ligation  (New England Biolabs)


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    Name:
    Salt T4 DNA Ligase
    Description:
    A salt tolerant variant of T4 DNA Ligase Salt T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex DNA or RNA and is designed to function at higher salt concentrations than wild type T4 DNA Ligase This enzyme will join cohesive end termini at salt concentrations as high as 300 mM without any loss in activity This enzyme is insensitive to salt carried over from other reaction components vector or insert DNA and allows ligation reactions to proceed in alternative reaction buffers with higher levels of salt e g NEBuffer 3 1
    Catalog Number:
    m0467s
    Price:
    64
    Size:
    20 000 units
    Category:
    DNA Ligases
    Buy from Supplier


    Structured Review

    New England Biolabs ligation
    Salt T4 DNA Ligase
    A salt tolerant variant of T4 DNA Ligase Salt T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex DNA or RNA and is designed to function at higher salt concentrations than wild type T4 DNA Ligase This enzyme will join cohesive end termini at salt concentrations as high as 300 mM without any loss in activity This enzyme is insensitive to salt carried over from other reaction components vector or insert DNA and allows ligation reactions to proceed in alternative reaction buffers with higher levels of salt e g NEBuffer 3 1
    https://www.bioz.com/result/ligation/product/New England Biolabs
    Average 95 stars, based on 55 article reviews
    Price from $9.99 to $1999.99
    ligation - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Clone Assay:

    Article Title: A Transcript Profiling Approach Reveals an Abscisic Acid-Specific Glycosyltransferase (UGT73C14) Induced in Developing Fiber of Ligon lintless-2 Mutant of Cotton (Gossypium hirsutum L.)
    Article Snippet: Paragraph title: Cloning and expression of recombinant UTG73C14 in E. coli ... Aliquots of the primers GhUGT-Ala2Malc5 ( 5’-GCTCAAGGTCACTTTGTCTTGATC-3’ ) and GhUGT-3NotI ( 5’-ATCATGCGGCCGCTAACGCATTTCCTGAGATTGTTGG-3’ ) were treated with T4 polynucleotide kinase in 1X T4 DNA ligase buffer (containing 10 mM ATP, New England Biolabs, Ipswich, MA) at 37°C for 1 hour.

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes
    Article Snippet: The Pyrite cloning steps are outline in Fig. . .. Briefly, 2 μl of 10 × T4 DNA ligase buffer (or standard NEB enzyme buffer supplemented with 1 mM ATP), 400 units (1 μl) of NEB T4 DNA ligase, 6 units (0.3 μl) of each NEB restriction enzyme, 0.045 pmol of vector, 10 times (0.450 pmol) of purified insert fragment, and water to bring the final volume to 20 μl are mixed on ice.

    Amplification:

    Article Title: Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection
    Article Snippet: The blunt-ended and phosphorylated DNA was purified with the Qiagen PCR Purification Kit according to the manufacturer’s protocol, diluted to 10 ng/μL in NEB T4 DNA Ligase Buffer (50 mM Tris-HCl, 10 mM MgCl2 , 10 mM dithiothreitol, 1 mM ATP, pH 7.5) and circularized by ligation with 200 units of T4 DNA ligase (NEB) for 15.5 hours at room temperature. .. 70 ng of circular monomer was used as a substrate for rolling-circle amplification at 30 °C for 20 hours in a 100 μL reaction using the Illustra TempliPhi 100 Amplification Kit (GE Healthcare).

    Molecular Cloning:

    Article Title: Designing Calcium-Binding Proteins for Molecular MR Imaging
    Article Snippet: Paragraph title: 2.2. Molecular Cloning Reagents ... Ligation: T4 DNA ligase and ligation buffer (New England Biolabs).

    Ligation:

    Article Title: A Transcript Profiling Approach Reveals an Abscisic Acid-Specific Glycosyltransferase (UGT73C14) Induced in Developing Fiber of Ligon lintless-2 Mutant of Cotton (Gossypium hirsutum L.)
    Article Snippet: Aliquots of the primers GhUGT-Ala2Malc5 ( 5’-GCTCAAGGTCACTTTGTCTTGATC-3’ ) and GhUGT-3NotI ( 5’-ATCATGCGGCCGCTAACGCATTTCCTGAGATTGTTGG-3’ ) were treated with T4 polynucleotide kinase in 1X T4 DNA ligase buffer (containing 10 mM ATP, New England Biolabs, Ipswich, MA) at 37°C for 1 hour. .. Ligation reactions were transformed into XL10-Gold Ultracompetent cells (Agilent Technologies) and selected on solid agar media containing 100 µg/mL ampicillin.

    Article Title: Designing Calcium-Binding Proteins for Molecular MR Imaging
    Article Snippet: .. Ligation: T4 DNA ligase and ligation buffer (New England Biolabs). .. ATP (Sigma).

