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Roche 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 ).
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Images

1) Product Images from "Early Necrotic DNA Degradation "

Article Title: Early Necrotic DNA Degradation

Journal: The American Journal of Pathology

doi:

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 ).
Figure Legend 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 ).

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

2) Product Images from "Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair"

Article Title: Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

Journal: DNA repair

doi: 10.1016/j.dnarep.2014.01.015

BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative
Figure Legend Snippet: BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative

Techniques Used:

3) Product Images from "Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair"

Article Title: Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

Journal: DNA repair

doi: 10.1016/j.dnarep.2014.01.015

BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative
Figure Legend Snippet: BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative

Techniques Used:

4) Product Images from "Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation"

Article Title: Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-7187-9_15

ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA
Figure Legend Snippet: ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA

Techniques Used: Labeling, Sequencing, Amplification

5) Product Images from "Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation"

Article Title: Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-7187-9_15

ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA
Figure Legend Snippet: ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA

Techniques Used: Labeling, Sequencing, Amplification

6) Product Images from "Early Necrotic DNA Degradation "

Article Title: Early Necrotic DNA Degradation

Journal: The American Journal of Pathology

doi:

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 ).
Figure Legend 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 ).

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

7) Product Images from "An efficient ORF selection system for DNA fragment libraries based on split beta-lactamase complementation"

Article Title: An efficient ORF selection system for DNA fragment libraries based on split beta-lactamase complementation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0235853

Schematic representation of pVMAKORF001 vector. Only relevant genes and restriction sites are shown. The map is not to scale. Kan r , kanamycin resistance gene; ColE1, origin of replication; Fori, phage M13 origin of replication; AraC and pBAD, Cassette encoding arabinose promoter and its regulator AraC; BlaSS, Native bla signal sequence; α 24–195, Bla-Alpha fragment encoding 24–195 amino acids of beta-lactamase; M182T, Methionine 182 to threonine mutation; NGR, Asparagine-Glycine-Arginine tripeptide; S1 and S2, (Gly4Ser)3 linker; Sp. Spacer sequence; TEV, Tobacco Etch Virus protease sequence; ω 196–286, Bla-Omega fragment encoding 196–286 amino acids of beta-lactamase; SacB and SacR, gene cassette encoding levansucrase of B . subtilis . (A)-(C) Sequence of different cassettes of the vector. (D) Sequence of insert to be cloned in the vector. (E) Insert carrying 4 base 5’ overhangs after T4 DNA polymerase treatment in the presence of dTTP.
Figure Legend Snippet: Schematic representation of pVMAKORF001 vector. Only relevant genes and restriction sites are shown. The map is not to scale. Kan r , kanamycin resistance gene; ColE1, origin of replication; Fori, phage M13 origin of replication; AraC and pBAD, Cassette encoding arabinose promoter and its regulator AraC; BlaSS, Native bla signal sequence; α 24–195, Bla-Alpha fragment encoding 24–195 amino acids of beta-lactamase; M182T, Methionine 182 to threonine mutation; NGR, Asparagine-Glycine-Arginine tripeptide; S1 and S2, (Gly4Ser)3 linker; Sp. Spacer sequence; TEV, Tobacco Etch Virus protease sequence; ω 196–286, Bla-Omega fragment encoding 196–286 amino acids of beta-lactamase; SacB and SacR, gene cassette encoding levansucrase of B . subtilis . (A)-(C) Sequence of different cassettes of the vector. (D) Sequence of insert to be cloned in the vector. (E) Insert carrying 4 base 5’ overhangs after T4 DNA polymerase treatment in the presence of dTTP.

