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    New England Biolabs t4 dna ligase new england biolabs
    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 New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 dna ligase
    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, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25057 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs r0147m t4 dna ligase new england biolabs
    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.
    R0147m T4 Dna Ligase New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs b0202 q5 high fidelity dna polymerase new england biolabs
    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.
    B0202 Q5 High Fidelity Dna Polymerase New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs 3195l t4 dna ligase reaction buffer new england biolabs cat
    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.
    3195l T4 Dna Ligase Reaction Buffer New England Biolabs Cat, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 dna ligase new england biolabs cat
    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 New England Biolabs Cat, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa t4 dna ligase
    Schematic illustration of noncanonical self-assembly and biomedical applications of multifunctional DNA NFs. ( a ) RCR-based assembly of NFs. The linear DNA template was first hybridized with a primer and then ligated to form a circular template by <t>T4</t> DNA
    T4 Dna Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher t4 dna ligase buffer
    Schematic illustration of noncanonical self-assembly and biomedical applications of multifunctional DNA NFs. ( a ) RCR-based assembly of NFs. The linear DNA template was first hybridized with a primer and then ligated to form a circular template by <t>T4</t> DNA
    T4 Dna Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 847 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

    Radioprotection of T4 DNA ligase by rationally-designed peptides. T4 DNA ligase was irradiated in the indicated mixtures and then tested for residual activity by incubation with linearized (L) pUC19 DNA (2.686 kbp). Formation of supercoiled (SC) and open-circular (OC) DNA forms were determined by agarose gel electrophoresis. Peptides were added to 25 mM Pi with or without 1 mM MnCl 2 to the following concentrations: DP1-L, 3 mM; DP1-D, 3 mM; DP2, 3 mM; OP1, 3.75 mM; HP1, 5 mM. Note, the final concentration of peptides in the reaction mixtures corresponded to 30 mM of total amino acid residues. Other abbreviations: Pi, potassium phosphate buffer (pH 7.4); M, DNA size standards (Mass Ruler DNA Ladder mix; Fermentas, Glen Burnie, MD).

    Journal: PLoS ONE

    Article Title: MDP: A Deinococcus Mn2+-Decapeptide Complex Protects Mice from Ionizing Radiation

    doi: 10.1371/journal.pone.0160575

    Figure Lengend Snippet: Radioprotection of T4 DNA ligase by rationally-designed peptides. T4 DNA ligase was irradiated in the indicated mixtures and then tested for residual activity by incubation with linearized (L) pUC19 DNA (2.686 kbp). Formation of supercoiled (SC) and open-circular (OC) DNA forms were determined by agarose gel electrophoresis. Peptides were added to 25 mM Pi with or without 1 mM MnCl 2 to the following concentrations: DP1-L, 3 mM; DP1-D, 3 mM; DP2, 3 mM; OP1, 3.75 mM; HP1, 5 mM. Note, the final concentration of peptides in the reaction mixtures corresponded to 30 mM of total amino acid residues. Other abbreviations: Pi, potassium phosphate buffer (pH 7.4); M, DNA size standards (Mass Ruler DNA Ladder mix; Fermentas, Glen Burnie, MD).

    Article Snippet: T4 DNA ligase T4 DNA ligase (2,000 U/μl) (New England Biolabs, Ipswich, MA) was diluted in the various reagent-mixtures to 2 U/μl for irradiation.

    Techniques: Irradiation, Activity Assay, Incubation, Agarose Gel Electrophoresis, Concentration Assay

    BRCA1 expression counteracts mutant p53 GOF activity on DNA repair assay (A) Comparison of ligation products of 5′-cohesive-ended linear DNA in the presence of T4 DNA ligase alone (lane 3) or following pre-incubation with whole protein extracts derived from H1299 cells transfected with mutp53R175H and BRCA1 expressing vectors in separate reactions (lanes 5 and 7, respectively) or in co-trasfection conditions (lane 6). (B) Whole protein extracts (40 μg) used in the T4 DNA ligase assay previously described were subjected to Western blot analysis and probed with the indicated antibodies. (C-E) SKBr3 cells were transiently transfected with ApaI-linearized pSI-CHECK2 vector (c) and with either siRNA oligos indicated in the figures (d) and (e). After 48 h from the transfection the cells were harvested and the functional changes in NHEJ were assessed measuring the Firefly Luciferase activity. Luciferase activity was expressed as (Firefly/protein amount) × (1/Renilla). Columns , means from two independent assays each of them was done in triplicate; bars , SD. P -values were calculated with two tailed t-test. Statistically significant results were with p -value

    Journal: Oncotarget

    Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression

    doi:

