t4 dna ligase Roche Search Results


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  • 99
    New England Biolabs t4 dna ligase
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 49148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher u t4 dna ligase
    U T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche t4 dna ligase roche
    A–C : Comparison of apoptotic ( A , B ) and necrotic ( C ) thymus stained by DAPI (blue fluorescence) and by in situ ligation using oligonucleotide probes with single dA overhangs (red fluorescence). A and C are dual-stained images, B is a single-stained (DAPI) image and is provided for the easy comparison of nuclear morphology with C . D and E : Comparison of apoptotic and necrotic thymus stained by TUNEL. D : Apoptotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). E : Necrotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). Insets show high-magnification images of apoptotic and necrotic nuclei labeled by TUNEL (side of the inset , 80 μm). Strong positive staining is present in both cases. However necrotic thymus is uniformly positive with the loss of the characteristic pattern of cell death seen in glucocorticoid-induced apoptosis. F and G : Comparison of apoptotic and necrotic thymus using in situ ligation with blunt-ended probes detecting double-strand DNA breaks bearing 5′ phosphates. F : Apoptotic thymus, blunt ends detection. G : Necrotic thymus, blunt ends detection. Note that in situ ligation does not detect double-strand DNA breaks bearing 5′ phosphates in necrotic thymus. H and I : DNA nicks relegation in necrotic thymus using <t>T4</t> DNA ligase. H : Necrotic thymus TUNEL-stained before T4 ligase pretreatment; I : the same thymus after T4 ligase pretreatment. DNA nicks do not contribute to the strong positive staining of necrotic thymus by TUNEL. No change in TUNEL signal intensity occurred after treatment of necrotic tissue with T4 DNA ligase to seal DNA nicks. Apoptosis was induced in the thymus by an intraperitoneal injection of 6 mg/kg of dexamethasone. Necrosis was induced in the thymus by freezing with liquid nitrogen. Scale bars: 200 μm ( C , E ); 300 μm ( G ); 150 μm ( I ).
    T4 Dna Ligase Roche, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim t4 dna ligase
    A–C : Comparison of apoptotic ( A , B ) and necrotic ( C ) thymus stained by DAPI (blue fluorescence) and by in situ ligation using oligonucleotide probes with single dA overhangs (red fluorescence). A and C are dual-stained images, B is a single-stained (DAPI) image and is provided for the easy comparison of nuclear morphology with C . D and E : Comparison of apoptotic and necrotic thymus stained by TUNEL. D : Apoptotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). E : Necrotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). Insets show high-magnification images of apoptotic and necrotic nuclei labeled by TUNEL (side of the inset , 80 μm). Strong positive staining is present in both cases. However necrotic thymus is uniformly positive with the loss of the characteristic pattern of cell death seen in glucocorticoid-induced apoptosis. F and G : Comparison of apoptotic and necrotic thymus using in situ ligation with blunt-ended probes detecting double-strand DNA breaks bearing 5′ phosphates. F : Apoptotic thymus, blunt ends detection. G : Necrotic thymus, blunt ends detection. Note that in situ ligation does not detect double-strand DNA breaks bearing 5′ phosphates in necrotic thymus. H and I : DNA nicks relegation in necrotic thymus using <t>T4</t> DNA ligase. H : Necrotic thymus TUNEL-stained before T4 ligase pretreatment; I : the same thymus after T4 ligase pretreatment. DNA nicks do not contribute to the strong positive staining of necrotic thymus by TUNEL. No change in TUNEL signal intensity occurred after treatment of necrotic tissue with T4 DNA ligase to seal DNA nicks. Apoptosis was induced in the thymus by an intraperitoneal injection of 6 mg/kg of dexamethasone. Necrosis was induced in the thymus by freezing with liquid nitrogen. Scale bars: 200 μm ( C , E ); 300 μm ( G ); 150 μm ( I ).
    T4 Dna Ligase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 908 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Roche t4 dna ligase kit
    A–C : Comparison of apoptotic ( A , B ) and necrotic ( C ) thymus stained by DAPI (blue fluorescence) and by in situ ligation using oligonucleotide probes with single dA overhangs (red fluorescence). A and C are dual-stained images, B is a single-stained (DAPI) image and is provided for the easy comparison of nuclear morphology with C . D and E : Comparison of apoptotic and necrotic thymus stained by TUNEL. D : Apoptotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). E : Necrotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). Insets show high-magnification images of apoptotic and necrotic nuclei labeled by TUNEL (side of the inset , 80 μm). Strong positive staining is present in both cases. However necrotic thymus is uniformly positive with the loss of the characteristic pattern of cell death seen in glucocorticoid-induced apoptosis. F and G : Comparison of apoptotic and necrotic thymus using in situ ligation with blunt-ended probes detecting double-strand DNA breaks bearing 5′ phosphates. F : Apoptotic thymus, blunt ends detection. G : Necrotic thymus, blunt ends detection. Note that in situ ligation does not detect double-strand DNA breaks bearing 5′ phosphates in necrotic thymus. H and I : DNA nicks relegation in necrotic thymus using <t>T4</t> DNA ligase. H : Necrotic thymus TUNEL-stained before T4 ligase pretreatment; I : the same thymus after T4 ligase pretreatment. DNA nicks do not contribute to the strong positive staining of necrotic thymus by TUNEL. No change in TUNEL signal intensity occurred after treatment of necrotic tissue with T4 DNA ligase to seal DNA nicks. Apoptosis was induced in the thymus by an intraperitoneal injection of 6 mg/kg of dexamethasone. Necrosis was induced in the thymus by freezing with liquid nitrogen. Scale bars: 200 μm ( C , E ); 300 μm ( G ); 150 μm ( I ).