Techniques Used: Plasmid Preparation, Sequencing, Mutagenesis, Clone Assay

8) Product Images from "Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair"

Article Title: Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

Journal: DNA repair

doi: 10.1016/j.dnarep.2014.01.015

BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative
Figure Legend Snippet: BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative

Techniques Used:

9) Product Images from "Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism"

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism

Journal: BMC Microbiology

doi: 10.1186/1471-2180-4-21

Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
Figure Legend Snippet: Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Size-exclusion Chromatography, Agarose Gel Electrophoresis, Purification, Incubation, Ligation, Clone Assay

10) Product Images from "Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair"

Article Title: Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

Journal: DNA repair

doi: 10.1016/j.dnarep.2014.01.015

BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative
Figure Legend Snippet: BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative

Techniques Used:

11) Product Images from "Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair"

Article Title: Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

Journal: DNA repair

doi: 10.1016/j.dnarep.2014.01.015

BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative
Figure Legend Snippet: BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative

Techniques Used:

12) Product Images from "Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation"

Article Title: Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-7187-9_15

ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA
Figure Legend Snippet: ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA

Techniques Used: Labeling, Sequencing, Amplification

13) Product Images from "Motor-like DNA motion due to an ATP-hydrolyzing protein under nanoconfinement"

Article Title: Motor-like DNA motion due to an ATP-hydrolyzing protein under nanoconfinement

Journal: Scientific Reports

doi: 10.1038/s41598-018-28278-0

( A ) Schematic of the device. ( B,C ) Kymographs showing the fluorescent intensity along nanochannel axis as a function of time for bare λ -DNA ( B ) and λ -DNA with T4 DNA ligase in a catalytically active buffer ( C ), respectively. ( D ) Center of mass of the molecules in ( B ) (red, diamonds) and ( C ) (blue, circles) as function of time.
Figure Legend Snippet: ( A ) Schematic of the device. ( B,C ) Kymographs showing the fluorescent intensity along nanochannel axis as a function of time for bare λ -DNA ( B ) and λ -DNA with T4 DNA ligase in a catalytically active buffer ( C ), respectively. ( D ) Center of mass of the molecules in ( B ) (red, diamonds) and ( C ) (blue, circles) as function of time.

Techniques Used:

Average mean-square displacement curves calculated from the center of mass position for 20 molecules for each condition. Blue circles correspond to data DNA with T4 DNA ligase in the presence of its cofactors, while red diamonds indicate bare DNA. Error bars are the standard deviation between molecules of the experimental set, and are depicted only for select data points to illustrate the trend. The continuous and dashed curves correspond to the fits for the conditions as described in the text.
Figure Legend Snippet: Average mean-square displacement curves calculated from the center of mass position for 20 molecules for each condition. Blue circles correspond to data DNA with T4 DNA ligase in the presence of its cofactors, while red diamonds indicate bare DNA. Error bars are the standard deviation between molecules of the experimental set, and are depicted only for select data points to illustrate the trend. The continuous and dashed curves correspond to the fits for the conditions as described in the text.

Techniques Used: Standard Deviation

14) Product Images from "Early Necrotic DNA Degradation "

Article Title: Early Necrotic DNA Degradation

Journal: The American Journal of Pathology

doi:

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 ).
Figure Legend 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 ).

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

15) Product Images from "Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation"

Article Title: Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-7187-9_15

ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA
Figure Legend Snippet: ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA

Techniques Used: Labeling, Sequencing, Amplification

16) Product Images from "Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation"

Article Title: Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-7187-9_15

ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA
Figure Legend Snippet: ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA

Techniques Used: Labeling, Sequencing, Amplification

17) Product Images from "Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction"

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

Journal: PLoS ONE

doi: 10.1371/journal.pone.0111538

Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).
Figure Legend Snippet: Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).

Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Amplification, Polymerase Chain Reaction, Ligation

18) Product Images from "Early Necrotic DNA Degradation "

Article Title: Early Necrotic DNA Degradation

Journal: The American Journal of Pathology

doi:

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 ).
Figure Legend 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 ).