    Figure Lengend Snippet: BRCA1 expression counteracts mutant p53 GOF activity on DNA repair assay (A) Comparison of ligation products of 5′-cohesive-ended linear DNA in the presence of T4 DNA ligase alone (lane 3) or following pre-incubation with whole protein extracts derived from H1299 cells transfected with mutp53R175H and BRCA1 expressing vectors in separate reactions (lanes 5 and 7, respectively) or in co-trasfection conditions (lane 6). (B) Whole protein extracts (40 μg) used in the T4 DNA ligase assay previously described were subjected to Western blot analysis and probed with the indicated antibodies. (C-E) SKBr3 cells were transiently transfected with ApaI-linearized pSI-CHECK2 vector (c) and with either siRNA oligos indicated in the figures (d) and (e). After 48 h from the transfection the cells were harvested and the functional changes in NHEJ were assessed measuring the Firefly Luciferase activity. Luciferase activity was expressed as (Firefly/protein amount) × (1/Renilla). Columns , means from two independent assays each of them was done in triplicate; bars , SD. P -values were calculated with two tailed t-test. Statistically significant results were with p -value

    Article Snippet: 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of T4 DNA ligase (200 U, New England Biolabs).

    Techniques: Expressing, Mutagenesis, Activity Assay, Ligation, Incubation, Derivative Assay, Transfection, Western Blot, Plasmid Preparation, Functional Assay, Non-Homologous End Joining, Luciferase, Two Tailed Test

    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

    Schematic illustration of noncanonical self-assembly and biomedical applications of multifunctional DNA NFs. ( a ) RCR-based assembly of NFs. The linear DNA template was first hybridized with a primer and then ligated to form a circular template by T4 DNA

    Journal: Nature protocols

    Article Title: Preparation and biomedical applications of programmable and multifunctional DNA nanoflowers

    doi: 10.1038/nprot.2015.078

    Figure Lengend Snippet: Schematic illustration of noncanonical self-assembly and biomedical applications of multifunctional DNA NFs. ( a ) RCR-based assembly of NFs. The linear DNA template was first hybridized with a primer and then ligated to form a circular template by T4 DNA

    Article Snippet: Acetonitrile (CH3 CN; Sigma-Aldrich, cat. no. L810045-06) Oxidizer (0.02 M, tetrahydrofuran:H2 O:pyridine:iodine = 90.54:9.05:0.41:0.43 (vol/vol/vol/wt); Sigma-Aldrich, cat. no. L860045-06) dA-CE phosphoramidite (Glen Research, cat. no. 10-1000-10e, 1.0g) dmf-dG-CE phosphoramidite (Glen Research, cat. no. 10-1020-10e, 1.0g) dT-CE phosphoramidite (Glen Research, cat. no. 10-1030-10e, 1.0g) ac-dC-CE phosphoramidite (Glen Research, cat. no. 10-1015-10e, 1.0g) Chemical phosphorylation reagent II (CPR II; Glen Research, cat. no. 10-1901-90, 100μmoles) Thymidine lcaa CPG (1,000 Å, ChemGenes, cat. no. N-5104, 5g) Deoxyadenosine ( n -bz) 3′-lcaa CPG (1,000 Å, ChemGenes, cat. no. N-5101, 5g) Ammonium hydroxide (Sinopharm Chemical Reagent, cat. no. 10002118) Methylamine water solution (Sinopharm Chemical Reagent, cat. no. 80081228) Absolute ethanol (General-Reagent, cat. no. P1090446) Glacial acetic acid (Sinopharm Chemical Reagent, cat. no. 10000218) Triethylamine ((C2 H5 )3 N; Sigma-Aldrich, cat. no. T0886-500ML) Sodium chloride (NaCl; Adamas, cat. no. 81793B) Magnesium chloride hexahydrate (MgCl2 ·6H2 O; Sinopharm Chemical Reagent, cat. no. 10012818) Acetonitrile (CH3 CN; Adamas, cat. no. 80988C) T4 DNA ligase (400,000 units/ml; New England Biolabs, cat. no. M0202S) DNA ligation buffer, 10× (10×(50 mM Tris-HCl, 1 mM MgCl2 , 1 mM ATP and 1 mM DTT); New England Biolabs, cat. no. M0202S) dNTP (10 mM; Takara, cat. no. 4019) Cyanine 3-dUTP (1 mM; Enzo Life Sciences, cat. no. ENZ-42501) Cyanine 5-dUTP (1 mM; Enzo Life Sciences, cat. no. ENZ-42502) Fluorescein-12-ddUTP (1 mM; Enzo Life Sciences, cat. no. ENZ-42833) (6) ROX-12-ddUTP (0.1 mM; Enzo Life Sciences, cat. no. ENZ-42857) BSA, 10× (New England Biolabs, cat. no. M0269S) Phi29 DNA polymerase (10,000 units/ml; New England Biolabs, cat. no. M0269S) phi29 DNA polymerase buffer, 10× (10×(50 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM MgCl2 and 4 mM DTT); New England Biolabs, cat. no. M0269S) Dulbecco’s PBS (1× DPBS basic; Gibco, cat. no. C14190500BT-500ml) Agarose (HydraGene, cat. no. R9012LE-100g) Ethidium bromide (Dingguo, cat. no. NEP028) !

    Techniques: