m0202  (New England Biolabs)


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    Name:
    T4 DNA Ligase
    Description:
    T4 DNA Ligase 100 000 units
    Catalog Number:
    M0202L
    Price:
    256
    Category:
    DNA Ligases
    Size:
    100 000 units
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    Structured Review

    New England Biolabs m0202
    T4 DNA Ligase
    T4 DNA Ligase 100 000 units
    https://www.bioz.com/result/m0202/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    m0202 - by Bioz Stars, 2021-06
    99/100 stars

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    Related Articles

    Plasmid Preparation:

    Article Title:
    Article Snippet: This result is suggestive of substrate inhibition, quite likely due to the nonspecific interaction of the ligase with the DNA. .. Indeed, reaction of 100 nm substrate 1 with 0.1 nm T4 DNA ligase in the presence of added carrier DNA (100 μg/ml pUC19 vector, preincubated 10 min with ligase, ∼150 μm in base pairs) gave a k obs value that was half that found when reacting 100 nm substrate 1 in the absence of added carrier (5 μm in base pairs), giving confidence that the small decrease in turnover rate seen at 1 μm substrate (50 μm base pairs) can be explained primarily by nonspecific inhibition by binding to dsDNA. ..

    Inhibition:

    Article Title:
    Article Snippet: This result is suggestive of substrate inhibition, quite likely due to the nonspecific interaction of the ligase with the DNA. .. Indeed, reaction of 100 nm substrate 1 with 0.1 nm T4 DNA ligase in the presence of added carrier DNA (100 μg/ml pUC19 vector, preincubated 10 min with ligase, ∼150 μm in base pairs) gave a k obs value that was half that found when reacting 100 nm substrate 1 in the absence of added carrier (5 μm in base pairs), giving confidence that the small decrease in turnover rate seen at 1 μm substrate (50 μm base pairs) can be explained primarily by nonspecific inhibition by binding to dsDNA. ..

    Binding Assay:

    Article Title:
    Article Snippet: This result is suggestive of substrate inhibition, quite likely due to the nonspecific interaction of the ligase with the DNA. .. Indeed, reaction of 100 nm substrate 1 with 0.1 nm T4 DNA ligase in the presence of added carrier DNA (100 μg/ml pUC19 vector, preincubated 10 min with ligase, ∼150 μm in base pairs) gave a k obs value that was half that found when reacting 100 nm substrate 1 in the absence of added carrier (5 μm in base pairs), giving confidence that the small decrease in turnover rate seen at 1 μm substrate (50 μm base pairs) can be explained primarily by nonspecific inhibition by binding to dsDNA. ..

    other:

    Article Title: Efficient assembly of very short oligonucleotides using T4 DNA Ligase
    Article Snippet: Specifically, T4 DNA Ligase has been known for some time to be capable of joining oligos as small as pentamers and hexamers on a complete template [ ], however ligases such as Tth DNA Ligase are severely inefficient at the hexamer level [ ].

    Article Title:
    Article Snippet: Given past reports of a weak affinity for non-nicked DNA by T4 DNA ligase, this result may seem surprising.

    Article Title: Circle ligation of in vitro assembled chromatin indicates a highly flexible structure
    Article Snippet: Mechanistic studies with naked DNA indicate that Eco RI cohesive ends associate and dissociate rapidly many times before covalent sealing by T4 DNA ligase ( ).

    Marker:

    Article Title: Efficient strategy for introducing large and multiple changes in plasmid DNA
    Article Snippet: .. Phusion® high-fidelity DNA polymerase, DNA marker, Taq DNA polymerase, T4-PNK, and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA, USA). .. Human cDNA library was purchased from Clontech Laboratories (Mountain View, CA, USA).

    Ligation:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). .. Ligation assays were performed with T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, PBCV1 DNA ligase, hLig3, and E . coli DNA ligase A. .. Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across a minimum of 3 replicates. (TIF).

    Activity Assay:

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends
    Article Snippet: The failure to observe ligation products in presence of HMO2 as well as the inability of exonuclease III to digest the DNA suggests that HMO2 may be binding to the ends of the DNA duplex to prevent access to both T4 DNA ligase and exonuclease III. .. Also note that failure to join DNA ends is not due to HMO2 merely interacting with the ligase to prevent its activity, as evidenced by the activity of T4 DNA ligase on an internal DNA nick in the presence of HMO2 ( C). ..

