Lambda Mini Kit, Qiagen, Hilden, Germany). .. Fragments between 1000 and 2000 bp were excised from the gel (Qiagen, Venlo, The Netherlands), end repaired using Klenow - T4 polymerase mixture (Fermentas, St.-Leon-Rot, Germany) and phosphorylated using T4 polynucleotide kinase (Roche, Vilvoorde, Belgium).

Labeling:

Article Title: The requirement for the highly conserved G−1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase
Article Snippet: .. Purified tRNAHis (20 pmol) was treated with calf intestinal phosphatase (Roche) to remove the 5′ phosphate, phenol-extracted, and ethanol-precipitated, and resuspended tRNA was labeled with 25 pmol of [γ-32 P]ATP (7000 Ci/mmol MP Biologicals) using T4 polynucleotide kinase (Roche), followed by removal of excess label using a Micro Bio-spin 6 chromatography column (Bio-Rad), and gel purification. .. His6-Hts1 was purified by immobilized metal ion affinity chromatography , and 5′ end-labeled tRNAHis species from wild-type or thg1 -Δ [ 2μ LEU2 tRNAHis A73, 2μ HIS3 HTS1 ] strains were incubated in a buffer containing 50 mM HEPES (pH 7.5), 4 mM DTT, 20 mM KCl, 10 mM MgCl2 , 2.5 mM ATP, 1 mM L-Histidine, and 2.5 μM yeast HisRS (His6-Hts1) for 30 min at 30°C, essentially as described ( ).

Article Title: Heterogeneity in Expression of DNA Polymerase β and DNA Repair Activity in Human Tumor Cell Lines
Article Snippet: .. The 51-bp template was labeled at the 5′ end by [γ-32 P]ATP (Amersham Pharmacia Biotech, Piscataway, NJ) and T4 polynucleotide kinase (Roche Molecular Biochemicals, Indianapolis, IN). ..

Article Title: Human Cytomegalovirus Reactivation during Lactation and Mother-to-Child Transmission in Preterm Infants
Article Snippet: .. The specificity of the amplified DNA fragments was confirmed by Southern hybridization with the internal oligonucleotide probe P2 terminally labeled with [γ-32 P]ATP (Hartmann Analytik, Braunschweig, Germany) and T4 polynucleotide kinase (Roche, Mannheim, Germany) (21-23). .. DNA samples negative for HCMV were probed for successful DNA preparation by beta-globin PCR according to Bauer et al. ( ).

Article Title: Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay
Article Snippet: .. For 5′-end labeling, degraded RNA fragments, γ-P32 -ATP and T4 polynucleotide kinase (Roche) were mixed and incubated at 37°C for 30 min. For 3′-end labeling, degraded RNA fragments, α-P32 ATP and T4 RNA ligase (NEB) were mixed and incubated at 37°C for 30 min. .. Reactions were stopped by sample loading buffers and the labeling results were analyzed on 15% TBE–PAGE.

Purification:

Article Title: The requirement for the highly conserved G−1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase
Article Snippet: .. Purified tRNAHis (20 pmol) was treated with calf intestinal phosphatase (Roche) to remove the 5′ phosphate, phenol-extracted, and ethanol-precipitated, and resuspended tRNA was labeled with 25 pmol of [γ-32 P]ATP (7000 Ci/mmol MP Biologicals) using T4 polynucleotide kinase (Roche), followed by removal of excess label using a Micro Bio-spin 6 chromatography column (Bio-Rad), and gel purification. .. His6-Hts1 was purified by immobilized metal ion affinity chromatography , and 5′ end-labeled tRNAHis species from wild-type or thg1 -Δ [ 2μ LEU2 tRNAHis A73, 2μ HIS3 HTS1 ] strains were incubated in a buffer containing 50 mM HEPES (pH 7.5), 4 mM DTT, 20 mM KCl, 10 mM MgCl2 , 2.5 mM ATP, 1 mM L-Histidine, and 2.5 μM yeast HisRS (His6-Hts1) for 30 min at 30°C, essentially as described ( ).

Article Title: On the mechanism of strand assimilation by the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8)
Article Snippet: .. PB11 was 5′ 32 P-labeled with T4 polynucleotide kinase and purified using Sephadex G-25 (fine) Quick spin columns (Roche Molecular Biochemicals). .. M13 and φX174 ssDNA were purchased from New England Biolabs.

