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  • 99
    New England Biolabs t4 dna ligase buffer
    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and <t>T4</t> DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    T4 Dna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7738 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 dna ligase reaction buffer
    Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, <t>T4</t> ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).
    T4 Dna Ligase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1372 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase reaction buffer/product/New England Biolabs
    Average 99 stars, based on 1372 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase reaction buffer - by Bioz Stars, 2020-09
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    99
    New England Biolabs t4 rna ligase buffer
    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using <t>T4</t> RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.
    T4 Rna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase buffer/product/New England Biolabs
    Average 99 stars, based on 479 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase buffer - by Bioz Stars, 2020-09
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    New England Biolabs phusion high fidelity pcr master mix
    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using <t>T4</t> RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.
    Phusion High Fidelity Pcr Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4975 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion high fidelity pcr master mix/product/New England Biolabs
    Average 99 stars, based on 4975 article reviews
    Price from $9.99 to $1999.99
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    New England Biolabs t4 rna ligase 2 buffer
    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using <t>T4</t> RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.
    T4 Rna Ligase 2 Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase 2 buffer/product/New England Biolabs
    Average 99 stars, based on 47 article reviews
    Price from $9.99 to $1999.99
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    New England Biolabs phusion high fidelity pcr master mix with hf buffer
    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using <t>T4</t> RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.
    Phusion High Fidelity Pcr Master Mix With Hf Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 567 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion high fidelity pcr master mix with hf buffer/product/New England Biolabs
    Average 99 stars, based on 567 article reviews
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    New England Biolabs t4 rna ligase 1 buffer
    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using <t>T4</t> RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.
    T4 Rna Ligase 1 Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 848 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase 1 buffer/product/New England Biolabs
    Average 99 stars, based on 848 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase 1 buffer - by Bioz Stars, 2020-09
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    New England Biolabs quick ligation reaction buffer
    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using <t>T4</t> RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.
    Quick Ligation Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quick ligation reaction buffer/product/New England Biolabs
    Average 99 stars, based on 357 article reviews
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    quick ligation reaction buffer - by Bioz Stars, 2020-09
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    Image Search Results


    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

    Journal: Plant Methods

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    doi: 10.1186/s13007-018-0359-7

    Figure Lengend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

    Article Snippet: Standard restriction enzymes are sufficient for Pyrite cloning, but they should be tested for functionality and fidelity in the T4 DNA ligase buffer.

    Techniques: Clone Assay, Plasmid Preparation, Purification, Polymerase Chain Reaction, Incubation, Transformation Assay

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M111.284992

    Figure Lengend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Article Snippet: RQF Assay for Enzyme Self-adenylylation The RQF was utilized as above with T4 DNA ligase (5 μm , < 5% adenylyated in ATP-free buffer) in syringe A and ATP (2 mm ATP added to 1× ATP-free buffer containing 200 μCi of [α-32 P]ATP/ml solution) in syringe B.

    Techniques:

    Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M111.284992

    Figure Lengend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Article Snippet: RQF Assay for Enzyme Self-adenylylation The RQF was utilized as above with T4 DNA ligase (5 μm , < 5% adenylyated in ATP-free buffer) in syringe A and ATP (2 mm ATP added to 1× ATP-free buffer containing 200 μCi of [α-32 P]ATP/ml solution) in syringe B.

    Techniques: Diffusion-based Assay, Binding Assay

    Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M111.284992

    Figure Lengend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Article Snippet: RQF Assay for Enzyme Self-adenylylation The RQF was utilized as above with T4 DNA ligase (5 μm , < 5% adenylyated in ATP-free buffer) in syringe A and ATP (2 mm ATP added to 1× ATP-free buffer containing 200 μCi of [α-32 P]ATP/ml solution) in syringe B.

    Techniques:

    Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).

    Journal: Nucleic Acids Research

    Article Title: Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro

    doi:

    Figure Lengend Snippet: Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).

    Article Snippet: The reaction was incubated for 10 min at 37°C in T4 ligase buffer (New England Biolabs) containing 100 µg/ml bovine serum albumin, 75 mM KCl and the heteroduplex oligo recovered after Dpn II digestion (estimated to be a ∼100-fold molar excess over the plasmid ends).

    Techniques: Plasmid Preparation, Ligation, Generated

    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.

    Journal: Genome Research

    Article Title: Strand-specific deep sequencing of the transcriptome

    doi: 10.1101/gr.094318.109

    Figure Lengend Snippet: DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.

    Article Snippet: We incubated the following reaction mixture for 30 min at 37°C: 10 μL of sample, 1 μL of 10× T4 RNA ligase buffer (as fresh ATP supply), 10 U of polynucleotide kinase (New England BioLabs), 3 μL of RNase free water.

    Techniques: De-Phosphorylation Assay, Ligation, Polyacrylamide Gel Electrophoresis, Selection, Amplification, Polymerase Chain Reaction, Sequencing

    tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Journal:

    Article Title: Loss of a Universal tRNA Feature ▿

    doi: 10.1128/JB.01203-06

    Figure Lengend Snippet: tRNA end sequence analysis. A-C. RNA ligation products. Joints formed by T4 RNA ligase (brackets), involving the circled 5′-monophosphate ends, as revealed by RT-PCR with the indicated primers. Sequences of precursor RNA sequences are shown with

    Article Snippet: For rephosphorylation, dephosphorylated RNA was incubated at 125 ng/μl for 50 min at 37°C in RNA ligase buffer with 0.5 U of T4 polynucleotide kinase (New England Biolabs)/μl, purifying the product RNA as described above.

    Techniques: Sequencing, Ligation, Reverse Transcription Polymerase Chain Reaction

    Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Article Snippet: The products were then treated with T4 Polynucleotide Kinase to add mono-phosphate to non-capped mRNA to ready it for ligation; a reaction mixture consisting of 1 µl of T4 Polynucleotide Kinase (Fermentas, # EK0032), 2 µl of RNA Ligase Reaction Buffer (New England Biolabs), 0.5 µl of RNaseOUT (Invitrogen, #10777-019), 1 µl of 100 mM ATP solution (Fermentas, #R0441), and 15.5 µl of alkaline phosphatase-treated RNA was incubated for 30 minutes at 37°C.

    Techniques: De-Phosphorylation Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing