t4 ligase  (Thermo Fisher)


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    Structured Review

    Thermo Fisher t4 ligase
    T4 Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase/product/Thermo Fisher
    Average 99 stars, based on 182 article reviews
    Price from $9.99 to $1999.99
    t4 ligase - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression
    Article Snippet: Paragraph title: PAS- or bHLH-deleted Clock and Bmal1 clones ... Primers were phosphorylated with T4 kinase (Invitrogen) for 20 min at 20 °C and purified on Sephadex G25 columns.

    Amplification:

    Article Title: Regulation of Tryptophan Operon Expression in the Archaeon Methanothermobacter thermautotrophicus
    Article Snippet: DNA molecules designated A56, B12, B13, B15, and B16 (see Fig. ) were PCR amplified from p7557 DNA using primer pairs MX64/MX97, MX91/MX96, MX103/MX96, MX92/MX124, and MX91/MX125 (Table ), respectively. .. Preparations of these DNAs were 32 P-end-labeled by incubation for 1 h at 37°C in reaction mixtures (20 μl) that contained T4 kinase (10 U), kinase buffer (Invitrogen), and 200 μCi [γ-32 P]ATP (∼7 kCi/mmol).

    Article Title: Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering
    Article Snippet: After restriction digestion with SmaI and SalI (Thermo Fisher Scientific), the sgRNA construct was ligated into the pUC19 vector and transformed into E. coli TOP10 cells for storage and amplification. .. After digestion, the sgRNA construct and the pBBR1MCS2 backbone were ligated with T4 ligase (Thermo Fisher Scientific) yielding the pBBR1MCS2-sgRNA.

    Article Title: Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression
    Article Snippet: Primers were phosphorylated with T4 kinase (Invitrogen) for 20 min at 20 °C and purified on Sephadex G25 columns. .. Conditions for PCR (Taq DNA polymerase High Fidelity) were as follows: 95 °C for 2 min, followed by 30 cycles of amplification at 95 °C for 30 s, 54 °C for 30 s and 68 °C for 8 min with a time increment of 20 s per cycle and a final extension at 68 °C for 10 min. PCR products were purified on gel (gel extraction kit; Qiagen) and ligated [25 min at room temperature (22–23 °C); T4 DNA ligase; New England Biolabs].

    Article Title: Characterization of MtsR, a New Metal Regulator in Group A Streptococcus, Involved in Iron Acquisition and Virulence
    Article Snippet: A 337-bp fragment containing the upstream region of the sia operon (Pshr fragment) was amplified from the NZ131 chromosome by use of siaGSF and siaGSR primers. .. The PCR products were purified using a Rapid PCR purification system (Marligen Bioscience Inc.) and end labeled with [γ-32 P]ATP by use of T4 kinase (Invitrogen).

    Autoradiography:

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: Probes were end labeled with [α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA). .. The samples were analyzed by electrophoresis on a 5% polyacrylamide (37.5:1 acrylamide/bisacrylamide) gel and then autoradiography.

    Article Title: Ca2+, CREB and Kr?ppel: A novel KLF7-binding element conserved in mouse and human TRKB promoters is required for CREB-dependent transcription
    Article Snippet: Single stranded oliogonucleotides (−339 to −361 with and without mutations) were radiolabeled with P-32 using T4 kinase (Invitrogen) prior to annealing. .. Binding reactions were incubated at room temperature for 20 min prior to separation on a 6% TBE gel and autoradiography.

    Cytometry:

    Article Title: Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression *
    Article Snippet: .. Phycoerythrin-labeled or FITC-labeled secondary antibodies for flow cytometry, TRIzol reagent, zysorbin, the TA cloning kit, and T4 ligase were supplied by Invitrogen. .. All reagents required for the synthesis of cDNA from RNA, including Moloney murine virus reverse transcriptase, and all plasmids not provided by Addgene or a collaborator plus Transfast reagent were provided by Promega.

    Construct:

    Article Title: Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering
    Article Snippet: .. After digestion, the sgRNA construct and the pBBR1MCS2 backbone were ligated with T4 ligase (Thermo Fisher Scientific) yielding the pBBR1MCS2-sgRNA. ..

    Article Title: Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression
    Article Snippet: Primers were phosphorylated with T4 kinase (Invitrogen) for 20 min at 20 °C and purified on Sephadex G25 columns. .. Constructs lacking both PAS core repeats were generated using clones lacking one PAS domain as PCR templates.

    Article Title: Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression *
    Article Snippet: All siRNA constructs and the associated reagents were provided by Santa Cruz Biotechnology. .. Phycoerythrin-labeled or FITC-labeled secondary antibodies for flow cytometry, TRIzol reagent, zysorbin, the TA cloning kit, and T4 ligase were supplied by Invitrogen.

    Real-time Polymerase Chain Reaction:

    Article Title: Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression *
    Article Snippet: Phycoerythrin-labeled or FITC-labeled secondary antibodies for flow cytometry, TRIzol reagent, zysorbin, the TA cloning kit, and T4 ligase were supplied by Invitrogen. .. Kapa Taq polymerase for use in QPCR reactions was supplied by Kapa Biosystems.

    Incubation:

    Article Title: Regulation of Tryptophan Operon Expression in the Archaeon Methanothermobacter thermautotrophicus
    Article Snippet: .. Preparations of these DNAs were 32 P-end-labeled by incubation for 1 h at 37°C in reaction mixtures (20 μl) that contained T4 kinase (10 U), kinase buffer (Invitrogen), and 200 μCi [γ-32 P]ATP (∼7 kCi/mmol). .. The labeled DNA molecules were separated from unincorporated nucleotides by passage through a Sephadex G-50 column, and aliquots (1 ng) were mixed with 50 ng of unlabeled poly(dI-dC) and incubated in transcription buffer (10 μl) with increasing amounts of TrpY and/or l -tryptophan, as stated in the figure legends.