    T4 Dna Ligase Kit, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche t4 dna ligase buffer
    A–C : Comparison of apoptotic ( A , B ) and necrotic ( C ) thymus stained by DAPI (blue fluorescence) and by in situ ligation using oligonucleotide probes with single dA overhangs (red fluorescence). A and C are dual-stained images, B is a single-stained (DAPI) image and is provided for the easy comparison of nuclear morphology with C . D and E : Comparison of apoptotic and necrotic thymus stained by TUNEL. D : Apoptotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). E : Necrotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). Insets show high-magnification images of apoptotic and necrotic nuclei labeled by TUNEL (side of the inset , 80 μm). Strong positive staining is present in both cases. However necrotic thymus is uniformly positive with the loss of the characteristic pattern of cell death seen in glucocorticoid-induced apoptosis. F and G : Comparison of apoptotic and necrotic thymus using in situ ligation with blunt-ended probes detecting double-strand DNA breaks bearing 5′ phosphates. F : Apoptotic thymus, blunt ends detection. G : Necrotic thymus, blunt ends detection. Note that in situ ligation does not detect double-strand DNA breaks bearing 5′ phosphates in necrotic thymus. H and I : DNA nicks relegation in necrotic thymus using <t>T4</t> DNA ligase. H : Necrotic thymus TUNEL-stained before T4 ligase pretreatment; I : the same thymus after T4 ligase pretreatment. DNA nicks do not contribute to the strong positive staining of necrotic thymus by TUNEL. No change in TUNEL signal intensity occurred after treatment of necrotic tissue with T4 DNA ligase to seal DNA nicks. Apoptosis was induced in the thymus by an intraperitoneal injection of 6 mg/kg of dexamethasone. Necrosis was induced in the thymus by freezing with liquid nitrogen. Scale bars: 200 μm ( C , E ); 300 μm ( G ); 150 μm ( I ).
    T4 Dna Ligase Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche t4 dna ligase enzyme
    A–C : Comparison of apoptotic ( A , B ) and necrotic ( C ) thymus stained by DAPI (blue fluorescence) and by in situ ligation using oligonucleotide probes with single dA overhangs (red fluorescence). A and C are dual-stained images, B is a single-stained (DAPI) image and is provided for the easy comparison of nuclear morphology with C . D and E : Comparison of apoptotic and necrotic thymus stained by TUNEL. D : Apoptotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). E : Necrotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). Insets show high-magnification images of apoptotic and necrotic nuclei labeled by TUNEL (side of the inset , 80 μm). Strong positive staining is present in both cases. However necrotic thymus is uniformly positive with the loss of the characteristic pattern of cell death seen in glucocorticoid-induced apoptosis. F and G : Comparison of apoptotic and necrotic thymus using in situ ligation with blunt-ended probes detecting double-strand DNA breaks bearing 5′ phosphates. F : Apoptotic thymus, blunt ends detection. G : Necrotic thymus, blunt ends detection. Note that in situ ligation does not detect double-strand DNA breaks bearing 5′ phosphates in necrotic thymus. H and I : DNA nicks relegation in necrotic thymus using <t>T4</t> DNA ligase. H : Necrotic thymus TUNEL-stained before T4 ligase pretreatment; I : the same thymus after T4 ligase pretreatment. DNA nicks do not contribute to the strong positive staining of necrotic thymus by TUNEL. No change in TUNEL signal intensity occurred after treatment of necrotic tissue with T4 DNA ligase to seal DNA nicks. Apoptosis was induced in the thymus by an intraperitoneal injection of 6 mg/kg of dexamethasone. Necrosis was induced in the thymus by freezing with liquid nitrogen. Scale bars: 200 μm ( C , E ); 300 μm ( G ); 150 μm ( I ).
    T4 Dna Ligase Enzyme, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche bacteriophage t4 dna ligase