Article Title: Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi
Article Snippet: For the cleavage 3′-product of the pGEM-T easy construct, one-half of the product was dephosphorylated at 37°C for 30 min with 1 U of calf intestinal alkaline phosphatase (Roche) and subsequently gel purified (3cGEMT*). .. Aliquots of each sample RNA were split into two tubes to insure the same radiolabeling level for samples with and without T4 polynucleotide kinase (T4 PNK, Roche).

Article Title: Quantitative Deep Sequencing Reveals Dynamic HIV-1 Escape and Large Population Shifts during CCR5 Antagonist Therapy In Vivo
Article Snippet: Deep Sequencing, Data Filtering and Alignment The V3 loop amplicons were first end-repaired with T4 DNA polymerase and 5′-phosphorylated with T4 Polynucleotide Kinase (454/Roche). .. Amplicons were then column purified (MinElute, Qiagen).

Article Title: Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials
Article Snippet: Purified DNA from bacteriophages 14/1, PNM and ISP was sonicated for 1 s at 20% intensity using a Sonics Vibracell and separated on a 1% agarose gel (Eurogentec, Seraing, Belgium). .. Fragments between 1000 and 2000 bp were excised from the gel (Qiagen, Venlo, The Netherlands), end repaired using Klenow - T4 polymerase mixture (Fermentas, St.-Leon-Rot, Germany) and phosphorylated using T4 polynucleotide kinase (Roche, Vilvoorde, Belgium).

Sequencing:

Article Title: Human Cytomegalovirus Reactivation during Lactation and Mother-to-Child Transmission in Preterm Infants
Article Snippet: These primers amplified a 123-bp fragment of the IE1 protein coding sequence as described earlier ( - ). .. The specificity of the amplified DNA fragments was confirmed by Southern hybridization with the internal oligonucleotide probe P2 terminally labeled with [γ-32 P]ATP (Hartmann Analytik, Braunschweig, Germany) and T4 polynucleotide kinase (Roche, Mannheim, Germany) (21-23).

Article Title: Quantitative Deep Sequencing Reveals Dynamic HIV-1 Escape and Large Population Shifts during CCR5 Antagonist Therapy In Vivo
Article Snippet: .. Deep Sequencing, Data Filtering and Alignment The V3 loop amplicons were first end-repaired with T4 DNA polymerase and 5′-phosphorylated with T4 Polynucleotide Kinase (454/Roche). .. Amplicons were then column purified (MinElute, Qiagen).

Article Title: Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials
Article Snippet: Paragraph title: Genome sequencing and proteomic analysis ... Fragments between 1000 and 2000 bp were excised from the gel (Qiagen, Venlo, The Netherlands), end repaired using Klenow - T4 polymerase mixture (Fermentas, St.-Leon-Rot, Germany) and phosphorylated using T4 polynucleotide kinase (Roche, Vilvoorde, Belgium).

Lysis:

Article Title: Genetic Characterization of a Multicomponent Signal Transduction System Controlling the Expression of Cable Pili in Burkholderia cenocepacia
Article Snippet: DNA-modifying enzymes, including restriction endonucleases, T4 polynucleotide kinase, T4 DNA ligase, T4 polymerase, and Taq polymerase, were obtained from Roche, New England Biolabs, Promega, and Invitrogen. .. Plasmid DNA was isolated by the boiling lysis method ( ) or using the QIAprep Spin Miniprep kit (QIAGEN, Inc.).

Plasmid Preparation:

Article Title: Genetic Characterization of a Multicomponent Signal Transduction System Controlling the Expression of Cable Pili in Burkholderia cenocepacia
Article Snippet: DNA-modifying enzymes, including restriction endonucleases, T4 polynucleotide kinase, T4 DNA ligase, T4 polymerase, and Taq polymerase, were obtained from Roche, New England Biolabs, Promega, and Invitrogen. .. Plasmid DNA was isolated by the boiling lysis method ( ) or using the QIAprep Spin Miniprep kit (QIAGEN, Inc.).

Article Title: BcsKC Is an Essential Protein for the Type VI Secretion System Activity in Burkholderia cenocepacia That Forms an Outer Membrane Complex with BcsLB *
Article Snippet: T4 polynucleotide kinase and T4 DNA ligase (Roche Applied Science) were used as recommended by the manufacturers. .. Mobilization of complementing plasmids and mutagenesis plasmids into B. cenocepacia K56-2 was performed by triparental mating using E. coli DH5α carrying the helper plasmid pRK2013 ( , ).