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: Probes were end labeled with [α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA). .. For the GC box 1 probes, 100 femtomoles of the 30-bp labeled probes containing wild-type and mutant GC box 1 (∼1 × 105 dpm) were incubated with 12 μg of uninfected or infected MeWo cell nuclear extract in a 10-μl reaction mixture in binding buffer A [40 mM HEPES (pH 7.9), 100 mM NaCl, 10 mM MgCl2 , 200 μg/ml bovine serum albumin, 12% glycerol, 0.05% NP-40, 1 mM dithiothreitol, and 3 μg poly(dI-dC)].

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: Proteinase K in TE buffer (10 mM Tris-HCl and 0.001 M EDTA) was added to a final concentration of 100 μg per ml, and the NaCl concentration was adjusted to 0.15 M. The mixture was then incubated at 50°C for 3 h. The DNA was isolated by phenol chloroform extraction, followed by ethanol precipitation. .. The blots were probed with a 400-bp PCR product prepared from pVO2 using primers 5′-GTGCTCCTGTCGTTGAGGACCCGG-3′ and 5′-CCTCTGACTTGAGCGTCGATTTTT-3′ and end labeled with[α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA).

    Article Title: Ca2+, CREB and Kr?ppel: A novel KLF7-binding element conserved in mouse and human TRKB promoters is required for CREB-dependent transcription
    Article Snippet: Single stranded oliogonucleotides (−339 to −361 with and without mutations) were radiolabeled with P-32 using T4 kinase (Invitrogen) prior to annealing. .. Binding reactions were incubated at room temperature for 20 min prior to separation on a 6% TBE gel and autoradiography.

    Article Title: Characterization of MtsR, a New Metal Regulator in Group A Streptococcus, Involved in Iron Acquisition and Virulence
    Article Snippet: The PCR products were purified using a Rapid PCR purification system (Marligen Bioscience Inc.) and end labeled with [γ-32 P]ATP by use of T4 kinase (Invitrogen). .. In the DNA binding assay, increasing concentrations of rMtsR were incubated with approximately 0.5 pmol labeled DNA fragment for 10 min at room temperature in a 24-μl reaction buffer containing 20 mM Na2 HPO4 , 50 mM NaCl, 5 mM MgCl2 , 2 mM dithiothreitol, 0.4 mg/ml bovine serum albumin, 0.2 mg/ml sheared salmon sperm DNA (Ambion), and 9.6% glycerol.

    Article Title: Birth of a chimeric primate gene by capture of the transposase gene from a mobile element
    Article Snippet: Complementary oligonucleotides were mixed at a concentration of 1.8 mM and end-labeled with gamma-P33 or gamma-P32 by using T4 kinase (Invitrogen). .. Reactions were incubated for 1 h at 25°C, and samples were separated by electrophoresis on 6% native polyacrylamide gels.

    Cell Culture:

    Article Title: Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression *
    Article Snippet: H647 cells were cultured in RPMI medium (Sigma-Aldrich) with 10% FCS, l -glutamine, penicillin, and streptomycin. .. Phycoerythrin-labeled or FITC-labeled secondary antibodies for flow cytometry, TRIzol reagent, zysorbin, the TA cloning kit, and T4 ligase were supplied by Invitrogen.

    Expressing:

    Article Title: Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression *
    Article Snippet: To assess binding of endogenous Elk1 to exogenous ITGB6 promoter constructs, we used the adenosquamous carcinoma cell line H647 after determining that these cells exhibit a high expression level of endogenous Elk1. .. Phycoerythrin-labeled or FITC-labeled secondary antibodies for flow cytometry, TRIzol reagent, zysorbin, the TA cloning kit, and T4 ligase were supplied by Invitrogen.

    Modification:

    Article Title: Tracing the Evolutionary History of Inositol, 1, 4, 5-trisphosphate receptor: Insights from Analyses of Capsaspora owczarzaki Ca2+ Release Channel Orthologs
    Article Snippet: T4 ligase and Dylight 700CW and Dylight 800CW secondary antibodies were from Thermo Scientific. .. DMEM (Dulbecco’s modified Eagle medium), Roswell Park Memorial Institute (RPMI) 1640 media, chicken serum, beta mercaptoethanol, G418 sulfate, and Trizol were from Invitrogen.

    Western Blot:

    Article Title: Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression *
    Article Snippet: Elk1 and GAPDH antibodies for Western blotting were supplied by Cell Signaling Technology. .. Phycoerythrin-labeled or FITC-labeled secondary antibodies for flow cytometry, TRIzol reagent, zysorbin, the TA cloning kit, and T4 ligase were supplied by Invitrogen.

    Article Title: Birth of a chimeric primate gene by capture of the transposase gene from a mobile element
    Article Snippet: Purified proteins were analyzed by Coomassie staining and Western blotting with an MBP monoclonal antibody (R29.6 from Santa Cruz Biotechnology). .. Complementary oligonucleotides were mixed at a concentration of 1.8 mM and end-labeled with gamma-P33 or gamma-P32 by using T4 kinase (Invitrogen).

    Transformation Assay:

    Article Title: Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering
    Article Snippet: After restriction digestion with SmaI and SalI (Thermo Fisher Scientific), the sgRNA construct was ligated into the pUC19 vector and transformed into E. coli TOP10 cells for storage and amplification. .. After digestion, the sgRNA construct and the pBBR1MCS2 backbone were ligated with T4 ligase (Thermo Fisher Scientific) yielding the pBBR1MCS2-sgRNA.