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

19) Product Images from "Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair"

Article Title: Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

Journal: DNA repair

doi: 10.1016/j.dnarep.2014.01.015

BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative
Figure Legend Snippet: BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative

Techniques Used:

20) Product Images from "Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation"

Article Title: Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-7187-9_15

ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA
Figure Legend Snippet: ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA

Techniques Used: Labeling, Sequencing, Amplification

21) Product Images from "An efficient ORF selection system for DNA fragment libraries based on split beta-lactamase complementation"

Article Title: An efficient ORF selection system for DNA fragment libraries based on split beta-lactamase complementation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0235853

Schematic representation of pVMAKORF001 vector. Only relevant genes and restriction sites are shown. The map is not to scale. Kan r , kanamycin resistance gene; ColE1, origin of replication; Fori, phage M13 origin of replication; AraC and pBAD, Cassette encoding arabinose promoter and its regulator AraC; BlaSS, Native bla signal sequence; α 24–195, Bla-Alpha fragment encoding 24–195 amino acids of beta-lactamase; M182T, Methionine 182 to threonine mutation; NGR, Asparagine-Glycine-Arginine tripeptide; S1 and S2, (Gly4Ser)3 linker; Sp. Spacer sequence; TEV, Tobacco Etch Virus protease sequence; ω 196–286, Bla-Omega fragment encoding 196–286 amino acids of beta-lactamase; SacB and SacR, gene cassette encoding levansucrase of B . subtilis . (A)-(C) Sequence of different cassettes of the vector. (D) Sequence of insert to be cloned in the vector. (E) Insert carrying 4 base 5’ overhangs after T4 DNA polymerase treatment in the presence of dTTP.
Figure Legend Snippet: Schematic representation of pVMAKORF001 vector. Only relevant genes and restriction sites are shown. The map is not to scale. Kan r , kanamycin resistance gene; ColE1, origin of replication; Fori, phage M13 origin of replication; AraC and pBAD, Cassette encoding arabinose promoter and its regulator AraC; BlaSS, Native bla signal sequence; α 24–195, Bla-Alpha fragment encoding 24–195 amino acids of beta-lactamase; M182T, Methionine 182 to threonine mutation; NGR, Asparagine-Glycine-Arginine tripeptide; S1 and S2, (Gly4Ser)3 linker; Sp. Spacer sequence; TEV, Tobacco Etch Virus protease sequence; ω 196–286, Bla-Omega fragment encoding 196–286 amino acids of beta-lactamase; SacB and SacR, gene cassette encoding levansucrase of B . subtilis . (A)-(C) Sequence of different cassettes of the vector. (D) Sequence of insert to be cloned in the vector. (E) Insert carrying 4 base 5’ overhangs after T4 DNA polymerase treatment in the presence of dTTP.

Techniques Used: Plasmid Preparation, Sequencing, Mutagenesis, Clone Assay

22) Product Images from "Probing transient protein-mediated DNA linkages using nanoconfinement"

Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

Journal: Biomicrofluidics

doi: 10.1063/1.4882775

AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with T4 DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.
Figure Legend Snippet: AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with T4 DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.

Techniques Used: Binding Assay

Mean aligned DNA molecule loop lengths as function of time for 22 molecules per dataset withtheir linear fits. Bare λ-DNA (blue), λ-DNA with T4 DNA ligase (green), and λ-DNA with T4 DNA ligaseand ATP (red).
Figure Legend Snippet: Mean aligned DNA molecule loop lengths as function of time for 22 molecules per dataset withtheir linear fits. Bare λ-DNA (blue), λ-DNA with T4 DNA ligase (green), and λ-DNA with T4 DNA ligaseand ATP (red).