    Article Title: Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection
    Article Snippet: .. The blunt-ended and phosphorylated DNA was purified with the Qiagen PCR Purification Kit according to the manufacturer’s protocol, diluted to 10 ng/μL in NEB T4 DNA Ligase Buffer (50 mM Tris-HCl, 10 mM MgCl2 , 10 mM dithiothreitol, 1 mM ATP, pH 7.5) and circularized by ligation with 200 units of T4 DNA ligase (NEB) for 15.5 hours at room temperature. .. Circular monomers were gel purified on 1% TAE-Agarose gels.

    Purification:

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes
    Article Snippet: .. Briefly, 2 μl of 10 × T4 DNA ligase buffer (or standard NEB enzyme buffer supplemented with 1 mM ATP), 400 units (1 μl) of NEB T4 DNA ligase, 6 units (0.3 μl) of each NEB restriction enzyme, 0.045 pmol of vector, 10 times (0.450 pmol) of purified insert fragment, and water to bring the final volume to 20 μl are mixed on ice. ..

    Article Title: Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection
    Article Snippet: .. The blunt-ended and phosphorylated DNA was purified with the Qiagen PCR Purification Kit according to the manufacturer’s protocol, diluted to 10 ng/μL in NEB T4 DNA Ligase Buffer (50 mM Tris-HCl, 10 mM MgCl2 , 10 mM dithiothreitol, 1 mM ATP, pH 7.5) and circularized by ligation with 200 units of T4 DNA ligase (NEB) for 15.5 hours at room temperature. .. Circular monomers were gel purified on 1% TAE-Agarose gels.

    Sequencing:

    Article Title: A Transcript Profiling Approach Reveals an Abscisic Acid-Specific Glycosyltransferase (UGT73C14) Induced in Developing Fiber of Ligon lintless-2 Mutant of Cotton (Gossypium hirsutum L.)
    Article Snippet: Cloning and expression of recombinant UTG73C14 in E. coli The coding sequence for cotton UGT73C14 was fused at its N-terminus to the C-terminus of maltose-binding protein (MBP) via introduction into the E. coli expression vector pMAL-c5X. .. Aliquots of the primers GhUGT-Ala2Malc5 ( 5’-GCTCAAGGTCACTTTGTCTTGATC-3’ ) and GhUGT-3NotI ( 5’-ATCATGCGGCCGCTAACGCATTTCCTGAGATTGTTGG-3’ ) were treated with T4 polynucleotide kinase in 1X T4 DNA ligase buffer (containing 10 mM ATP, New England Biolabs, Ipswich, MA) at 37°C for 1 hour.

    Article Title: Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection
    Article Snippet: Library Construction Libraries of target sites were incorporated into double-stranded DNA by PCR with Taq DNA Polymerase (NEB) on a pUC19 starting template with primers “N5-PvuI” and “CCR5-224-N4,” “CCR5-224-N5,” “CCR5-224-N6,” “CCR5-224-N7,” “VF2468-N4,” “VF2468-N5,” “VF2468-N6,” or “VF2468-N7,” yielding an approximately 545-bp product with a Pvu I restriction site adjacent to the library sequence, and purified with the Qiagen PCR Purification Kit. .. The blunt-ended and phosphorylated DNA was purified with the Qiagen PCR Purification Kit according to the manufacturer’s protocol, diluted to 10 ng/μL in NEB T4 DNA Ligase Buffer (50 mM Tris-HCl, 10 mM MgCl2 , 10 mM dithiothreitol, 1 mM ATP, pH 7.5) and circularized by ligation with 200 units of T4 DNA ligase (NEB) for 15.5 hours at room temperature.

    Expressing:

    Article Title: A Transcript Profiling Approach Reveals an Abscisic Acid-Specific Glycosyltransferase (UGT73C14) Induced in Developing Fiber of Ligon lintless-2 Mutant of Cotton (Gossypium hirsutum L.)
    Article Snippet: Paragraph title: Cloning and expression of recombinant UTG73C14 in E. coli ... Aliquots of the primers GhUGT-Ala2Malc5 ( 5’-GCTCAAGGTCACTTTGTCTTGATC-3’ ) and GhUGT-3NotI ( 5’-ATCATGCGGCCGCTAACGCATTTCCTGAGATTGTTGG-3’ ) were treated with T4 polynucleotide kinase in 1X T4 DNA ligase buffer (containing 10 mM ATP, New England Biolabs, Ipswich, MA) at 37°C for 1 hour.

    Polymerase Chain Reaction:

    Article Title: A Transcript Profiling Approach Reveals an Abscisic Acid-Specific Glycosyltransferase (UGT73C14) Induced in Developing Fiber of Ligon lintless-2 Mutant of Cotton (Gossypium hirsutum L.)
    Article Snippet: Aliquots of the primers GhUGT-Ala2Malc5 ( 5’-GCTCAAGGTCACTTTGTCTTGATC-3’ ) and GhUGT-3NotI ( 5’-ATCATGCGGCCGCTAACGCATTTCCTGAGATTGTTGG-3’ ) were treated with T4 polynucleotide kinase in 1X T4 DNA ligase buffer (containing 10 mM ATP, New England Biolabs, Ipswich, MA) at 37°C for 1 hour. .. The resulting PCR product was digested with Not I and ligated into pMAL-c5X that had been previously digested with Xmn I and Not I and treated with calf alkaline intestinal phosphatase.