    Hybridization:

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: The DNA was digested with DpnI and EcoRI as described by Stow and Davison ( ) and analyzed by Southern blot hybridization. .. The blots were probed with a 400-bp PCR product prepared from pVO2 using primers 5′-GTGCTCCTGTCGTTGAGGACCCGG-3′ and 5′-CCTCTGACTTGAGCGTCGATTTTT-3′ and end labeled with[α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA).

    Flow Cytometry:

    Article Title: Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression *
    Article Snippet: .. Phycoerythrin-labeled or FITC-labeled secondary antibodies for flow cytometry, TRIzol reagent, zysorbin, the TA cloning kit, and T4 ligase were supplied by Invitrogen. .. All reagents required for the synthesis of cDNA from RNA, including Moloney murine virus reverse transcriptase, and all plasmids not provided by Addgene or a collaborator plus Transfast reagent were provided by Promega.

    Southern Blot:

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: The DNA was digested with DpnI and EcoRI as described by Stow and Davison ( ) and analyzed by Southern blot hybridization. .. The blots were probed with a 400-bp PCR product prepared from pVO2 using primers 5′-GTGCTCCTGTCGTTGAGGACCCGG-3′ and 5′-CCTCTGACTTGAGCGTCGATTTTT-3′ and end labeled with[α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA).

    Infection:

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: Probes were end labeled with [α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA). .. For the GC box 1 probes, 100 femtomoles of the 30-bp labeled probes containing wild-type and mutant GC box 1 (∼1 × 105 dpm) were incubated with 12 μg of uninfected or infected MeWo cell nuclear extract in a 10-μl reaction mixture in binding buffer A [40 mM HEPES (pH 7.9), 100 mM NaCl, 10 mM MgCl2 , 200 μg/ml bovine serum albumin, 12% glycerol, 0.05% NP-40, 1 mM dithiothreitol, and 3 μg poly(dI-dC)].

    Generated:

    Article Title: Regulation of Tryptophan Operon Expression in the Archaeon Methanothermobacter thermautotrophicus
    Article Snippet: The double-stranded DNA molecules generated were designated A12 through A19 (see Fig. for sequences). .. Preparations of these DNAs were 32 P-end-labeled by incubation for 1 h at 37°C in reaction mixtures (20 μl) that contained T4 kinase (10 U), kinase buffer (Invitrogen), and 200 μCi [γ-32 P]ATP (∼7 kCi/mmol).

    Article Title: Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression
    Article Snippet: Mutants lacking either PAS A (ΔPAS A), PAS B (ΔPAS B), or PAS A and PAS B (ΔPAS AB) core repeats or bHLH (ΔbHLH) were generated by PCR using FLAG-mClock and 5×Myc-mBmal1b plasmids as templates. .. Primers were phosphorylated with T4 kinase (Invitrogen) for 20 min at 20 °C and purified on Sephadex G25 columns.

    Article Title: Ca2+, CREB and Kr?ppel: A novel KLF7-binding element conserved in mouse and human TRKB promoters is required for CREB-dependent transcription
    Article Snippet: Nuclear cortical neuron extracts were generated and EMSA conducted as previously described ( ). .. Single stranded oliogonucleotides (−339 to −361 with and without mutations) were radiolabeled with P-32 using T4 kinase (Invitrogen) prior to annealing.

    Polymerase Chain Reaction:

    Article Title: Regulation of Tryptophan Operon Expression in the Archaeon Methanothermobacter thermautotrophicus
    Article Snippet: DNA molecules designated A56, B12, B13, B15, and B16 (see Fig. ) were PCR amplified from p7557 DNA using primer pairs MX64/MX97, MX91/MX96, MX103/MX96, MX92/MX124, and MX91/MX125 (Table ), respectively. .. Preparations of these DNAs were 32 P-end-labeled by incubation for 1 h at 37°C in reaction mixtures (20 μl) that contained T4 kinase (10 U), kinase buffer (Invitrogen), and 200 μCi [γ-32 P]ATP (∼7 kCi/mmol).

    Article Title: Tracing the Evolutionary History of Inositol, 1, 4, 5-trisphosphate receptor: Insights from Analyses of Capsaspora owczarzaki Ca2+ Release Channel Orthologs
    Article Snippet: Clone ID 1X Colony PCR Master Mix was from Lucigen. .. T4 ligase and Dylight 700CW and Dylight 800CW secondary antibodies were from Thermo Scientific.

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: .. The blots were probed with a 400-bp PCR product prepared from pVO2 using primers 5′-GTGCTCCTGTCGTTGAGGACCCGG-3′ and 5′-CCTCTGACTTGAGCGTCGATTTTT-3′ and end labeled with[α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA). .. This fragment includes a portion (nucleotide positions 1465 to 1864) of the pVO2 plasmid containing the ColE1 origin of replication and was designed to detect both intact DpnI-resistant linearized pVO2 (3.9 kb) resulting from replication of the input plasmid and an 872-bp fragment resulting from the DpnI-sensitive unreplicated input plasmid.

    Article Title: Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering
    Article Snippet: After digestion, the sgRNA construct and the pBBR1MCS2 backbone were ligated with T4 ligase (Thermo Fisher Scientific) yielding the pBBR1MCS2-sgRNA. .. The cas9 gene was PCR amplified using the pUC57hSpCas9 vector as template and primers BG10937/BG10938.

    Article Title: Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression
    Article Snippet: Mutants lacking either PAS A (ΔPAS A), PAS B (ΔPAS B), or PAS A and PAS B (ΔPAS AB) core repeats or bHLH (ΔbHLH) were generated by PCR using FLAG-mClock and 5×Myc-mBmal1b plasmids as templates. .. Primers were phosphorylated with T4 kinase (Invitrogen) for 20 min at 20 °C and purified on Sephadex G25 columns.