Techniques Used:

Histogram of end-to-end lengths of extended DNA molecules, bare λ-DNA (solid bars), λ-DNA with T4DNA ligase (gray bars), and λ-DNA with T4 DNA ligase and ATP (white bars). A Gaussian was fit toeach distribution to determine the
Figure Legend Snippet: Histogram of end-to-end lengths of extended DNA molecules, bare λ-DNA (solid bars), λ-DNA with T4DNA ligase (gray bars), and λ-DNA with T4 DNA ligase and ATP (white bars). A Gaussian was fit toeach distribution to determine the

Techniques Used:

Histograms of heights of DNA-DNA crossings. (a) Bare DNA (N = 41). (b) DNA with T4 DNA ligase andATP (N = 174). The red dotted line corresponds to unoccupied crossings, the blue dashed line tooccupied crossings, and the
Figure Legend Snippet: Histograms of heights of DNA-DNA crossings. (a) Bare DNA (N = 41). (b) DNA with T4 DNA ligase andATP (N = 174). The red dotted line corresponds to unoccupied crossings, the blue dashed line tooccupied crossings, and the

Techniques Used:

23) Product Images from "Motor-like DNA motion due to an ATP-hydrolyzing protein under nanoconfinement"

Article Title: Motor-like DNA motion due to an ATP-hydrolyzing protein under nanoconfinement

Journal: Scientific Reports

doi: 10.1038/s41598-018-28278-0

( A ) Schematic of the device. ( B,C ) Kymographs showing the fluorescent intensity along nanochannel axis as a function of time for bare λ -DNA ( B ) and λ -DNA with T4 DNA ligase in a catalytically active buffer ( C ), respectively. ( D ) Center of mass of the molecules in ( B ) (red, diamonds) and ( C ) (blue, circles) as function of time.
Figure Legend Snippet: ( A ) Schematic of the device. ( B,C ) Kymographs showing the fluorescent intensity along nanochannel axis as a function of time for bare λ -DNA ( B ) and λ -DNA with T4 DNA ligase in a catalytically active buffer ( C ), respectively. ( D ) Center of mass of the molecules in ( B ) (red, diamonds) and ( C ) (blue, circles) as function of time.

Techniques Used:

Average mean-square displacement curves calculated from the center of mass position for 20 molecules for each condition. Blue circles correspond to data DNA with T4 DNA ligase in the presence of its cofactors, while red diamonds indicate bare DNA. Error bars are the standard deviation between molecules of the experimental set, and are depicted only for select data points to illustrate the trend. The continuous and dashed curves correspond to the fits for the conditions as described in the text.
Figure Legend Snippet: Average mean-square displacement curves calculated from the center of mass position for 20 molecules for each condition. Blue circles correspond to data DNA with T4 DNA ligase in the presence of its cofactors, while red diamonds indicate bare DNA. Error bars are the standard deviation between molecules of the experimental set, and are depicted only for select data points to illustrate the trend. The continuous and dashed curves correspond to the fits for the conditions as described in the text.

Techniques Used: Standard Deviation

24) Product Images from "Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction"

Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

Journal: PLoS ONE

doi: 10.1371/journal.pone.0111538

Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).
Figure Legend Snippet: Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).

Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Amplification, Polymerase Chain Reaction, Ligation

Related Articles

Selection:

Article Title: An efficient ORF selection system for DNA fragment libraries based on split beta-lactamase complementation
Article Snippet: .. The inserts are then ligated to BsaI-digested pVMAKORF001 vector (for ORF selection) using T4 DNA ligase. .. The ligation mix is then electroporated in E . coli host TOP10F’ to obtain M . tuberculosis DNA fragment library. (PDF).

Ligation:

Article Title: Early Necrotic DNA Degradation
Article Snippet: .. The buffer was aspirated, and the full ligation mix containing the ligation buffer with the hairpin probe (35 μg/ml) and T4 DNA ligase (250 U/ml, Roche Molecular Biochemicals) was applied to the sections. .. In a mock control reaction solution, an equal volume of 50% glycerol in water was substituted for T4 DNA ligase.

Purification:

Article Title: Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair
Article Snippet: .. The DNA was incubated in the mitochondrial extracts, and then purified from the extracts by phenol/chloroform purification and divided into two aliquots, one was incubated with T4 DNA ligase (to determine the level of unligated but ligatable BER intermediates after repair) and one was left untreated. .. T4 DNA ligase treatment of the DNA resulted in ~2-fold increase in the intensity of the fully repaired 24 nucleotide band at the 40 and 60 min incubation times, indicating the presence of substantial amounts of unligated, but ligatable, BER intermediates in the samples ( ).