    Article Title: Designing Calcium-Binding Proteins for Molecular MR Imaging
    Article Snippet: PCR: KOD hot start DNA polymerase and buffer (Novagen). dNTP (Novagen). .. Ligation: T4 DNA ligase and ligation buffer (New England Biolabs).

    Article Title: Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection
    Article Snippet: .. The blunt-ended and phosphorylated DNA was purified with the Qiagen PCR Purification Kit according to the manufacturer’s protocol, diluted to 10 ng/μL in NEB T4 DNA Ligase Buffer (50 mM Tris-HCl, 10 mM MgCl2 , 10 mM dithiothreitol, 1 mM ATP, pH 7.5) and circularized by ligation with 200 units of T4 DNA ligase (NEB) for 15.5 hours at room temperature. .. Circular monomers were gel purified on 1% TAE-Agarose gels.

    Transformation Assay:

    Article Title: A Transcript Profiling Approach Reveals an Abscisic Acid-Specific Glycosyltransferase (UGT73C14) Induced in Developing Fiber of Ligon lintless-2 Mutant of Cotton (Gossypium hirsutum L.)
    Article Snippet: Aliquots of the primers GhUGT-Ala2Malc5 ( 5’-GCTCAAGGTCACTTTGTCTTGATC-3’ ) and GhUGT-3NotI ( 5’-ATCATGCGGCCGCTAACGCATTTCCTGAGATTGTTGG-3’ ) were treated with T4 polynucleotide kinase in 1X T4 DNA ligase buffer (containing 10 mM ATP, New England Biolabs, Ipswich, MA) at 37°C for 1 hour. .. Ligation reactions were transformed into XL10-Gold Ultracompetent cells (Agilent Technologies) and selected on solid agar media containing 100 µg/mL ampicillin.

    Article Title: Designing Calcium-Binding Proteins for Molecular MR Imaging
    Article Snippet: Ligation: T4 DNA ligase and ligation buffer (New England Biolabs). .. Transformation: Competent cells: DH5α and BL21(DE3) pLysS (Invitrogen).

    Recombinant:

    Article Title: A Transcript Profiling Approach Reveals an Abscisic Acid-Specific Glycosyltransferase (UGT73C14) Induced in Developing Fiber of Ligon lintless-2 Mutant of Cotton (Gossypium hirsutum L.)
    Article Snippet: Paragraph title: Cloning and expression of recombinant UTG73C14 in E. coli ... Aliquots of the primers GhUGT-Ala2Malc5 ( 5’-GCTCAAGGTCACTTTGTCTTGATC-3’ ) and GhUGT-3NotI ( 5’-ATCATGCGGCCGCTAACGCATTTCCTGAGATTGTTGG-3’ ) were treated with T4 polynucleotide kinase in 1X T4 DNA ligase buffer (containing 10 mM ATP, New England Biolabs, Ipswich, MA) at 37°C for 1 hour.

    Plasmid Preparation:

    Article Title: A Transcript Profiling Approach Reveals an Abscisic Acid-Specific Glycosyltransferase (UGT73C14) Induced in Developing Fiber of Ligon lintless-2 Mutant of Cotton (Gossypium hirsutum L.)
    Article Snippet: Cloning and expression of recombinant UTG73C14 in E. coli The coding sequence for cotton UGT73C14 was fused at its N-terminus to the C-terminus of maltose-binding protein (MBP) via introduction into the E. coli expression vector pMAL-c5X. .. Aliquots of the primers GhUGT-Ala2Malc5 ( 5’-GCTCAAGGTCACTTTGTCTTGATC-3’ ) and GhUGT-3NotI ( 5’-ATCATGCGGCCGCTAACGCATTTCCTGAGATTGTTGG-3’ ) were treated with T4 polynucleotide kinase in 1X T4 DNA ligase buffer (containing 10 mM ATP, New England Biolabs, Ipswich, MA) at 37°C for 1 hour.

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes
    Article Snippet: .. Briefly, 2 μl of 10 × T4 DNA ligase buffer (or standard NEB enzyme buffer supplemented with 1 mM ATP), 400 units (1 μl) of NEB T4 DNA ligase, 6 units (0.3 μl) of each NEB restriction enzyme, 0.045 pmol of vector, 10 times (0.450 pmol) of purified insert fragment, and water to bring the final volume to 20 μl are mixed on ice. ..