    A–C : Comparison of apoptotic ( A , B ) and necrotic ( C ) thymus stained by DAPI (blue fluorescence) and by in situ ligation using oligonucleotide probes with single dA overhangs (red fluorescence). A and C are dual-stained images, B is a single-stained (DAPI) image and is provided for the easy comparison of nuclear morphology with C . D and E : Comparison of apoptotic and necrotic thymus stained by TUNEL. D : Apoptotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). E : Necrotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). Insets show high-magnification images of apoptotic and necrotic nuclei labeled by TUNEL (side of the inset , 80 μm). Strong positive staining is present in both cases. However necrotic thymus is uniformly positive with the loss of the characteristic pattern of cell death seen in glucocorticoid-induced apoptosis. F and G : Comparison of apoptotic and necrotic thymus using in situ ligation with blunt-ended probes detecting double-strand DNA breaks bearing 5′ phosphates. F : Apoptotic thymus, blunt ends detection. G : Necrotic thymus, blunt ends detection. Note that in situ ligation does not detect double-strand DNA breaks bearing 5′ phosphates in necrotic thymus. H and I : DNA nicks relegation in necrotic thymus using <t>T4</t> DNA ligase. H : Necrotic thymus TUNEL-stained before T4 ligase pretreatment; I : the same thymus after T4 ligase pretreatment. DNA nicks do not contribute to the strong positive staining of necrotic thymus by TUNEL. No change in TUNEL signal intensity occurred after treatment of necrotic tissue with T4 DNA ligase to seal DNA nicks. Apoptosis was induced in the thymus by an intraperitoneal injection of 6 mg/kg of dexamethasone. Necrosis was induced in the thymus by freezing with liquid nitrogen. Scale bars: 200 μm ( C , E ); 300 μm ( G ); 150 μm ( I ).
    Bacteriophage T4 Dna Ligase, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche 11004760001 t4 dna ligase
    A–C : Comparison of apoptotic ( A , B ) and necrotic ( C ) thymus stained by DAPI (blue fluorescence) and by in situ ligation using oligonucleotide probes with single dA overhangs (red fluorescence). A and C are dual-stained images, B is a single-stained (DAPI) image and is provided for the easy comparison of nuclear morphology with C . D and E : Comparison of apoptotic and necrotic thymus stained by TUNEL. D : Apoptotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). E : Necrotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). Insets show high-magnification images of apoptotic and necrotic nuclei labeled by TUNEL (side of the inset , 80 μm). Strong positive staining is present in both cases. However necrotic thymus is uniformly positive with the loss of the characteristic pattern of cell death seen in glucocorticoid-induced apoptosis. F and G : Comparison of apoptotic and necrotic thymus using in situ ligation with blunt-ended probes detecting double-strand DNA breaks bearing 5′ phosphates. F : Apoptotic thymus, blunt ends detection. G : Necrotic thymus, blunt ends detection. Note that in situ ligation does not detect double-strand DNA breaks bearing 5′ phosphates in necrotic thymus. H and I : DNA nicks relegation in necrotic thymus using <t>T4</t> DNA ligase. H : Necrotic thymus TUNEL-stained before T4 ligase pretreatment; I : the same thymus after T4 ligase pretreatment. DNA nicks do not contribute to the strong positive staining of necrotic thymus by TUNEL. No change in TUNEL signal intensity occurred after treatment of necrotic tissue with T4 DNA ligase to seal DNA nicks. Apoptosis was induced in the thymus by an intraperitoneal injection of 6 mg/kg of dexamethasone. Necrosis was induced in the thymus by freezing with liquid nitrogen. Scale bars: 200 μm ( C , E ); 300 μm ( G ); 150 μm ( I ).
    11004760001 T4 Dna Ligase, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche t4 dna ligase buffer 1×
    A–C : Comparison of apoptotic ( A , B ) and necrotic ( C ) thymus stained by DAPI (blue fluorescence) and by in situ ligation using oligonucleotide probes with single dA overhangs (red fluorescence). A and C are dual-stained images, B is a single-stained (DAPI) image and is provided for the easy comparison of nuclear morphology with C . D and E : Comparison of apoptotic and necrotic thymus stained by TUNEL. D : Apoptotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). E : Necrotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). Insets show high-magnification images of apoptotic and necrotic nuclei labeled by TUNEL (side of the inset , 80 μm). Strong positive staining is present in both cases. However necrotic thymus is uniformly positive with the loss of the characteristic pattern of cell death seen in glucocorticoid-induced apoptosis. F and G : Comparison of apoptotic and necrotic thymus using in situ ligation with blunt-ended probes detecting double-strand DNA breaks bearing 5′ phosphates. F : Apoptotic thymus, blunt ends detection. G : Necrotic thymus, blunt ends detection. Note that in situ ligation does not detect double-strand DNA breaks bearing 5′ phosphates in necrotic thymus. H and I : DNA nicks relegation in necrotic thymus using <t>T4</t> DNA ligase. H : Necrotic thymus TUNEL-stained before T4 ligase pretreatment; I : the same thymus after T4 ligase pretreatment. DNA nicks do not contribute to the strong positive staining of necrotic thymus by TUNEL. No change in TUNEL signal intensity occurred after treatment of necrotic tissue with T4 DNA ligase to seal DNA nicks. Apoptosis was induced in the thymus by an intraperitoneal injection of 6 mg/kg of dexamethasone. Necrosis was induced in the thymus by freezing with liquid nitrogen. Scale bars: 200 μm ( C , E ); 300 μm ( G ); 150 μm ( I ).
    T4 Dna Ligase Buffer 1×, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche dna ligase