Article Title: Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials
Article Snippet: Fragments between 1000 and 2000 bp were excised from the gel (Qiagen, Venlo, The Netherlands), end repaired using Klenow - T4 polymerase mixture (Fermentas, St.-Leon-Rot, Germany) and phosphorylated using T4 polynucleotide kinase (Roche, Vilvoorde, Belgium). .. Plasmids from individual clones were isolated and sequenced using the standard M13f vector primer.

Hybridization:

Article Title: Human Cytomegalovirus Reactivation during Lactation and Mother-to-Child Transmission in Preterm Infants
Article Snippet: .. The specificity of the amplified DNA fragments was confirmed by Southern hybridization with the internal oligonucleotide probe P2 terminally labeled with [γ-32 P]ATP (Hartmann Analytik, Braunschweig, Germany) and T4 polynucleotide kinase (Roche, Mannheim, Germany) (21-23). .. DNA samples negative for HCMV were probed for successful DNA preparation by beta-globin PCR according to Bauer et al. ( ).

Agarose Gel Electrophoresis:

Article Title: Human Cytomegalovirus Reactivation during Lactation and Mother-to-Child Transmission in Preterm Infants
Article Snippet: The PCR products were separated by 3% agarose gel electrophoresis and stained with ethidium bromide. .. The specificity of the amplified DNA fragments was confirmed by Southern hybridization with the internal oligonucleotide probe P2 terminally labeled with [γ-32 P]ATP (Hartmann Analytik, Braunschweig, Germany) and T4 polynucleotide kinase (Roche, Mannheim, Germany) (21-23).

Article Title: Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials
Article Snippet: Purified DNA from bacteriophages 14/1, PNM and ISP was sonicated for 1 s at 20% intensity using a Sonics Vibracell and separated on a 1% agarose gel (Eurogentec, Seraing, Belgium). .. Fragments between 1000 and 2000 bp were excised from the gel (Qiagen, Venlo, The Netherlands), end repaired using Klenow - T4 polymerase mixture (Fermentas, St.-Leon-Rot, Germany) and phosphorylated using T4 polynucleotide kinase (Roche, Vilvoorde, Belgium).

Ethanol Precipitation:

Article Title: On the mechanism of strand assimilation by the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8)
Article Snippet: PB11 was 5′ 32 P-labeled with T4 polynucleotide kinase and purified using Sephadex G-25 (fine) Quick spin columns (Roche Molecular Biochemicals). .. Unless otherwise stated, pUC18 form I DNA was prepared from E.coli JM109 with the Promega Wizard Plus DNA purification system followed by ethanol precipitation.

Incubation:

Article Title: RNA-binding Site of Escherichia coli Peptidyl-tRNA Hydrolase
Article Snippet: .. To aminoacylate the above duplex with histidyl-tRNA synthetase, phosphorylation at the two 5′-ends was ensured by a 30-min incubation at 37 °C in the presence of 50 m m Tris-HCl (pH 8.0), 10 m m MgCl2 , 5 m m dithiothreitol, 2 m m ATP and 0.5 units/μl of T4 polynucleotide kinase (Roche Applied Science). ..

Article Title: Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay
Article Snippet: .. For 5′-end labeling, degraded RNA fragments, γ-P32 -ATP and T4 polynucleotide kinase (Roche) were mixed and incubated at 37°C for 30 min. For 3′-end labeling, degraded RNA fragments, α-P32 ATP and T4 RNA ligase (NEB) were mixed and incubated at 37°C for 30 min. .. Reactions were stopped by sample loading buffers and the labeling results were analyzed on 15% TBE–PAGE.

Concentration Assay:

Article Title: RNA-binding Site of Escherichia coli Peptidyl-tRNA Hydrolase
Article Snippet: Before use, oligonucleotides were dissolved in NMR buffer (50 m m sodium acetate (pH 6.0), 200 m m NaCl) at a final concentration of 1040 μ m each, incubated for 1 h at 60 °C, and slowly cooled down to room temperature. .. To aminoacylate the above duplex with histidyl-tRNA synthetase, phosphorylation at the two 5′-ends was ensured by a 30-min incubation at 37 °C in the presence of 50 m m Tris-HCl (pH 8.0), 10 m m MgCl2 , 5 m m dithiothreitol, 2 m m ATP and 0.5 units/μl of T4 polynucleotide kinase (Roche Applied Science).