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    New England Biolabs t4 dna ligase ligation protocol
    Effect of Inhibiting dsDNA on Enzyme Self-Adenylylation Rate. The determined rates for self-adenylylation of an uninhibited reaction, 2.5 μM <t>T4</t> DNA ligase (red) and 2.5 μM T4 DNA ligase and inhibited reactions 2.5 μM DNA (blue) and 10 μM DNA (green). The reactions were fit to a single exponential equation ( Eq 6 ) to determine the reaction rate. The uninhibited reaction was determined to have a single turnover rate of 20 s -1 ± 2 s -1 . While the 2.5 μM inhibited reaction had a single turnover rate of 2.8 s -1 ± 0.5 s -1 and the 10 μM inhibited reaction had a single turnover rate of 1.0 s -1 ± 1 s -1 . All reactions were performed a minimum of three times at 16°C. Error reported is the standard error for the replicates.
    T4 Dna Ligase Ligation Protocol, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase ligation protocol/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
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    77
    New England Biolabs ligation reactions in vitro ligation reactions
    Scheme of <t>in</t> <t>vitro</t> <t>ligation</t> selection and sequencing library preparation. For each ligase selected library, an equal amount of 4 random RNA oligos containing a constant region (solid line), a randomized region (wavy line) and a known 3′-nt were combined to make a random oligo pool and used as substrates in a ligation reaction with pre-adenylated SR1 DNA adapter using a specific T4 RNA ligase. The ligated products were reverse transcribed and amplified to introduce the required primer regions for Ion Torrent sequencing. To determine the sequence content of the random RNA oligo pool, each of the four RNA oligos was sequenced independently. First, the oligos were poly A tailed for the random RNA oligo U, C and G or poly C tailed for the random RNA oligo A using poly(A) polymerase. The tailed RNA oligos were then reverse transcribed using primers complementary to the polymer tails ( Supplementary Table S1 ). The cDNA libraries were amplified and processed in the same manner as the ligase selected libraries described above.
    Ligation Reactions In Vitro Ligation Reactions, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs ligation cloning procedures pcr reactions
    Improvement of Ty1 transposition. ( a ) Substitution of alternative promoters in retroelement. ‘Distinct transposants' refers to the number of unique cells in which Ty1 underwent a full retrotransposition cycle at least once. This uniqueness explicitly excludes daughter cells arising from the original transposed variant. ( b ) Transposition rates for BY4741 knockout strains. Transcript ( c ) and cDNA levels ( d ) of engineered Ty1 retroelements. ( e ) Transposition rate of strains overexpressing the initiator methionine tRNA IMT4. ( f ) tRNA iMet overexpression improves cDNA synthesis. Low-copy and high-copy data were collected on different days and hence are normalized to their respective ‘with tRNA Glc' values. ( g ) Transposition rate and mutation rate conferred by retroelements containing cargo of various sizes. ‘Max distinct mutants' refers to the maximum number of mutants attainable in a cargo of a particular length, given a 0.15 kb −1 mutation rate and the maximum number of distinct transposants attainable for a particular cargo size (maximum is calculated over 3, 5 and 7-day induction times). Strains containing the appropriate retroelement were exposed to galactose at high OD for ( g ) and low OD for a and b for 3 days and then plated on uracil-deficient media to count transposants. For c and d , cells were exposed to the appropriate carbon source at high OD for 3 days. Total <t>DNA</t> and RNA was extracted after induction and nucleic acid levels were quantified using quantitative reverse <t>transcriptase–PCR.</t> For e and f , strains containing a genomically integrated retroelement were exposed to galactose at 22 °C at high OD for 3 days. Error bars in a and b represent 95% confidence intervals from biological triplicates. Error bars in e and g represent the s.d. of biological triplicates. For c , d and f error bars represent the s.d. of technical triplicates.
    Ligation Cloning Procedures Pcr Reactions, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of Inhibiting dsDNA on Enzyme Self-Adenylylation Rate. The determined rates for self-adenylylation of an uninhibited reaction, 2.5 μM T4 DNA ligase (red) and 2.5 μM T4 DNA ligase and inhibited reactions 2.5 μM DNA (blue) and 10 μM DNA (green). The reactions were fit to a single exponential equation ( Eq 6 ) to determine the reaction rate. The uninhibited reaction was determined to have a single turnover rate of 20 s -1 ± 2 s -1 . While the 2.5 μM inhibited reaction had a single turnover rate of 2.8 s -1 ± 0.5 s -1 and the 10 μM inhibited reaction had a single turnover rate of 1.0 s -1 ± 1 s -1 . All reactions were performed a minimum of three times at 16°C. Error reported is the standard error for the replicates.

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: Effect of Inhibiting dsDNA on Enzyme Self-Adenylylation Rate. The determined rates for self-adenylylation of an uninhibited reaction, 2.5 μM T4 DNA ligase (red) and 2.5 μM T4 DNA ligase and inhibited reactions 2.5 μM DNA (blue) and 10 μM DNA (green). The reactions were fit to a single exponential equation ( Eq 6 ) to determine the reaction rate. The uninhibited reaction was determined to have a single turnover rate of 20 s -1 ± 2 s -1 . While the 2.5 μM inhibited reaction had a single turnover rate of 2.8 s -1 ± 0.5 s -1 and the 10 μM inhibited reaction had a single turnover rate of 1.0 s -1 ± 1 s -1 . All reactions were performed a minimum of three times at 16°C. Error reported is the standard error for the replicates.