    RecA⋅ATP-mediated recombination of a short heterologous (77-bp, ∼54% sequence divergence) substrate ( lds het ) in the presence of MutSL. (A) Scheme of the reaction between css hom and the heterologous lds het (black square) substrates. The 77-bp heterologous segment was restricted to an internal region toward the 3′ end. The css hom <t>DNA</t> (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A with 5 mM <t>ATP,</t> followed by RecA, (MutS, MutL or MutSL) and the lds het substrate, and incubated (60 min, 37°C). Band positions correspond to substrates as in Figure 2 . The minus (–) symbol denotes absence of MutS or MutL. C denotes the DNA substrates control. The percentage of jm intermediates and nc products are shown beneath the gel. Results shown as mean ± 5% SEM of ≥3 independent experiments. (B) Experiments with dATP and increasing RecA concentrations. The 3276-nt css hom DNA was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM dATP. The lds het substrate, increasing RecA concentrations and, where indicated, a fixed MutS concentration were then added and incubated (60 min, 37°C). (C) The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A with 5 mM ATP, followed by RecA, the [γ 32 P]- lds hom (lanes 2–5) or [γ 32 P]- lds het lanes 7–10 substrate, in the presence or absence of increasing MutS concentrations, and incubated (60 min, 37°C). Band positions correspond to substrates as in Figure 2 . The minus (–) symbol denotes absence of MutS.
    Dna Ligase, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Roche t7 dna ligase
    RecA⋅ATP-mediated recombination of a short heterologous (77-bp, ∼54% sequence divergence) substrate ( lds het ) in the presence of MutSL. (A) Scheme of the reaction between css hom and the heterologous lds het (black square) substrates. The 77-bp heterologous segment was restricted to an internal region toward the 3′ end. The css hom <t>DNA</t> (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A with 5 mM <t>ATP,</t> followed by RecA, (MutS, MutL or MutSL) and the lds het substrate, and incubated (60 min, 37°C). Band positions correspond to substrates as in Figure 2 . The minus (–) symbol denotes absence of MutS or MutL. C denotes the DNA substrates control. The percentage of jm intermediates and nc products are shown beneath the gel. Results shown as mean ± 5% SEM of ≥3 independent experiments. (B) Experiments with dATP and increasing RecA concentrations. The 3276-nt css hom DNA was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM dATP. The lds het substrate, increasing RecA concentrations and, where indicated, a fixed MutS concentration were then added and incubated (60 min, 37°C). (C) The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A with 5 mM ATP, followed by RecA, the [γ 32 P]- lds hom (lanes 2–5) or [γ 32 P]- lds het lanes 7–10 substrate, in the presence or absence of increasing MutS concentrations, and incubated (60 min, 37°C). Band positions correspond to substrates as in Figure 2 . The minus (–) symbol denotes absence of MutS.
    T7 Dna Ligase, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche rapid dna ligation kit
    Schematic representation of the expression constructs ( A ) pLgals1-hum-β3GN-T2 and ( B ) pLgals1-hum-MFng . The <t>PCR-amplified</t> <t>DNA</t> sequences were inserted in the pLgals1-Tev vector construct at the multicloning site, which is preceded by the 6HisTag
    Rapid Dna Ligation Kit, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 3335 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche dna ligase kit
    Schematic representation of the expression constructs ( A ) pLgals1-hum-β3GN-T2 and ( B ) pLgals1-hum-MFng . The <t>PCR-amplified</t> <t>DNA</t> sequences were inserted in the pLgals1-Tev vector construct at the multicloning site, which is preceded by the 6HisTag
    Dna Ligase Kit, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A–C : Comparison of apoptotic ( A , B ) and necrotic ( C ) thymus stained by DAPI (blue fluorescence) and by in situ ligation using oligonucleotide probes with single dA overhangs (red fluorescence). A and C are dual-stained images, B is a single-stained (DAPI) image and is provided for the easy comparison of nuclear morphology with C . D and E : Comparison of apoptotic and necrotic thymus stained by TUNEL. D : Apoptotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). E : Necrotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). Insets show high-magnification images of apoptotic and necrotic nuclei labeled by TUNEL (side of the inset , 80 μm). Strong positive staining is present in both cases. However necrotic thymus is uniformly positive with the loss of the characteristic pattern of cell death seen in glucocorticoid-induced apoptosis. F and G : Comparison of apoptotic and necrotic thymus using in situ ligation with blunt-ended probes detecting double-strand DNA breaks bearing 5′ phosphates. F : Apoptotic thymus, blunt ends detection. G : Necrotic thymus, blunt ends detection. Note that in situ ligation does not detect double-strand DNA breaks bearing 5′ phosphates in necrotic thymus. H and I : DNA nicks relegation in necrotic thymus using T4 DNA ligase. H : Necrotic thymus TUNEL-stained before T4 ligase pretreatment; I : the same thymus after T4 ligase pretreatment. DNA nicks do not contribute to the strong positive staining of necrotic thymus by TUNEL. No change in TUNEL signal intensity occurred after treatment of necrotic tissue with T4 DNA ligase to seal DNA nicks. Apoptosis was induced in the thymus by an intraperitoneal injection of 6 mg/kg of dexamethasone. Necrosis was induced in the thymus by freezing with liquid nitrogen. Scale bars: 200 μm ( C , E ); 300 μm ( G ); 150 μm ( I ).