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    New England Biolabs t4 dna ligase
    Mutations in larger plasmids. ( A ) Schematic representation of LFEAP mutagenesis in large plasmids. The first-round PCRs cut large plasmids into small pieces (~5 kb) with mutagenic ends. The second-round PCRs and the subsequent annealing yield multi-part DNAs with sticky ends, which can be seamlessly joined by <t>T4</t> DNA ligase simultaneously. ( B ) Introduction of mutations in a 25 kb plasmid. Electrophoresis on a 1% agarose gel shows the DNA products generated by the procedure described in the Supplementary Methods. Lanes 1–5: DNA fragments 1 to 5 generated by first-round PCRs. Lane 6: mixture of annealed multi-part DNAs with sticky ends generated by second-round PCRs and the subsequent annealing. Lane 7: the mixture as shown in lane 6 treated with T4 DNA ligase. Lane 8: 1 kb DNA ladder. ( C ) Introduction of mutations in a 50 kb plasmid. Electrophoresis on a 1% agarose gel shows the DNA products generated by the procedure shown in the Supplementary Methods. Lanes 1–10: DNA fragments 1 to 10 generated by first-round PCRs. Lane 11: mixture of annealed multi-part DNAs with sticky ends generated by second-round PCRs and the subsequent annealing. Lane 12: the mixture as shown in lane 11 treated with T4 DNA ligase. Lane 13: 1 kb DNA ladder. ( D ) Electrophoresis on a 0.5% agarose gel of a 25 kb plasmid. Lane 1: 25 kb plasmid before introducing mutations. Lanes 2–6: 25 kb plasmids after introducing mutations propagated from five single colonies. Lane 7: GeneRuler high range DNA ladder (Thermo Fisher Scientific). Incorrect patterns are marked with a ‘×’. ( E .
    T4 Dna Ligase, 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
    https://www.bioz.com/result/t4 dna ligase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Mutations in larger plasmids. ( A ) Schematic representation of LFEAP mutagenesis in large plasmids. The first-round PCRs cut large plasmids into small pieces (~5 kb) with mutagenic ends. The second-round PCRs and the subsequent annealing yield multi-part DNAs with sticky ends, which can be seamlessly joined by T4 DNA ligase simultaneously. ( B ) Introduction of mutations in a 25 kb plasmid. Electrophoresis on a 1% agarose gel shows the DNA products generated by the procedure described in the Supplementary Methods. Lanes 1–5: DNA fragments 1 to 5 generated by first-round PCRs. Lane 6: mixture of annealed multi-part DNAs with sticky ends generated by second-round PCRs and the subsequent annealing. Lane 7: the mixture as shown in lane 6 treated with T4 DNA ligase. Lane 8: 1 kb DNA ladder. ( C ) Introduction of mutations in a 50 kb plasmid. Electrophoresis on a 1% agarose gel shows the DNA products generated by the procedure shown in the Supplementary Methods. Lanes 1–10: DNA fragments 1 to 10 generated by first-round PCRs. Lane 11: mixture of annealed multi-part DNAs with sticky ends generated by second-round PCRs and the subsequent annealing. Lane 12: the mixture as shown in lane 11 treated with T4 DNA ligase. Lane 13: 1 kb DNA ladder. ( D ) Electrophoresis on a 0.5% agarose gel of a 25 kb plasmid. Lane 1: 25 kb plasmid before introducing mutations. Lanes 2–6: 25 kb plasmids after introducing mutations propagated from five single colonies. Lane 7: GeneRuler high range DNA ladder (Thermo Fisher Scientific). Incorrect patterns are marked with a ‘×’. ( E .

    Journal: Scientific Reports

    Article Title: Efficient strategy for introducing large and multiple changes in plasmid DNA