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques:

    T4 DNA Ligase Reaction Model. Modified reaction pathway to include the newly observed reactions in the previously described DNA ligation pathway that are inhibited by the presence of non-nicked dsDNA. A . Non-nicked dsDNA can bind to the deadenylylated form of the enzyme inhibition formation of the adenylylated form of the enzyme. B . Non-nicked dsDNA binds to the Lig-AMP form, preventing complexation with its preferred ds-nDNA substrate.

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: T4 DNA Ligase Reaction Model. Modified reaction pathway to include the newly observed reactions in the previously described DNA ligation pathway that are inhibited by the presence of non-nicked dsDNA. A . Non-nicked dsDNA can bind to the deadenylylated form of the enzyme inhibition formation of the adenylylated form of the enzyme. B . Non-nicked dsDNA binds to the Lig-AMP form, preventing complexation with its preferred ds-nDNA substrate.

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques: Modification, DNA Ligation, Enzyme Inhibition Assay

    k cat /K m Curve for T4 DNA Ligase. The data was obtained through titration of increasing concentrations of a 75mer-ds-nDNA substrate, reacted at 16°C to determine initial reaction rates. T4 DNA ligase concentrations used were 25 pM– 100 pM. The initial rates were plotted against their respective substrate concentrations and fit by: A . a classical uncompetitive substrate inhibition model ( Eq 2 ), where k cat and K m Values of 0.44 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 590 nM ± 170 nM. B . A competitive substrate inhibition for a Bi-Bi Ping-Pong mechanism ( Eq 3 ) k cat and K m values of 0.48 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 54 nM ± 15 nM. All data points are the average of at least three independent experiments, and the error reported is the standard deviation for the replicates.

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: k cat /K m Curve for T4 DNA Ligase. The data was obtained through titration of increasing concentrations of a 75mer-ds-nDNA substrate, reacted at 16°C to determine initial reaction rates. T4 DNA ligase concentrations used were 25 pM– 100 pM. The initial rates were plotted against their respective substrate concentrations and fit by: A . a classical uncompetitive substrate inhibition model ( Eq 2 ), where k cat and K m Values of 0.44 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 590 nM ± 170 nM. B . A competitive substrate inhibition for a Bi-Bi Ping-Pong mechanism ( Eq 3 ) k cat and K m values of 0.48 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 54 nM ± 15 nM. All data points are the average of at least three independent experiments, and the error reported is the standard deviation for the replicates.

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques: Titration, Inhibition, Standard Deviation

    Various Inhibitors Effects on Rate of Nick Sealing. Various concentrations of dsDNA substrates were utilized as potential inhibitors of the T4 DNA ligase steady state ligation reaction on 20 nM of the 75mer-ds-nDNA substrate. All reactions were performed in the presence of 25 pM of T4 DNA ligase, a minimum of three times at 16°C. Error reported is the standard deviation for the replicates.

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: Various Inhibitors Effects on Rate of Nick Sealing. Various concentrations of dsDNA substrates were utilized as potential inhibitors of the T4 DNA ligase steady state ligation reaction on 20 nM of the 75mer-ds-nDNA substrate. All reactions were performed in the presence of 25 pM of T4 DNA ligase, a minimum of three times at 16°C. Error reported is the standard deviation for the replicates.

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques: Ligation, Standard Deviation

    Competition for ds-nDNA-Binding by dsDNA. Lane one contains 4 nM of the 75mer-ds-nDNA substrate alone, lanes 2–6 show shifting of the 4 nM substrate into a completely bound state as the concentration of T4 DNA ligase is increased from 100 nM– 1000 nM. Lanes 7–11 are of a titration of increasing concentrations of the unlabeled I-75-dsDNA oligo into a reaction containing 4 nM labeled nicked substrate and 1000 nM T4 DNA ligase. EMSA reactions were all performed and electrophoresed at room temperature (22°C).

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: Competition for ds-nDNA-Binding by dsDNA. Lane one contains 4 nM of the 75mer-ds-nDNA substrate alone, lanes 2–6 show shifting of the 4 nM substrate into a completely bound state as the concentration of T4 DNA ligase is increased from 100 nM– 1000 nM. Lanes 7–11 are of a titration of increasing concentrations of the unlabeled I-75-dsDNA oligo into a reaction containing 4 nM labeled nicked substrate and 1000 nM T4 DNA ligase. EMSA reactions were all performed and electrophoresed at room temperature (22°C).

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques: Binding Assay, Concentration Assay, Titration, Labeling

    Scheme of in vitro ligation selection and sequencing library preparation. For each ligase selected library, an equal amount of 4 random RNA oligos containing a constant region (solid line), a randomized region (wavy line) and a known 3′-nt were combined to make a random oligo pool and used as substrates in a ligation reaction with pre-adenylated SR1 DNA adapter using a specific T4 RNA ligase. The ligated products were reverse transcribed and amplified to introduce the required primer regions for Ion Torrent sequencing. To determine the sequence content of the random RNA oligo pool, each of the four RNA oligos was sequenced independently. First, the oligos were poly A tailed for the random RNA oligo U, C and G or poly C tailed for the random RNA oligo A using poly(A) polymerase. The tailed RNA oligos were then reverse transcribed using primers complementary to the polymer tails ( Supplementary Table S1 ). The cDNA libraries were amplified and processed in the same manner as the ligase selected libraries described above.