Incubation:

Article Title: Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair
Article Snippet: .. DNA was resuspended in 10 mM Tris–HCl pH 8.5 and divided into two aliquots; one was incubated with 1 μl T4 DNA ligase (Roche) at 37 °C for 60 min and one was left untreated. .. T4 DNA ligase was inactivated at 70 °C for 10 min and DNA was digested with XbaI and HindIII restriction enzymes and separated in 12% denaturing polyacrylamide gel at 300 V for 1 h.

Article Title: Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair
Article Snippet: .. The DNA was incubated in the mitochondrial extracts, and then purified from the extracts by phenol/chloroform purification and divided into two aliquots, one was incubated with T4 DNA ligase (to determine the level of unligated but ligatable BER intermediates after repair) and one was left untreated. .. T4 DNA ligase treatment of the DNA resulted in ~2-fold increase in the intensity of the fully repaired 24 nucleotide band at the 40 and 60 min incubation times, indicating the presence of substantial amounts of unligated, but ligatable, BER intermediates in the samples ( ).

other:

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

Article Title: Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair
Article Snippet: T4 DNA ligase was inactivated at 70 °C for 10 min and DNA was digested with XbaI and HindIII restriction enzymes and separated in 12% denaturing polyacrylamide gel at 300 V for 1 h.

Article Title: Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation
Article Snippet: In the mock reaction an equal volume of 50% glycerol in water is substituted for T4 DNA ligase.

Article Title: Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation
Article Snippet: 10× reaction buffer for T4 DNA ligase: 660 mM-Tris–HCl, 50 mM MgCl2 , 10 mM dithioerythritol, 10 mM ATP, pH 7.5 (20 °C) (Roche Molecular Biochemicals, Indianapolis, IN).

Plasmid Preparation:

Article Title: An efficient ORF selection system for DNA fragment libraries based on split beta-lactamase complementation
Article Snippet: .. The inserts are then ligated to BsaI-digested pVMAKORF001 vector (for ORF selection) using T4 DNA ligase. .. The ligation mix is then electroporated in E . coli host TOP10F’ to obtain M . tuberculosis DNA fragment library. (PDF).

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    Roche 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 ).
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    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

    BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative

    Journal: DNA repair

    Article Title: Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

    doi: 10.1016/j.dnarep.2014.01.015

    Figure Lengend Snippet: BER analysis of mitochondrial extracts. BER analysis was carried out in duplicate using two independently prepared mitochondrial extracts from HeLa (A) and U2OS (B) cell lines. The increased level of the upper band after T4 DNA ligase treatment relative

    Article Snippet: T4 DNA ligase was inactivated at 70 °C for 10 min and DNA was digested with XbaI and HindIII restriction enzymes and separated in 12% denaturing polyacrylamide gel at 300 V for 1 h.

    Techniques:

    ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation

    doi: 10.1007/978-1-4939-7187-9_15

    Figure Lengend Snippet: ISL probe and regular ISL labeling. ( a ) A regular ISL probe is a blunt-ended DNA fragment with 3′OH/5′OH at the ends. It can be tagged with either digoxigenin or fluorophores. The probe's actual sequence is unimportant for the regular ISL procedure. The probe can be used in the isothermal signal amplification procedure if it contains a single recognition site for a blunt-end restrictase (Sma I in this example). ( b ) In the regular ISL, probes are ligated to 5′PO 4 blunt-ended apoptotic DNA breaks in tissue sections. They cannot ligate to each other due to the absence of 5′PO 4 termini required by T4 DNA ligase. Therefore, the regular ISL reaction stops when all DNA probes are ligated to 5′PO 4 breaks in apoptotic DNA

    Article Snippet: In the mock reaction an equal volume of 50% glycerol in water is substituted for T4 DNA ligase.

    Techniques: Labeling, Sequencing, Amplification