    Journal: The American Journal of Pathology

    Article Title: Early Necrotic DNA Degradation

    doi:

    Figure Lengend Snippet: A–C : Comparison of apoptotic ( A , B ) and necrotic ( C ) thymus stained by DAPI (blue fluorescence) and by in situ ligation using oligonucleotide probes with single dA overhangs (red fluorescence). A and C are dual-stained images, B is a single-stained (DAPI) image and is provided for the easy comparison of nuclear morphology with C . D and E : Comparison of apoptotic and necrotic thymus stained by TUNEL. D : Apoptotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). E : Necrotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). Insets show high-magnification images of apoptotic and necrotic nuclei labeled by TUNEL (side of the inset , 80 μm). Strong positive staining is present in both cases. However necrotic thymus is uniformly positive with the loss of the characteristic pattern of cell death seen in glucocorticoid-induced apoptosis. F and G : Comparison of apoptotic and necrotic thymus using in situ ligation with blunt-ended probes detecting double-strand DNA breaks bearing 5′ phosphates. F : Apoptotic thymus, blunt ends detection. G : Necrotic thymus, blunt ends detection. Note that in situ ligation does not detect double-strand DNA breaks bearing 5′ phosphates in necrotic thymus. H and I : DNA nicks relegation in necrotic thymus using T4 DNA ligase. H : Necrotic thymus TUNEL-stained before T4 ligase pretreatment; I : the same thymus after T4 ligase pretreatment. DNA nicks do not contribute to the strong positive staining of necrotic thymus by TUNEL. No change in TUNEL signal intensity occurred after treatment of necrotic tissue with T4 DNA ligase to seal DNA nicks. Apoptosis was induced in the thymus by an intraperitoneal injection of 6 mg/kg of dexamethasone. Necrosis was induced in the thymus by freezing with liquid nitrogen. Scale bars: 200 μm ( C , E ); 300 μm ( G ); 150 μm ( I ).

    Article Snippet: In a mock control reaction solution, an equal volume of 50% glycerol in water was substituted for T4 DNA ligase.