    Journal: Nucleic Acids Research

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation

    doi: 10.1093/nar/gkr1263

    Figure Lengend Snippet: Scheme of in vitro ligation selection and sequencing library preparation. For each ligase selected library, an equal amount of 4 random RNA oligos containing a constant region (solid line), a randomized region (wavy line) and a known 3′-nt were combined to make a random oligo pool and used as substrates in a ligation reaction with pre-adenylated SR1 DNA adapter using a specific T4 RNA ligase. The ligated products were reverse transcribed and amplified to introduce the required primer regions for Ion Torrent sequencing. To determine the sequence content of the random RNA oligo pool, each of the four RNA oligos was sequenced independently. First, the oligos were poly A tailed for the random RNA oligo U, C and G or poly C tailed for the random RNA oligo A using poly(A) polymerase. The tailed RNA oligos were then reverse transcribed using primers complementary to the polymer tails ( Supplementary Table S1 ). The cDNA libraries were amplified and processed in the same manner as the ligase selected libraries described above.

    Article Snippet: Ligation reactions In vitro ligation reactions containing defined RNA substrates were carried out in 10 µl reactions containing 0.5 µM miRNA, 1 µM adenylated DNA adapter, 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 40 U of Murine RNase Inhibitor (New England Biolabs), 12.5% PEG8000 and 0.1 µM ( , and ) or 1.3 µM ligase ( ).

    Techniques: In Vitro, Ligation, Selection, Sequencing, Amplification, Introduce

    Comparison of miRNA ligation efficiencies using SR1 versus SR1-S adapter. ( A ) Sequences and predicted secondary structures of SR1 and SR1-S adapters. The underlined sequence is shared by both adapters. The secondary structures of SR1 and SR1-S are presented in bracket and dot form where brackets represent base paired nucleotides and dots represent unpaired nucleotides. ( B ) Ligation reactions of miRNAs with the SR1 or SR1-S adapter were performed using Rnl2tr. Ligation products were resolved in 15% TBE–urea gels and stained with SYBR Gold. ( C ) Ligation efficiency was calculated and plotted. The data points plotted represent average ligation efficiency ± standard deviation from two independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation

    doi: 10.1093/nar/gkr1263

    Figure Lengend Snippet: Comparison of miRNA ligation efficiencies using SR1 versus SR1-S adapter. ( A ) Sequences and predicted secondary structures of SR1 and SR1-S adapters. The underlined sequence is shared by both adapters. The secondary structures of SR1 and SR1-S are presented in bracket and dot form where brackets represent base paired nucleotides and dots represent unpaired nucleotides. ( B ) Ligation reactions of miRNAs with the SR1 or SR1-S adapter were performed using Rnl2tr. Ligation products were resolved in 15% TBE–urea gels and stained with SYBR Gold. ( C ) Ligation efficiency was calculated and plotted. The data points plotted represent average ligation efficiency ± standard deviation from two independent experiments.

    Article Snippet: Ligation reactions In vitro ligation reactions containing defined RNA substrates were carried out in 10 µl reactions containing 0.5 µM miRNA, 1 µM adenylated DNA adapter, 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 40 U of Murine RNase Inhibitor (New England Biolabs), 12.5% PEG8000 and 0.1 µM ( , and ) or 1.3 µM ligase ( ).

    Techniques: Ligation, Sequencing, Staining, Standard Deviation

    Improving miRNA ligation efficiency using redesigned adapters. ( A ) Ligation of miRNAs with SR1 adapter or a new adapter specifically designed for each miRNA. Ligation reactions were performed using Rnl2tr. Ligation products were resolved in 15% TBE–urea gels, stained with SYBR Gold to visualize the nucleic acids. ( B ) Ligation efficiency was determined and plotted. The data points represent average ligation efficiency ± standard deviation from two independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation

    doi: 10.1093/nar/gkr1263

    Figure Lengend Snippet: Improving miRNA ligation efficiency using redesigned adapters. ( A ) Ligation of miRNAs with SR1 adapter or a new adapter specifically designed for each miRNA. Ligation reactions were performed using Rnl2tr. Ligation products were resolved in 15% TBE–urea gels, stained with SYBR Gold to visualize the nucleic acids. ( B ) Ligation efficiency was determined and plotted. The data points represent average ligation efficiency ± standard deviation from two independent experiments.

    Article Snippet: Ligation reactions In vitro ligation reactions containing defined RNA substrates were carried out in 10 µl reactions containing 0.5 µM miRNA, 1 µM adenylated DNA adapter, 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 40 U of Murine RNase Inhibitor (New England Biolabs), 12.5% PEG8000 and 0.1 µM ( , and ) or 1.3 µM ligase ( ).