    Article Title: Characterization of MtsR, a New Metal Regulator in Group A Streptococcus, Involved in Iron Acquisition and Virulence
    Article Snippet: .. The PCR products were purified using a Rapid PCR purification system (Marligen Bioscience Inc.) and end labeled with [γ-32 P]ATP by use of T4 kinase (Invitrogen). .. The 32 P-labeled DNA fragments were purified from an 8% polyacrylamide gel, using the Rapid PCR purification system kit.

    Binding Assay:

    Article Title: Regulation of Tryptophan Operon Expression in the Archaeon Methanothermobacter thermautotrophicus
    Article Snippet: Paragraph title: Preparation of 32 P-labeled TRP boxes containing DNA molecules and use in electrophoretic mobility shift assays (EMSA) of TrpY binding. ... Preparations of these DNAs were 32 P-end-labeled by incubation for 1 h at 37°C in reaction mixtures (20 μl) that contained T4 kinase (10 U), kinase buffer (Invitrogen), and 200 μCi [γ-32 P]ATP (∼7 kCi/mmol).

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: Thirty-base-pair oligonucleotide probes (IDT, Coralville, IA) containing wild-type and mutant GC box 1 elements and 40-bp probes containing wild-type and mutant YY1 and GC box 2 binding site elements were used in electrophoretic mobility shift assays (EMSAs). .. Probes were end labeled with [α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA).

    Article Title: Ca2+, CREB and Kr?ppel: A novel KLF7-binding element conserved in mouse and human TRKB promoters is required for CREB-dependent transcription
    Article Snippet: Single stranded oliogonucleotides (−339 to −361 with and without mutations) were radiolabeled with P-32 using T4 kinase (Invitrogen) prior to annealing. .. Binding reactions were incubated at room temperature for 20 min prior to separation on a 6% TBE gel and autoradiography.

    Article Title: Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression *
    Article Snippet: To assess binding of endogenous Elk1 to exogenous ITGB6 promoter constructs, we used the adenosquamous carcinoma cell line H647 after determining that these cells exhibit a high expression level of endogenous Elk1. .. Phycoerythrin-labeled or FITC-labeled secondary antibodies for flow cytometry, TRIzol reagent, zysorbin, the TA cloning kit, and T4 ligase were supplied by Invitrogen.

    Article Title: Characterization of MtsR, a New Metal Regulator in Group A Streptococcus, Involved in Iron Acquisition and Virulence
    Article Snippet: The PCR products were purified using a Rapid PCR purification system (Marligen Bioscience Inc.) and end labeled with [γ-32 P]ATP by use of T4 kinase (Invitrogen). .. In some cases the binding assays were done in the presence of 250 μM EDTA with or without FeSO4 (made fresh).

    Mutagenesis:

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: Thirty-base-pair oligonucleotide probes (IDT, Coralville, IA) containing wild-type and mutant GC box 1 elements and 40-bp probes containing wild-type and mutant YY1 and GC box 2 binding site elements were used in electrophoretic mobility shift assays (EMSAs). .. Probes were end labeled with [α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA).

    Article Title: Genetic Manipulation of Campylobacter jejuni
    Article Snippet: .. 5× salt buffer (see recipe)10 mg/ml bovine serum albumin (BSA) 100 mM dithiothreitol (DTT) Donor DNA (containing transposon) Recipient DNA (containing target of mutagenesis or total C. jejuni genomic DNA prepared as in Basic Protocol 6) Transposase: (available from Dr. David Lampe ( ) TE buffer, pH 8.0 ( APPENDIX 2A ) dNTP mix: 1.25 mM each dNTP ( APPENDIX 2A ) T4 DNA polymerase and T4 DNA polymerase buffer (Invitrogen) T4 ligase and T4 ligase buffer (Invitrogen) 30° and 70°C water baths and 11° and 16°C recirculating water baths (or thermal cycler) Nitrocellulose membrane (0.025 μm pore size VSWP (Millipore, cat. no. VSWP04700) Additional reagents and equipment for phenol:chloroform extraction and ethanol precipitation of DNA ( ) .. 1 Prepare transposition reaction by combining the following: 16.0 μl 5× salt buffer 2.0 μl 10 mg/ml BSA 1.6 μl 100 mM DTT 1.0 μg donor DNA 2.0 μg recipient DNA 500 ng transposase Distilled deionized H2 O to 80 μl Incubate reactions at 30°C for 4 hr.

    Isolation:

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: Proteinase K in TE buffer (10 mM Tris-HCl and 0.001 M EDTA) was added to a final concentration of 100 μg per ml, and the NaCl concentration was adjusted to 0.15 M. The mixture was then incubated at 50°C for 3 h. The DNA was isolated by phenol chloroform extraction, followed by ethanol precipitation. .. The blots were probed with a 400-bp PCR product prepared from pVO2 using primers 5′-GTGCTCCTGTCGTTGAGGACCCGG-3′ and 5′-CCTCTGACTTGAGCGTCGATTTTT-3′ and end labeled with[α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA).

    Transfection:

    Article Title: Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression *
    Article Snippet: All cells were growth-arrested in supplement-free medium for 24 h prior to the start of experiments, except in the case of transfection experiments. .. Phycoerythrin-labeled or FITC-labeled secondary antibodies for flow cytometry, TRIzol reagent, zysorbin, the TA cloning kit, and T4 ligase were supplied by Invitrogen.