    Techniques: Staining, Fluorescence, In Situ, Ligation, TUNEL Assay, Labeling, Injection

    RecA⋅ATP-mediated recombination of a short heterologous (77-bp, ∼54% sequence divergence) substrate ( lds het ) in the presence of MutSL. (A) Scheme of the reaction between css hom and the heterologous lds het (black square) substrates. The 77-bp heterologous segment was restricted to an internal region toward the 3′ end. The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A with 5 mM ATP, followed by RecA, (MutS, MutL or MutSL) and the lds het substrate, and incubated (60 min, 37°C). Band positions correspond to substrates as in Figure 2 . The minus (–) symbol denotes absence of MutS or MutL. C denotes the DNA substrates control. The percentage of jm intermediates and nc products are shown beneath the gel. Results shown as mean ± 5% SEM of ≥3 independent experiments. (B) Experiments with dATP and increasing RecA concentrations. The 3276-nt css hom DNA was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM dATP. The lds het substrate, increasing RecA concentrations and, where indicated, a fixed MutS concentration were then added and incubated (60 min, 37°C). (C) The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A with 5 mM ATP, followed by RecA, the [γ 32 P]- lds hom (lanes 2–5) or [γ 32 P]- lds het lanes 7–10 substrate, in the presence or absence of increasing MutS concentrations, and incubated (60 min, 37°C). Band positions correspond to substrates as in Figure 2 . The minus (–) symbol denotes absence of MutS.

    Journal: Frontiers in Microbiology

    Article Title: Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences

    doi: 10.3389/fmicb.2019.00237

    Figure Lengend Snippet: RecA⋅ATP-mediated recombination of a short heterologous (77-bp, ∼54% sequence divergence) substrate ( lds het ) in the presence of MutSL. (A) Scheme of the reaction between css hom and the heterologous lds het (black square) substrates. The 77-bp heterologous segment was restricted to an internal region toward the 3′ end. The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A with 5 mM ATP, followed by RecA, (MutS, MutL or MutSL) and the lds het substrate, and incubated (60 min, 37°C). Band positions correspond to substrates as in Figure 2 . The minus (–) symbol denotes absence of MutS or MutL. C denotes the DNA substrates control. The percentage of jm intermediates and nc products are shown beneath the gel. Results shown as mean ± 5% SEM of ≥3 independent experiments. (B) Experiments with dATP and increasing RecA concentrations. The 3276-nt css hom DNA was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM dATP. The lds het substrate, increasing RecA concentrations and, where indicated, a fixed MutS concentration were then added and incubated (60 min, 37°C). (C) The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A with 5 mM ATP, followed by RecA, the [γ 32 P]- lds hom (lanes 2–5) or [γ 32 P]- lds het lanes 7–10 substrate, in the presence or absence of increasing MutS concentrations, and incubated (60 min, 37°C). Band positions correspond to substrates as in Figure 2 . The minus (–) symbol denotes absence of MutS.

    Article Snippet: IPTG was from Calbiochem; DNA restriction enzymes and DNA ligase were from Roche, and polyethyleneimine, DTT, ATP, dATP were from Sigma.

    Techniques: Sequencing, Incubation, Concentration Assay

    A short homeologous (77-bp segment, ∼16% sequence divergence) region on an otherwise homologous substrate ( lds mis ) delays RecA⋅ATP-mediated DNA strand exchange. The scheme shows the three-strand exchange reaction between css hom (+ strand) and the homeologous lds mis (filled circle) or lds hom ( open circle) substrates. The 77-bp homeologous/homologous segment was restricted to an internal region toward the 3′ end. (A) The 3276-nt css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM ATP, followed by 3276-bp kpn I-linearized lds mis (lanes 1–7) or lds hom DNA (lanes 8–14) and RecA, and the reaction was incubated for various times (min) at 37°C. The reaction was separated by 0.8% agarose gel electrophoresis. Band positions correspond to substrates ( lds and css ), intermediates ( jm ) and product ( nc ). C denotes the DNA substrates control. The amount of recombination intermediates ( jm ) and products ( nc ) are expressed as a percentage of total substrate added. MutSL affects RecA⋅ATP rather than RecA⋅dATP-mediated DNA strand exchange. (B,C) The 3,277-nt homologous circular ssDNA ( css hom ) was pre-incubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM ATP or dATP. Then, RecA and increasing MutS and the 3,276-bp lds mis (B) or lds hom (C) DNA (20 μM in nt) substrate were added. The reaction was incubated (60 min, 37°C) and separated by 0.8% agarose gel electrophoresis. The amount of recombination products is expressed as a percentage of total substrate added. Quantification of intermediate/products beneath the gel shown as mean ± SEM of ≥3 independent experiments.