    Techniques: Ligation, Staining, Standard Deviation

    Improvement of miRNA ligation efficiency using a randomized adapter in the presence of mouse ES cell small RNAs. ( A ) Scheme of ligation reactions in the presence of mouse ES cell small RNAs. Each reaction contained 0.75 fmol of a 5′- 32 P labeled miRNA mixed with a 500-fold excess of mouse ES cell small RNAs and either the SR1 or the SR1-R adapter. Gray lines represent the ES cell small RNAs and the black line with an asterisk represents the radio-labeled miRNA. The SR1-R adapter is shown in black with a wavy line representing the random region at the 5′-end. Ligation products were resolved on 15% TBE–urea acrylamide gels, exposed to phosphor storage screens, and scanned. The ligated radio-labeled miRNA appears as a higher molecular weight band than unligated miRNA. ( B ) Representative results of ligation gels as described in A. ( C ) Comparison of ligation efficiency of miRNAs with the SR1 and SR1-R adapters. The intensity of ligated and unligated bands in each lane was quantified and ligation efficiencies were determined by calculating the percentage of ligated miRNA from the total miRNA. The data are represented as the average ± standard deviation ligation efficiency from two independent experimental replicates.

    Journal: Nucleic Acids Research

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation

    doi: 10.1093/nar/gkr1263

    Figure Lengend Snippet: Improvement of miRNA ligation efficiency using a randomized adapter in the presence of mouse ES cell small RNAs. ( A ) Scheme of ligation reactions in the presence of mouse ES cell small RNAs. Each reaction contained 0.75 fmol of a 5′- 32 P labeled miRNA mixed with a 500-fold excess of mouse ES cell small RNAs and either the SR1 or the SR1-R adapter. Gray lines represent the ES cell small RNAs and the black line with an asterisk represents the radio-labeled miRNA. The SR1-R adapter is shown in black with a wavy line representing the random region at the 5′-end. Ligation products were resolved on 15% TBE–urea acrylamide gels, exposed to phosphor storage screens, and scanned. The ligated radio-labeled miRNA appears as a higher molecular weight band than unligated miRNA. ( B ) Representative results of ligation gels as described in A. ( C ) Comparison of ligation efficiency of miRNAs with the SR1 and SR1-R adapters. The intensity of ligated and unligated bands in each lane was quantified and ligation efficiencies were determined by calculating the percentage of ligated miRNA from the total miRNA. The data are represented as the average ± standard deviation ligation efficiency from two independent experimental replicates.

    Article Snippet: Ligation reactions In vitro ligation reactions containing defined RNA substrates were carried out in 10 µl reactions containing 0.5 µM miRNA, 1 µM adenylated DNA adapter, 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 40 U of Murine RNase Inhibitor (New England Biolabs), 12.5% PEG8000 and 0.1 µM ( , and ) or 1.3 µM ligase ( ).

    Techniques: Ligation, Labeling, Molecular Weight, Standard Deviation

    Improvement of miRNA ligation efficiencies using a randomized adapter, SR1-R. ( A ) Ligation reactions were performed with Rnl2tr and the SR1 or SR1-R adapter. Ligation products were resolved in 15% TBE–urea gels and stained with SYBR Gold to visualize the nucleic acids. ( B ) Ligation efficiencies of 24 miRNAs with the SR1 or SR1-R adapter were determined and plotted. The data are represented as the average ± standard deviation from two experimental replicates.

    Journal: Nucleic Acids Research

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation

    doi: 10.1093/nar/gkr1263

    Figure Lengend Snippet: Improvement of miRNA ligation efficiencies using a randomized adapter, SR1-R. ( A ) Ligation reactions were performed with Rnl2tr and the SR1 or SR1-R adapter. Ligation products were resolved in 15% TBE–urea gels and stained with SYBR Gold to visualize the nucleic acids. ( B ) Ligation efficiencies of 24 miRNAs with the SR1 or SR1-R adapter were determined and plotted. The data are represented as the average ± standard deviation from two experimental replicates.

    Article Snippet: Ligation reactions In vitro ligation reactions containing defined RNA substrates were carried out in 10 µl reactions containing 0.5 µM miRNA, 1 µM adenylated DNA adapter, 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 40 U of Murine RNase Inhibitor (New England Biolabs), 12.5% PEG8000 and 0.1 µM ( , and ) or 1.3 µM ligase ( ).

    Techniques: Ligation, Staining, Standard Deviation

    3′-adapter ligation efficiencies of miRNAs. ( A ) Each miRNA was incubated in a ligation reaction containing Rnl2tr with or without SR1 adapter. The ligation products were separated on 15% TBE–urea gels and visualized with SYBR Gold. Ligated products correspond to high molecular weight bands, which only appear in reactions with SR1 adapter. Unligated miRNAs and SR1 adapters remain as lower molecular weight bands. ( B ) The ligation efficiency of each miRNA was determined and plotted. The data are represented as the average ± standard deviation from two experimental replicates.