    Electrophoretic Mobility Shift Assay:

    Article Title: Regulation of Tryptophan Operon Expression in the Archaeon Methanothermobacter thermautotrophicus
    Article Snippet: Paragraph title: Preparation of 32 P-labeled TRP boxes containing DNA molecules and use in electrophoretic mobility shift assays (EMSA) of TrpY binding. ... Preparations of these DNAs were 32 P-end-labeled by incubation for 1 h at 37°C in reaction mixtures (20 μl) that contained T4 kinase (10 U), kinase buffer (Invitrogen), and 200 μCi [γ-32 P]ATP (∼7 kCi/mmol).

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: Thirty-base-pair oligonucleotide probes (IDT, Coralville, IA) containing wild-type and mutant GC box 1 elements and 40-bp probes containing wild-type and mutant YY1 and GC box 2 binding site elements were used in electrophoretic mobility shift assays (EMSAs). .. Probes were end labeled with [α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA).

    Article Title: Ca2+, CREB and Kr?ppel: A novel KLF7-binding element conserved in mouse and human TRKB promoters is required for CREB-dependent transcription
    Article Snippet: Paragraph title: Electrophoretic Mobility Shift Assays (EMSAs) ... Single stranded oliogonucleotides (−339 to −361 with and without mutations) were radiolabeled with P-32 using T4 kinase (Invitrogen) prior to annealing.

    Article Title: Characterization of MtsR, a New Metal Regulator in Group A Streptococcus, Involved in Iron Acquisition and Virulence
    Article Snippet: The electrophoretic mobility gel shift assay (EMSA) was done according to Schmitt et al. ( ). .. The PCR products were purified using a Rapid PCR purification system (Marligen Bioscience Inc.) and end labeled with [γ-32 P]ATP by use of T4 kinase (Invitrogen).

    TA Cloning:

    Article Title: Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression *
    Article Snippet: .. Phycoerythrin-labeled or FITC-labeled secondary antibodies for flow cytometry, TRIzol reagent, zysorbin, the TA cloning kit, and T4 ligase were supplied by Invitrogen. .. All reagents required for the synthesis of cDNA from RNA, including Moloney murine virus reverse transcriptase, and all plasmids not provided by Addgene or a collaborator plus Transfast reagent were provided by Promega.

    Labeling:

    Article Title: Regulation of Tryptophan Operon Expression in the Archaeon Methanothermobacter thermautotrophicus
    Article Snippet: Preparations of these DNAs were 32 P-end-labeled by incubation for 1 h at 37°C in reaction mixtures (20 μl) that contained T4 kinase (10 U), kinase buffer (Invitrogen), and 200 μCi [γ-32 P]ATP (∼7 kCi/mmol). .. The labeled DNA molecules were separated from unincorporated nucleotides by passage through a Sephadex G-50 column, and aliquots (1 ng) were mixed with 50 ng of unlabeled poly(dI-dC) and incubated in transcription buffer (10 μl) with increasing amounts of TrpY and/or l -tryptophan, as stated in the figure legends.

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: .. Probes were end labeled with [α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA). .. For the GC box 1 probes, 100 femtomoles of the 30-bp labeled probes containing wild-type and mutant GC box 1 (∼1 × 105 dpm) were incubated with 12 μg of uninfected or infected MeWo cell nuclear extract in a 10-μl reaction mixture in binding buffer A [40 mM HEPES (pH 7.9), 100 mM NaCl, 10 mM MgCl2 , 200 μg/ml bovine serum albumin, 12% glycerol, 0.05% NP-40, 1 mM dithiothreitol, and 3 μg poly(dI-dC)].

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: .. The blots were probed with a 400-bp PCR product prepared from pVO2 using primers 5′-GTGCTCCTGTCGTTGAGGACCCGG-3′ and 5′-CCTCTGACTTGAGCGTCGATTTTT-3′ and end labeled with[α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA). .. This fragment includes a portion (nucleotide positions 1465 to 1864) of the pVO2 plasmid containing the ColE1 origin of replication and was designed to detect both intact DpnI-resistant linearized pVO2 (3.9 kb) resulting from replication of the input plasmid and an 872-bp fragment resulting from the DpnI-sensitive unreplicated input plasmid.

    Article Title: Characterization of MtsR, a New Metal Regulator in Group A Streptococcus, Involved in Iron Acquisition and Virulence
    Article Snippet: .. The PCR products were purified using a Rapid PCR purification system (Marligen Bioscience Inc.) and end labeled with [γ-32 P]ATP by use of T4 kinase (Invitrogen). .. The 32 P-labeled DNA fragments were purified from an 8% polyacrylamide gel, using the Rapid PCR purification system kit.

    Article Title: Birth of a chimeric primate gene by capture of the transposase gene from a mobile element
    Article Snippet: Complementary oligonucleotides were mixed at a concentration of 1.8 mM and end-labeled with gamma-P33 or gamma-P32 by using T4 kinase (Invitrogen). .. The labeling reaction was stopped by heating to 95°C for 7 min, followed by slow cooling at room temperature to allow the annealing of the oligonucleotides.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Tracing the Evolutionary History of Inositol, 1, 4, 5-trisphosphate receptor: Insights from Analyses of Capsaspora owczarzaki Ca2+ Release Channel Orthologs
    Article Snippet: T4 ligase and Dylight 700CW and Dylight 800CW secondary antibodies were from Thermo Scientific. .. Reagents used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Bio-Rad.