    Journal: Frontiers in Microbiology

    Article Title: Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences

    doi: 10.3389/fmicb.2019.00237

    Figure Lengend Snippet: A short homeologous (77-bp segment, ∼16% sequence divergence) region on an otherwise homologous substrate ( lds mis ) delays RecA⋅ATP-mediated DNA strand exchange. The scheme shows the three-strand exchange reaction between css hom (+ strand) and the homeologous lds mis (filled circle) or lds hom ( open circle) substrates. The 77-bp homeologous/homologous segment was restricted to an internal region toward the 3′ end. (A) The 3276-nt css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM ATP, followed by 3276-bp kpn I-linearized lds mis (lanes 1–7) or lds hom DNA (lanes 8–14) and RecA, and the reaction was incubated for various times (min) at 37°C. The reaction was separated by 0.8% agarose gel electrophoresis. Band positions correspond to substrates ( lds and css ), intermediates ( jm ) and product ( nc ). C denotes the DNA substrates control. The amount of recombination intermediates ( jm ) and products ( nc ) are expressed as a percentage of total substrate added. MutSL affects RecA⋅ATP rather than RecA⋅dATP-mediated DNA strand exchange. (B,C) The 3,277-nt homologous circular ssDNA ( css hom ) was pre-incubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM ATP or dATP. Then, RecA and increasing MutS and the 3,276-bp lds mis (B) or lds hom (C) DNA (20 μM in nt) substrate were added. The reaction was incubated (60 min, 37°C) and separated by 0.8% agarose gel electrophoresis. The amount of recombination products is expressed as a percentage of total substrate added. Quantification of intermediate/products beneath the gel shown as mean ± SEM of ≥3 independent experiments.

    Article Snippet: IPTG was from Calbiochem; DNA restriction enzymes and DNA ligase were from Roche, and polyethyleneimine, DTT, ATP, dATP were from Sigma.

    Techniques: Sequencing, Incubation, Agarose Gel Electrophoresis

    RecA⋅ATP-mediated strand exchange with short internal heterology and at the 5′-end in the presence of MutSL. (A) Scheme of the reaction between css hom and the lds het–ins substrate with internal region toward the 3′ end and at the 5′-end (black and gray squares, respectively). The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM ATP, after which RecA, (MutS, MutL or MutSL) and the lds het–ins substrate were added and incubated (60 min, 37°C). The percentage of jm intermediates and nc products are shown beneath the gel. Lane 1, the css and lds substrates (termed C). Results shown as the mean ±5% SEM of ≥3 independent experiments. (B) Experiments with dATP and increasing RecA concentrations. The 3276-nt css hom DNA was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM dATP, followed by the 3353-bp lds het–ins substrate, increasing RecA concentrations and, where indicated, a fixed MutS concentration, and incubated (60 min, 37°C). The reaction was separated by gel electrophoresis and quantified. The left-hand side bar denotes the fraction of jm intermediates (gray bar) and the right-hand side scale denotes the fraction of unreactive lds het–ins substrate (black bars). The minus (–) symbol indicates lack of MutS or MutL.

    Journal: Frontiers in Microbiology

    Article Title: Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences

    doi: 10.3389/fmicb.2019.00237

    Figure Lengend Snippet: RecA⋅ATP-mediated strand exchange with short internal heterology and at the 5′-end in the presence of MutSL. (A) Scheme of the reaction between css hom and the lds het–ins substrate with internal region toward the 3′ end and at the 5′-end (black and gray squares, respectively). The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM ATP, after which RecA, (MutS, MutL or MutSL) and the lds het–ins substrate were added and incubated (60 min, 37°C). The percentage of jm intermediates and nc products are shown beneath the gel. Lane 1, the css and lds substrates (termed C). Results shown as the mean ±5% SEM of ≥3 independent experiments. (B) Experiments with dATP and increasing RecA concentrations. The 3276-nt css hom DNA was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM dATP, followed by the 3353-bp lds het–ins substrate, increasing RecA concentrations and, where indicated, a fixed MutS concentration, and incubated (60 min, 37°C). The reaction was separated by gel electrophoresis and quantified. The left-hand side bar denotes the fraction of jm intermediates (gray bar) and the right-hand side scale denotes the fraction of unreactive lds het–ins substrate (black bars). The minus (–) symbol indicates lack of MutS or MutL.

    Article Snippet: IPTG was from Calbiochem; DNA restriction enzymes and DNA ligase were from Roche, and polyethyleneimine, DTT, ATP, dATP were from Sigma.

    Techniques: Incubation, Concentration Assay, Nucleic Acid Electrophoresis

    Quantification of species from AFM images of reactions containing lds het and css hom DNA. (A) Scheme of the reaction between css hom (open circle) and the lds het (black square) substrates, showing the jm intermediates, and the final nc product. The 3276-nt css hom DNA (10 μM in nt) was preincubated with SsbA, RecO, and RecA (60 min, 37°C) in buffer A containing 5 mM ATP, followed by linearized 3276-bp lds het DNA, alone (B) or with MutS (C) . Quantification of the different DNA species in the absence of MutS ( n = 284 molecules) (D) or in the presence of MutS ( n = 301 molecules) (E) . Quantification only includes dsDNA molecules with the original length and ssDNA molecules with a volume corresponding to the original length. Agg, lds het DNA with css hom at both ends or more complex aggregates. Arrows point to examples of the different species (black: ssDNA; red: dsDNA; dark blue: joint molecules; light blue: nicked circular; green: Agg).