    Journal: Nucleic Acids Research

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation

    doi: 10.1093/nar/gkr1263

    Figure Lengend Snippet: 3′-adapter ligation efficiencies of miRNAs. ( A ) Each miRNA was incubated in a ligation reaction containing Rnl2tr with or without SR1 adapter. The ligation products were separated on 15% TBE–urea gels and visualized with SYBR Gold. Ligated products correspond to high molecular weight bands, which only appear in reactions with SR1 adapter. Unligated miRNAs and SR1 adapters remain as lower molecular weight bands. ( B ) The ligation efficiency of each miRNA was determined and plotted. The data are represented as the average ± standard deviation from two experimental replicates.

    Article Snippet: Ligation reactions In vitro ligation reactions containing defined RNA substrates were carried out in 10 µl reactions containing 0.5 µM miRNA, 1 µM adenylated DNA adapter, 50 mM Tris–HCl pH 7.5, 10 mM MgCl2 , 1 mM DTT, 40 U of Murine RNase Inhibitor (New England Biolabs), 12.5% PEG8000 and 0.1 µM ( , and ) or 1.3 µM ligase ( ).

    Techniques: Ligation, Incubation, Molecular Weight, Standard Deviation

    Improvement of Ty1 transposition. ( a ) Substitution of alternative promoters in retroelement. ‘Distinct transposants' refers to the number of unique cells in which Ty1 underwent a full retrotransposition cycle at least once. This uniqueness explicitly excludes daughter cells arising from the original transposed variant. ( b ) Transposition rates for BY4741 knockout strains. Transcript ( c ) and cDNA levels ( d ) of engineered Ty1 retroelements. ( e ) Transposition rate of strains overexpressing the initiator methionine tRNA IMT4. ( f ) tRNA iMet overexpression improves cDNA synthesis. Low-copy and high-copy data were collected on different days and hence are normalized to their respective ‘with tRNA Glc' values. ( g ) Transposition rate and mutation rate conferred by retroelements containing cargo of various sizes. ‘Max distinct mutants' refers to the maximum number of mutants attainable in a cargo of a particular length, given a 0.15 kb −1 mutation rate and the maximum number of distinct transposants attainable for a particular cargo size (maximum is calculated over 3, 5 and 7-day induction times). Strains containing the appropriate retroelement were exposed to galactose at high OD for ( g ) and low OD for a and b for 3 days and then plated on uracil-deficient media to count transposants. For c and d , cells were exposed to the appropriate carbon source at high OD for 3 days. Total DNA and RNA was extracted after induction and nucleic acid levels were quantified using quantitative reverse transcriptase–PCR. For e and f , strains containing a genomically integrated retroelement were exposed to galactose at 22 °C at high OD for 3 days. Error bars in a and b represent 95% confidence intervals from biological triplicates. Error bars in e and g represent the s.d. of biological triplicates. For c , d and f error bars represent the s.d. of technical triplicates.

    Journal: Nature Communications

    Article Title: In vivo continuous evolution of genes and pathways in yeast

    doi: 10.1038/ncomms13051

    Figure Lengend Snippet: Improvement of Ty1 transposition. ( a ) Substitution of alternative promoters in retroelement. ‘Distinct transposants' refers to the number of unique cells in which Ty1 underwent a full retrotransposition cycle at least once. This uniqueness explicitly excludes daughter cells arising from the original transposed variant. ( b ) Transposition rates for BY4741 knockout strains. Transcript ( c ) and cDNA levels ( d ) of engineered Ty1 retroelements. ( e ) Transposition rate of strains overexpressing the initiator methionine tRNA IMT4. ( f ) tRNA iMet overexpression improves cDNA synthesis. Low-copy and high-copy data were collected on different days and hence are normalized to their respective ‘with tRNA Glc' values. ( g ) Transposition rate and mutation rate conferred by retroelements containing cargo of various sizes. ‘Max distinct mutants' refers to the maximum number of mutants attainable in a cargo of a particular length, given a 0.15 kb −1 mutation rate and the maximum number of distinct transposants attainable for a particular cargo size (maximum is calculated over 3, 5 and 7-day induction times). Strains containing the appropriate retroelement were exposed to galactose at high OD for ( g ) and low OD for a and b for 3 days and then plated on uracil-deficient media to count transposants. For c and d , cells were exposed to the appropriate carbon source at high OD for 3 days. Total DNA and RNA was extracted after induction and nucleic acid levels were quantified using quantitative reverse transcriptase–PCR. For e and f , strains containing a genomically integrated retroelement were exposed to galactose at 22 °C at high OD for 3 days. Error bars in a and b represent 95% confidence intervals from biological triplicates. Error bars in e and g represent the s.d. of biological triplicates. For c , d and f error bars represent the s.d. of technical triplicates.

    Article Snippet: Ligation cloning procedures PCR reactions were performed with Q5 Hot-Start DNA Polymerase (NEB) according to the manufacturer's specifications.

    Techniques: Variant Assay, Knock-Out, Over Expression, Gas Chromatography, Mutagenesis, Polymerase Chain Reaction