Article Title: On the mechanism of strand assimilation by the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8)
Article Snippet: Its concentration was determined using an extinction coefficient of 939 208.1 M–1 cm–1 at 260 nm. .. PB11 was 5′ 32 P-labeled with T4 polynucleotide kinase and purified using Sephadex G-25 (fine) Quick spin columns (Roche Molecular Biochemicals).

DNA Purification:

Article Title: On the mechanism of strand assimilation by the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8)
Article Snippet: PB11 was 5′ 32 P-labeled with T4 polynucleotide kinase and purified using Sephadex G-25 (fine) Quick spin columns (Roche Molecular Biochemicals). .. Unless otherwise stated, pUC18 form I DNA was prepared from E.coli JM109 with the Promega Wizard Plus DNA purification system followed by ethanol precipitation.

End Labeling:

Article Title: The requirement for the highly conserved G−1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase
Article Snippet: Paragraph title: 5′ end labeling of tRNA ... Purified tRNAHis (20 pmol) was treated with calf intestinal phosphatase (Roche) to remove the 5′ phosphate, phenol-extracted, and ethanol-precipitated, and resuspended tRNA was labeled with 25 pmol of [γ-32 P]ATP (7000 Ci/mmol MP Biologicals) using T4 polynucleotide kinase (Roche), followed by removal of excess label using a Micro Bio-spin 6 chromatography column (Bio-Rad), and gel purification.

Staining:

Article Title: Human Cytomegalovirus Reactivation during Lactation and Mother-to-Child Transmission in Preterm Infants
Article Snippet: The PCR products were separated by 3% agarose gel electrophoresis and stained with ethidium bromide. .. The specificity of the amplified DNA fragments was confirmed by Southern hybridization with the internal oligonucleotide probe P2 terminally labeled with [γ-32 P]ATP (Hartmann Analytik, Braunschweig, Germany) and T4 polynucleotide kinase (Roche, Mannheim, Germany) (21-23).

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    Roche t4 polynucleotide kinase
    Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The <t>T4</t> polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).
    T4 Polynucleotide Kinase, supplied by Roche, used in various techniques. Bioz Stars score: 98/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The T4 polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).

    Journal: Nucleic Acids Research

    Article Title: Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi

    doi: 10.1093/nar/gkr478

    Figure Lengend Snippet: Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The T4 polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).

    Article Snippet: Aliquots of each sample RNA were split into two tubes to insure the same radiolabeling level for samples with and without T4 polynucleotide kinase (T4 PNK, Roche).

    Techniques: Clone Assay, Construct, Negative Control, Labeling

    cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).

    Journal: RNA

    Article Title: Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay

    doi: 10.1261/rna.064501.117

    Figure Lengend Snippet: cTSN is a Ca 2+ -dependent endonuclease cleaving at the 5′-side of phosphodiester bonds. ( A ) cTSN (100 nM) degraded the 5′-fluorescein-labeled pre-miR142 RNA (500 nM) in the presence of Ca 2+ at concentrations of 0.1–1 mM. The sizes of pre-miR142 (68 nt) and an RNA marker (28 nt) were labeled in the left of the gel. ( B ) cTSN cleaved at the 5′-side of phosphodiester bonds to produce degraded fragments with 3′-phosphate and 5′-OH ends that could be labeled by T4 polynucleotide kinase (T4 PNK) but not by T4 RNA ligase (T4 ligase).

    Article Snippet: For 5′-end labeling, degraded RNA fragments, γ-P32 -ATP and T4 polynucleotide kinase (Roche) were mixed and incubated at 37°C for 30 min. For 3′-end labeling, degraded RNA fragments, α-P32 ATP and T4 RNA ligase (NEB) were mixed and incubated at 37°C for 30 min.

    Techniques: Labeling, Marker

    ) end labeled by T4 polynucleotide kinase and [γ-

    Journal:

    Article Title: Human Cytomegalovirus Reactivation during Lactation and Mother-to-Child Transmission in Preterm Infants

    doi: 10.1128/JCM.43.3.1318-1324.2005

    Figure Lengend Snippet: ) end labeled by T4 polynucleotide kinase and [γ-

    Article Snippet: The specificity of the amplified DNA fragments was confirmed by Southern hybridization with the internal oligonucleotide probe P2 terminally labeled with [γ-32 P]ATP (Hartmann Analytik, Braunschweig, Germany) and T4 polynucleotide kinase (Roche, Mannheim, Germany) (21-23).

    Techniques: Labeling