    Gel Extraction:

    Article Title: Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression
    Article Snippet: Primers were phosphorylated with T4 kinase (Invitrogen) for 20 min at 20 °C and purified on Sephadex G25 columns. .. Conditions for PCR (Taq DNA polymerase High Fidelity) were as follows: 95 °C for 2 min, followed by 30 cycles of amplification at 95 °C for 30 s, 54 °C for 30 s and 68 °C for 8 min with a time increment of 20 s per cycle and a final extension at 68 °C for 10 min. PCR products were purified on gel (gel extraction kit; Qiagen) and ligated [25 min at room temperature (22–23 °C); T4 DNA ligase; New England Biolabs].

    IA:

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: Thirty-base-pair oligonucleotide probes (IDT, Coralville, IA) containing wild-type and mutant GC box 1 elements and 40-bp probes containing wild-type and mutant YY1 and GC box 2 binding site elements were used in electrophoretic mobility shift assays (EMSAs). .. Probes were end labeled with [α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA).

    Article Title: Birth of a chimeric primate gene by capture of the transposase gene from a mobile element
    Article Snippet: EMSAs were performed by using synthetic double-stranded oligonucleotides (Integrated DNA Technologies, Coralville, IA), whose sequences are shown in Fig. 3. .. Complementary oligonucleotides were mixed at a concentration of 1.8 mM and end-labeled with gamma-P33 or gamma-P32 by using T4 kinase (Invitrogen).

    Purification:

    Article Title: Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression
    Article Snippet: .. Primers were phosphorylated with T4 kinase (Invitrogen) for 20 min at 20 °C and purified on Sephadex G25 columns. .. Conditions for PCR (Taq DNA polymerase High Fidelity) were as follows: 95 °C for 2 min, followed by 30 cycles of amplification at 95 °C for 30 s, 54 °C for 30 s and 68 °C for 8 min with a time increment of 20 s per cycle and a final extension at 68 °C for 10 min. PCR products were purified on gel (gel extraction kit; Qiagen) and ligated [25 min at room temperature (22–23 °C); T4 DNA ligase; New England Biolabs].

    Article Title: Ca2+, CREB and Kr?ppel: A novel KLF7-binding element conserved in mouse and human TRKB promoters is required for CREB-dependent transcription
    Article Snippet: GST or GST-KLF7c were purified from E. coli . .. Single stranded oliogonucleotides (−339 to −361 with and without mutations) were radiolabeled with P-32 using T4 kinase (Invitrogen) prior to annealing.

    Article Title: Characterization of MtsR, a New Metal Regulator in Group A Streptococcus, Involved in Iron Acquisition and Virulence
    Article Snippet: .. The PCR products were purified using a Rapid PCR purification system (Marligen Bioscience Inc.) and end labeled with [γ-32 P]ATP by use of T4 kinase (Invitrogen). .. The 32 P-labeled DNA fragments were purified from an 8% polyacrylamide gel, using the Rapid PCR purification system kit.

    Article Title: Birth of a chimeric primate gene by capture of the transposase gene from a mobile element
    Article Snippet: This analysis shows that each purification procedure yielded a major peptide species corresponding in molecular mass to full-length MBP (42.5 kDa), MBP-MAR (83 kDa), MBP-MAR-N92 (53 kDa), and MAR-N126 (57 kDa) fusion proteins (data not shown). .. Complementary oligonucleotides were mixed at a concentration of 1.8 mM and end-labeled with gamma-P33 or gamma-P32 by using T4 kinase (Invitrogen).

    Chromatin Immunoprecipitation:

    Article Title: Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression *
    Article Snippet: Phycoerythrin-labeled or FITC-labeled secondary antibodies for flow cytometry, TRIzol reagent, zysorbin, the TA cloning kit, and T4 ligase were supplied by Invitrogen. .. All ChIP antibodies were obtained from Abcam.

    SDS Page:

    Article Title: Tracing the Evolutionary History of Inositol, 1, 4, 5-trisphosphate receptor: Insights from Analyses of Capsaspora owczarzaki Ca2+ Release Channel Orthologs
    Article Snippet: T4 ligase and Dylight 700CW and Dylight 800CW secondary antibodies were from Thermo Scientific. .. Reagents used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Bio-Rad.

    Plasmid Preparation:

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: The blots were probed with a 400-bp PCR product prepared from pVO2 using primers 5′-GTGCTCCTGTCGTTGAGGACCCGG-3′ and 5′-CCTCTGACTTGAGCGTCGATTTTT-3′ and end labeled with[α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA). .. This fragment includes a portion (nucleotide positions 1465 to 1864) of the pVO2 plasmid containing the ColE1 origin of replication and was designed to detect both intact DpnI-resistant linearized pVO2 (3.9 kb) resulting from replication of the input plasmid and an 872-bp fragment resulting from the DpnI-sensitive unreplicated input plasmid.

    Article Title: Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering
    Article Snippet: Paragraph title: Generation of the non-targeting Cas9 plasmid ... After digestion, the sgRNA construct and the pBBR1MCS2 backbone were ligated with T4 ligase (Thermo Fisher Scientific) yielding the pBBR1MCS2-sgRNA.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Antioxidants protect against diabetes by improving glucose homeostasis in mouse models of inducible insulin resistance and obesity
    Article Snippet: .. Leptin concentrations were measured with the leptin Mouse/Rat ELISA (BioVendor, RD291001200R, Heidelberg, Germany) and T4 concentrations with the T4 ELISA kit (Invitrogen, EIAT4C, Carlsbad, CA, USA). .. Hyperinsulinaemic–euglycaemic clamp Before the hyperinsulinaemic–euglycaemic clamp, mice were fasted for 5 h. The surgery as well as overall clamp procedure were performed as previously published [ ].

    Functional Assay:

    Article Title: Birth of a chimeric primate gene by capture of the transposase gene from a mobile element
    Article Snippet: Paragraph title: Functional Experiments and Analyses. ... Complementary oligonucleotides were mixed at a concentration of 1.8 mM and end-labeled with gamma-P33 or gamma-P32 by using T4 kinase (Invitrogen).