    doi: 10.1038/s41598-018-20169-8

    Figure Lengend Snippet: Mutations in larger plasmids. ( A ) Schematic representation of LFEAP mutagenesis in large plasmids. The first-round PCRs cut large plasmids into small pieces (~5 kb) with mutagenic ends. The second-round PCRs and the subsequent annealing yield multi-part DNAs with sticky ends, which can be seamlessly joined by T4 DNA ligase simultaneously. ( B ) Introduction of mutations in a 25 kb plasmid. Electrophoresis on a 1% agarose gel shows the DNA products generated by the procedure described in the Supplementary Methods. Lanes 1–5: DNA fragments 1 to 5 generated by first-round PCRs. Lane 6: mixture of annealed multi-part DNAs with sticky ends generated by second-round PCRs and the subsequent annealing. Lane 7: the mixture as shown in lane 6 treated with T4 DNA ligase. Lane 8: 1 kb DNA ladder. ( C ) Introduction of mutations in a 50 kb plasmid. Electrophoresis on a 1% agarose gel shows the DNA products generated by the procedure shown in the Supplementary Methods. Lanes 1–10: DNA fragments 1 to 10 generated by first-round PCRs. Lane 11: mixture of annealed multi-part DNAs with sticky ends generated by second-round PCRs and the subsequent annealing. Lane 12: the mixture as shown in lane 11 treated with T4 DNA ligase. Lane 13: 1 kb DNA ladder. ( D ) Electrophoresis on a 0.5% agarose gel of a 25 kb plasmid. Lane 1: 25 kb plasmid before introducing mutations. Lanes 2–6: 25 kb plasmids after introducing mutations propagated from five single colonies. Lane 7: GeneRuler high range DNA ladder (Thermo Fisher Scientific). Incorrect patterns are marked with a ‘×’. ( E .

    Article Snippet: Phusion® high-fidelity DNA polymerase, DNA marker, Taq DNA polymerase, T4-PNK, and T4 DNA ligase were purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Mutagenesis, Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis, Generated

    Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.

    Journal: Nucleic Acids Research

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    doi: 10.1093/nar/gkp695

    Figure Lengend Snippet: Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.

    Article Snippet: Also note that failure to join DNA ends is not due to HMO2 merely interacting with the ligase to prevent its activity, as evidenced by the activity of T4 DNA ligase on an internal DNA nick in the presence of HMO2 ( C).

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis

    HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.

    Journal: Nucleic Acids Research

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    doi: 10.1093/nar/gkp695

    Figure Lengend Snippet: HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.

    Article Snippet: Also note that failure to join DNA ends is not due to HMO2 merely interacting with the ligase to prevent its activity, as evidenced by the activity of T4 DNA ligase on an internal DNA nick in the presence of HMO2 ( C).

    Techniques: Ligation

    HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.

    Journal: Nucleic Acids Research

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    doi: 10.1093/nar/gkp695

    Figure Lengend Snippet: HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.

    Article Snippet: Also note that failure to join DNA ends is not due to HMO2 merely interacting with the ligase to prevent its activity, as evidenced by the activity of T4 DNA ligase on an internal DNA nick in the presence of HMO2 ( C).

    Techniques:

    DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.

    Journal: Nucleic Acids Research

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    doi: 10.1093/nar/gkp695

    Figure Lengend Snippet: DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.

    Article Snippet: Also note that failure to join DNA ends is not due to HMO2 merely interacting with the ligase to prevent its activity, as evidenced by the activity of T4 DNA ligase on an internal DNA nick in the presence of HMO2 ( C).

    Techniques: Sequencing, Incubation, Ligation

    DNA ligation products obtained from chromatin assembled on a linear 2.27 kb DNA fragment (pUC19 with a 420 bp deletion) with Ava I ends. The chromatin sample in the absence (H5–) or presence (H5+) of histone H5 was incubated with T4 DNA ligase at a DNA concentration of 25 µg/ml. Lane M denotes linear λ Hin dIII DNA markers; lane I denotes supercoiled plasmid (pUC19 with a 420 bp deletion) containing one supercoil per ∼200 bp. The gel contained 0.2 µg/ml chloroquine sulfate. Linear and monomer circle DNA forms are indicated.

    Journal: Nucleic Acids Research

    Article Title: Circle ligation of in vitro assembled chromatin indicates a highly flexible structure

    doi:

    Figure Lengend Snippet: DNA ligation products obtained from chromatin assembled on a linear 2.27 kb DNA fragment (pUC19 with a 420 bp deletion) with Ava I ends. The chromatin sample in the absence (H5–) or presence (H5+) of histone H5 was incubated with T4 DNA ligase at a DNA concentration of 25 µg/ml. Lane M denotes linear λ Hin dIII DNA markers; lane I denotes supercoiled plasmid (pUC19 with a 420 bp deletion) containing one supercoil per ∼200 bp. The gel contained 0.2 µg/ml chloroquine sulfate. Linear and monomer circle DNA forms are indicated.

    Article Snippet: Mechanistic studies with naked DNA indicate that Eco RI cohesive ends associate and dissociate rapidly many times before covalent sealing by T4 DNA ligase ( ).