    Journal: Frontiers in Microbiology

    Article Title: Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences

    doi: 10.3389/fmicb.2019.00237

    Figure Lengend Snippet: Quantification of species from AFM images of reactions containing lds het and css hom DNA. (A) Scheme of the reaction between css hom (open circle) and the lds het (black square) substrates, showing the jm intermediates, and the final nc product. The 3276-nt css hom DNA (10 μM in nt) was preincubated with SsbA, RecO, and RecA (60 min, 37°C) in buffer A containing 5 mM ATP, followed by linearized 3276-bp lds het DNA, alone (B) or with MutS (C) . Quantification of the different DNA species in the absence of MutS ( n = 284 molecules) (D) or in the presence of MutS ( n = 301 molecules) (E) . Quantification only includes dsDNA molecules with the original length and ssDNA molecules with a volume corresponding to the original length. Agg, lds het DNA with css hom at both ends or more complex aggregates. Arrows point to examples of the different species (black: ssDNA; red: dsDNA; dark blue: joint molecules; light blue: nicked circular; green: Agg).

    Article Snippet: IPTG was from Calbiochem; DNA restriction enzymes and DNA ligase were from Roche, and polyethyleneimine, DTT, ATP, dATP were from Sigma.

    Techniques:

    Quantification of species from AFM images of reactions containing lds het–ins and css hom DNA. (A) Scheme of RecA-mediated recombination reaction between css hom (open circle) and lds het–ins (internal black square and a gray square at 5′-end) substrates, showing the jm intermediates and the hypothetical final nc product (in gray). The 3276-nt css hom DNA (10 μM in nt) was preincubated with SsbA, RecO and RecA (60 min, 37°C) in buffer A with 5 mM ATP. Then, the 3353-bp lds het–ins DNA was added without (B) or with MutS (C) . Quantification of the different DNA species observed in the absence of MutS ( n = 293 molecules) (D) or in the presence of MutS ( n = 419 molecules) (E) . Quantification only includes dsDNA molecules with the original length and ssDNA molecules with a volume corresponding to the original length. Agg, css hom with lds het–ins DNA at one end and internal regions or more complex aggregates. Arrows point to examples of the different species (black: ssDNA; red: dsDNA; light blue: nicked circular; green: Agg).

    Journal: Frontiers in Microbiology

    Article Title: Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences

    doi: 10.3389/fmicb.2019.00237

    Figure Lengend Snippet: Quantification of species from AFM images of reactions containing lds het–ins and css hom DNA. (A) Scheme of RecA-mediated recombination reaction between css hom (open circle) and lds het–ins (internal black square and a gray square at 5′-end) substrates, showing the jm intermediates and the hypothetical final nc product (in gray). The 3276-nt css hom DNA (10 μM in nt) was preincubated with SsbA, RecO and RecA (60 min, 37°C) in buffer A with 5 mM ATP. Then, the 3353-bp lds het–ins DNA was added without (B) or with MutS (C) . Quantification of the different DNA species observed in the absence of MutS ( n = 293 molecules) (D) or in the presence of MutS ( n = 419 molecules) (E) . Quantification only includes dsDNA molecules with the original length and ssDNA molecules with a volume corresponding to the original length. Agg, css hom with lds het–ins DNA at one end and internal regions or more complex aggregates. Arrows point to examples of the different species (black: ssDNA; red: dsDNA; light blue: nicked circular; green: Agg).

    Article Snippet: IPTG was from Calbiochem; DNA restriction enzymes and DNA ligase were from Roche, and polyethyleneimine, DTT, ATP, dATP were from Sigma.

    Techniques:

    Schematic representation of the expression constructs ( A ) pLgals1-hum-β3GN-T2 and ( B ) pLgals1-hum-MFng . The PCR-amplified DNA sequences were inserted in the pLgals1-Tev vector construct at the multicloning site, which is preceded by the 6HisTag

    Journal: Glycobiology

    Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2

    doi: 10.1093/glycob/cwr110

    Figure Lengend Snippet: Schematic representation of the expression constructs ( A ) pLgals1-hum-β3GN-T2 and ( B ) pLgals1-hum-MFng . The PCR-amplified DNA sequences were inserted in the pLgals1-Tev vector construct at the multicloning site, which is preceded by the 6HisTag

    Article Snippet: The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem.

    Techniques: Expressing, Construct, Polymerase Chain Reaction, Amplification, Plasmid Preparation