    Electrophoresis:

    Article Title: Regulation of Tryptophan Operon Expression in the Archaeon Methanothermobacter thermautotrophicus
    Article Snippet: Preparations of these DNAs were 32 P-end-labeled by incubation for 1 h at 37°C in reaction mixtures (20 μl) that contained T4 kinase (10 U), kinase buffer (Invitrogen), and 200 μCi [γ-32 P]ATP (∼7 kCi/mmol). .. The products were mixed with 1 μl of gel loading buffer (40% sucrose, 0.4% bromophenol blue, 0.4% xylene cyanol), subjected to electrophoresis through nondenaturing 6% polyacrylamide gels run in 90 mM Tris-borate, 2 mM EDTA (pH 8) buffer at 8 V/cm, and visualized by phosphorimaging.

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: Probes were end labeled with [α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA). .. The samples were analyzed by electrophoresis on a 5% polyacrylamide (37.5:1 acrylamide/bisacrylamide) gel and then autoradiography.

    Article Title: Birth of a chimeric primate gene by capture of the transposase gene from a mobile element
    Article Snippet: Complementary oligonucleotides were mixed at a concentration of 1.8 mM and end-labeled with gamma-P33 or gamma-P32 by using T4 kinase (Invitrogen). .. Reactions were incubated for 1 h at 25°C, and samples were separated by electrophoresis on 6% native polyacrylamide gels.

    Ethanol Precipitation:

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: Proteinase K in TE buffer (10 mM Tris-HCl and 0.001 M EDTA) was added to a final concentration of 100 μg per ml, and the NaCl concentration was adjusted to 0.15 M. The mixture was then incubated at 50°C for 3 h. The DNA was isolated by phenol chloroform extraction, followed by ethanol precipitation. .. The blots were probed with a 400-bp PCR product prepared from pVO2 using primers 5′-GTGCTCCTGTCGTTGAGGACCCGG-3′ and 5′-CCTCTGACTTGAGCGTCGATTTTT-3′ and end labeled with[α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA).

    Article Title: Genetic Manipulation of Campylobacter jejuni
    Article Snippet: .. 5× salt buffer (see recipe)10 mg/ml bovine serum albumin (BSA) 100 mM dithiothreitol (DTT) Donor DNA (containing transposon) Recipient DNA (containing target of mutagenesis or total C. jejuni genomic DNA prepared as in Basic Protocol 6) Transposase: (available from Dr. David Lampe ( ) TE buffer, pH 8.0 ( APPENDIX 2A ) dNTP mix: 1.25 mM each dNTP ( APPENDIX 2A ) T4 DNA polymerase and T4 DNA polymerase buffer (Invitrogen) T4 ligase and T4 ligase buffer (Invitrogen) 30° and 70°C water baths and 11° and 16°C recirculating water baths (or thermal cycler) Nitrocellulose membrane (0.025 μm pore size VSWP (Millipore, cat. no. VSWP04700) Additional reagents and equipment for phenol:chloroform extraction and ethanol precipitation of DNA ( ) .. 1 Prepare transposition reaction by combining the following: 16.0 μl 5× salt buffer 2.0 μl 10 mg/ml BSA 1.6 μl 100 mM DTT 1.0 μg donor DNA 2.0 μg recipient DNA 500 ng transposase Distilled deionized H2 O to 80 μl Incubate reactions at 30°C for 4 hr.

    Concentration Assay:

    Article Title: Antioxidants protect against diabetes by improving glucose homeostasis in mouse models of inducible insulin resistance and obesity
    Article Snippet: Blood plasma content measurement Insulin concentration was measured using the Mouse/Rat Insulin kit (Meso Scale Discovery, Rockville, MD, USA). .. Leptin concentrations were measured with the leptin Mouse/Rat ELISA (BioVendor, RD291001200R, Heidelberg, Germany) and T4 concentrations with the T4 ELISA kit (Invitrogen, EIAT4C, Carlsbad, CA, USA).

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: Proteinase K in TE buffer (10 mM Tris-HCl and 0.001 M EDTA) was added to a final concentration of 100 μg per ml, and the NaCl concentration was adjusted to 0.15 M. The mixture was then incubated at 50°C for 3 h. The DNA was isolated by phenol chloroform extraction, followed by ethanol precipitation. .. The blots were probed with a 400-bp PCR product prepared from pVO2 using primers 5′-GTGCTCCTGTCGTTGAGGACCCGG-3′ and 5′-CCTCTGACTTGAGCGTCGATTTTT-3′ and end labeled with[α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA).

    Article Title: Birth of a chimeric primate gene by capture of the transposase gene from a mobile element
    Article Snippet: .. Complementary oligonucleotides were mixed at a concentration of 1.8 mM and end-labeled with gamma-P33 or gamma-P32 by using T4 kinase (Invitrogen). .. The labeling reaction was stopped by heating to 95°C for 7 min, followed by slow cooling at room temperature to allow the annealing of the oligonucleotides.

    Lysis:

    Article Title: Cellular Transcription Factors Sp1 and Sp3 Suppress Varicella-Zoster Virus Origin-Dependent DNA Replication ▿
    Article Snippet: Total cellular DNA was prepared 48 h after superinfection by the addition of 5 ml of lysis buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA, 0.5% sodium dodecyl sulfate, 20 μg/ml RNase I) per dish. .. The blots were probed with a 400-bp PCR product prepared from pVO2 using primers 5′-GTGCTCCTGTCGTTGAGGACCCGG-3′ and 5′-CCTCTGACTTGAGCGTCGATTTTT-3′ and end labeled with[α-32 P]ATP using T4 kinase (Invitrogen, Carlsbad, CA).