    Techniques: DNA Ligation, Antiviral Assay, Incubation, Concentration Assay, Plasmid Preparation

    DNA products obtained from a linear 2.7 kb pUC19 DNA fragment treated with T4 DNA ligase in the form of naked DNA or chromatin at increasing DNA concentrations. The 2% agarose gels contained 0.2 µg/ml chloroquine sulfate in order to resolve the topoisomers resulting from the closed circular DNA forms. Monomer circle and linear DNA forms are indicated. ( A ) Lanes labeled 1–64 µg DNA/ml show the ligation products resulting from the naked DNA. M denotes linear λ Hin dIII DNA size markers; the 2.0 and 2.3 kb bands are resolved, whereas the 6.6, 9.4 and 23 kb bands run together. The lane labeled U denotes the unligated 2.7 kb DNA. ( B ) Lanes labeled 1–64 µg DNA/ml show the DNA products obtained from the H5+ chromatin. One chromatin sample lacking histone H5 (H5–) ligated at 16 µg DNA/ml is shown. The unligated DNA starting material used in this experiment is shown in A, lane U. Additionally, a core histone reconstituted DNA sample at a concentration of 16 µg/ml that was incubated overnight under ligation conditions, but without the T4 DNA ligase, ran indistinguishably from the starting DNA (not shown). M denotes linear unresolved 4.4–23 kb λ Hin dIII DNA markers; I denotes the (Form I) supercoiled form of pUC19 having one supercoil per ∼200 bp. ( C ) Quantitation of the percentage monomer circles formed for DNA or chromatin ligated over a range of DNA concentrations. The single black triangle at 16 µg/ml denotes the sample lacking histone H5.

    Journal: Nucleic Acids Research

    Article Title: Circle ligation of in vitro assembled chromatin indicates a highly flexible structure

    doi:

    Figure Lengend Snippet: DNA products obtained from a linear 2.7 kb pUC19 DNA fragment treated with T4 DNA ligase in the form of naked DNA or chromatin at increasing DNA concentrations. The 2% agarose gels contained 0.2 µg/ml chloroquine sulfate in order to resolve the topoisomers resulting from the closed circular DNA forms. Monomer circle and linear DNA forms are indicated. ( A ) Lanes labeled 1–64 µg DNA/ml show the ligation products resulting from the naked DNA. M denotes linear λ Hin dIII DNA size markers; the 2.0 and 2.3 kb bands are resolved, whereas the 6.6, 9.4 and 23 kb bands run together. The lane labeled U denotes the unligated 2.7 kb DNA. ( B ) Lanes labeled 1–64 µg DNA/ml show the DNA products obtained from the H5+ chromatin. One chromatin sample lacking histone H5 (H5–) ligated at 16 µg DNA/ml is shown. The unligated DNA starting material used in this experiment is shown in A, lane U. Additionally, a core histone reconstituted DNA sample at a concentration of 16 µg/ml that was incubated overnight under ligation conditions, but without the T4 DNA ligase, ran indistinguishably from the starting DNA (not shown). M denotes linear unresolved 4.4–23 kb λ Hin dIII DNA markers; I denotes the (Form I) supercoiled form of pUC19 having one supercoil per ∼200 bp. ( C ) Quantitation of the percentage monomer circles formed for DNA or chromatin ligated over a range of DNA concentrations. The single black triangle at 16 µg/ml denotes the sample lacking histone H5.

    Article Snippet: Mechanistic studies with naked DNA indicate that Eco RI cohesive ends associate and dissociate rapidly many times before covalent sealing by T4 DNA ligase ( ).

    Techniques: Labeling, Ligation, Concentration Assay, Incubation, Quantitation Assay

    Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.

    Journal: BMC Research Notes

    Article Title: Efficient assembly of very short oligonucleotides using T4 DNA Ligase

    doi: 10.1186/1756-0500-3-291

    Figure Lengend Snippet: Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.

    Article Snippet: Specifically, T4 DNA Ligase has been known for some time to be capable of joining oligos as small as pentamers and hexamers on a complete template [ ], however ligases such as Tth DNA Ligase are severely inefficient at the hexamer level [ ].

    Techniques: Activity Assay, Ligation

    Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).

    Journal: BMC Research Notes

    Article Title: Efficient assembly of very short oligonucleotides using T4 DNA Ligase

    doi: 10.1186/1756-0500-3-291

    Figure Lengend Snippet: Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).

    Article Snippet: Specifically, T4 DNA Ligase has been known for some time to be capable of joining oligos as small as pentamers and hexamers on a complete template [ ], however ligases such as Tth DNA Ligase are severely inefficient at the hexamer level [ ].

    Techniques: Ligation, Labeling