    DNA Binding Assay:

    Article Title: Characterization of MtsR, a New Metal Regulator in Group A Streptococcus, Involved in Iron Acquisition and Virulence
    Article Snippet: The PCR products were purified using a Rapid PCR purification system (Marligen Bioscience Inc.) and end labeled with [γ-32 P]ATP by use of T4 kinase (Invitrogen). .. In the DNA binding assay, increasing concentrations of rMtsR were incubated with approximately 0.5 pmol labeled DNA fragment for 10 min at room temperature in a 24-μl reaction buffer containing 20 mM Na2 HPO4 , 50 mM NaCl, 5 mM MgCl2 , 2 mM dithiothreitol, 0.4 mg/ml bovine serum albumin, 0.2 mg/ml sheared salmon sperm DNA (Ambion), and 9.6% glycerol.

    Staining:

    Article Title: Birth of a chimeric primate gene by capture of the transposase gene from a mobile element
    Article Snippet: Purified proteins were analyzed by Coomassie staining and Western blotting with an MBP monoclonal antibody (R29.6 from Santa Cruz Biotechnology). .. Complementary oligonucleotides were mixed at a concentration of 1.8 mM and end-labeled with gamma-P33 or gamma-P32 by using T4 kinase (Invitrogen).

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  • 99
    Thermo Fisher t4 rna ligase
    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using <t>T4</t> RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    T4 Rna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase/product/Thermo Fisher
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    99
    Thermo Fisher t4 dna ligase
    Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using <t>T4</t> DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
    Average 99 stars, based on 382 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-04
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    Image Search Results


    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).

    Journal: RNA Biology

    Article Title: Identification of host RNAs that interact with EBV noncoding RNA EBER2

    doi: 10.1080/15476286.2018.1518854

    Figure Lengend Snippet: Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).

    Article Snippet: RNA was resuspended in 14.5 µl H2 O and subjected to T4 RNA Ligase reaction by adding 1 µl of 20 µM 5′-phosporylated RL3 (5′-P -GUGUCAGUCACUUCCAGCGG-Puromycin-3′), 2 µl 10× T4 Ligase Buffer, 2 µl BSA, 0.5 µl T4 RNA Ligase (ThermoFisher), and incubated overnight at 16°C.

    Techniques: Allele-specific Oligonucleotide, Selection, In Vivo, Generated, Sequencing, Combined Bisulfite Restriction Analysis Assay, Activity Assay, shRNA, Protein Concentration, Size-exclusion Chromatography, Ligation, Staining, Purification, Sample Prep, Labeling, Autoradiography

    Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.

    Journal: Chembiochem

    Article Title: Practical Synthesis of Cap‐4 RNA

    doi: 10.1002/cbic.201900590

    Figure Lengend Snippet: Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.

    Article Snippet: The 38‐nt T. cruzi cap‐4 RNA was prepared by splinted enzymatic ligation of an 11‐nt cap‐4 RNA and a chemically synthesized 5′‐phosphorylated 27‐nt RNA by using T4 DNA ligase (Thermo Fisher) in analogy to ref. .

    Techniques: Ligation, Sequencing, High Performance Liquid Chromatography, Purification

    DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay

    doi: 10.1016/j.bpj.2018.08.013

    Figure Lengend Snippet: DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.

    Article Snippet: For ligation, T4 DNA ligase was used in standard conditions (25°C, 10 mM MgCl2 ), and the GC content of the target site was ∼50%.

    Techniques: Microscopy, Labeling, Ligation, Binding Assay, Imaging

    Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.

    Journal: Nucleic Acids Research

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    doi: 10.1093/nar/gkx033

    Figure Lengend Snippet: Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.

    Article Snippet: Splinted end-to-end ligation of single-stranded DNA using T4 DNA ligase To explore the efficiency of splinted end-to-end ligation of single stranded DNA with T4 DNA ligase in the absence of hair-pin structures, we designed a ligation scheme where the splinter oligonucleotide is hybridized to a biotinylated adapter oligonucleotide (the donor), allowing for subsequent immobilization of ligation products on beads and removal of the splinter by mild heat treatment.

    Techniques: DNA Ligation, Ligation, Staining, Marker

    Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.

    Journal: Nucleic Acids Research

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    doi: 10.1093/nar/gkx033

    Figure Lengend Snippet: Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.

    Article Snippet: Splinted end-to-end ligation of single-stranded DNA using T4 DNA ligase To explore the efficiency of splinted end-to-end ligation of single stranded DNA with T4 DNA ligase in the absence of hair-pin structures, we designed a ligation scheme where the splinter oligonucleotide is hybridized to a biotinylated adapter oligonucleotide (the donor), allowing for subsequent immobilization of ligation products on beads and removal of the splinter by mild heat treatment.

    Techniques: Hybridization, Ligation, Modification, DNA Ligation, DNA Purification, Mutagenesis, Purification

    Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.

    Journal: Nucleic Acids Research

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    doi: 10.1093/nar/gkx033

    Figure Lengend Snippet: Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.

    Article Snippet: Splinted end-to-end ligation of single-stranded DNA using T4 DNA ligase To explore the efficiency of splinted end-to-end ligation of single stranded DNA with T4 DNA ligase in the absence of hair-pin structures, we designed a ligation scheme where the splinter oligonucleotide is hybridized to a biotinylated adapter oligonucleotide (the donor), allowing for subsequent immobilization of ligation products on beads and removal of the splinter by mild heat treatment.

    Techniques: Ligation, Sequencing, Ancient DNA Assay