vl damaged dna  (New England Biolabs)


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    New England Biolabs vl damaged dna
    Repair of UVC-induced CPDs from the 3-kb <t>EcoRI</t> fragment of the Ad-encoded lacZ gene in human and rodent cells measured by loss of T4pdg-sensitive sites. ( A ) Southern blot analysis of the repair of UVC-induced CPDs in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad <t>DNA,</t> while lanes 3 and 4 contain Ad DNA exposed to 240 J/m 2 UVC. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with T4pdg (lanes 1 and 3) represent ssDNA breaks generated by other sources. Following UVC exposure, a large number of T4pdg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, NER removes CPDs resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as CPDs persist in the lacZ DNA, T4pdg will induce ssDNA breaks resulting in less full-length signal compared to the control. ( B ) Quantification of the percent removal of CPDs from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents an arithmetic mean ± SE of the percent removal of UVC-induced T4pdg-sensitive sites from three independent experiments. A significant increase in the percent removal of UVC-induced T4pdg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of T4pdg-sensitive sites was observed between GM637F and CHO-AA8 24 h (indicated by a cross/plus sign).
    Vl Damaged Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vl damaged dna/product/New England Biolabs
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vl damaged dna - by Bioz Stars, 2020-08
    85/100 stars

    Related Products / Commonly Used Together

    ecori-digested
    fpg
    endonuclease treatment

    Images

    1) Product Images from "Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine"

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine

    Journal: Mutagenesis

    doi: 10.1093/mutage/get027

    Repair of UVC-induced CPDs from the 3-kb EcoRI fragment of the Ad-encoded lacZ gene in human and rodent cells measured by loss of T4pdg-sensitive sites. ( A ) Southern blot analysis of the repair of UVC-induced CPDs in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 240 J/m 2 UVC. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with T4pdg (lanes 1 and 3) represent ssDNA breaks generated by other sources. Following UVC exposure, a large number of T4pdg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, NER removes CPDs resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as CPDs persist in the lacZ DNA, T4pdg will induce ssDNA breaks resulting in less full-length signal compared to the control. ( B ) Quantification of the percent removal of CPDs from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents an arithmetic mean ± SE of the percent removal of UVC-induced T4pdg-sensitive sites from three independent experiments. A significant increase in the percent removal of UVC-induced T4pdg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of T4pdg-sensitive sites was observed between GM637F and CHO-AA8 24 h (indicated by a cross/plus sign).
    Figure Legend Snippet: Repair of UVC-induced CPDs from the 3-kb EcoRI fragment of the Ad-encoded lacZ gene in human and rodent cells measured by loss of T4pdg-sensitive sites. ( A ) Southern blot analysis of the repair of UVC-induced CPDs in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 240 J/m 2 UVC. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with T4pdg (lanes 1 and 3) represent ssDNA breaks generated by other sources. Following UVC exposure, a large number of T4pdg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, NER removes CPDs resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as CPDs persist in the lacZ DNA, T4pdg will induce ssDNA breaks resulting in less full-length signal compared to the control. ( B ) Quantification of the percent removal of CPDs from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents an arithmetic mean ± SE of the percent removal of UVC-induced T4pdg-sensitive sites from three independent experiments. A significant increase in the percent removal of UVC-induced T4pdg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of T4pdg-sensitive sites was observed between GM637F and CHO-AA8 24 h (indicated by a cross/plus sign).

    Techniques Used: Southern Blot, Generated, Incubation

    Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).
    Figure Legend Snippet: Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Techniques Used: Southern Blot, Incubation, Generated

    Related Articles

    Electrophoresis:

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine
    Article Snippet: .. For endonuclease treatment of MB + VL-damaged DNA, the EcoRI-digested samples were divided in half and incubated with or without 16 units of Fpg (1× NEB buffer 1, 100 µg/ml BSA) for 5 h. Following incubation, the samples were loaded directly into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V). .. Ad DNA was separated on denaturing 8% alkaline agarose gels run at 10 V for 20–24 h. Following gel electrophoresis, two 30-min washes in neutralisation buffer [1.5 M Tris (pH 7.4), 1.5 M NaCl] were performed after which DNA was transferred to a neutral nylon membrane (Hybond N, Amersham) by upward capillary transfer of 10× SSC.

    Incubation:

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine
    Article Snippet: .. For endonuclease treatment of MB + VL-damaged DNA, the EcoRI-digested samples were divided in half and incubated with or without 16 units of Fpg (1× NEB buffer 1, 100 µg/ml BSA) for 5 h. Following incubation, the samples were loaded directly into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V). .. Ad DNA was separated on denaturing 8% alkaline agarose gels run at 10 V for 20–24 h. Following gel electrophoresis, two 30-min washes in neutralisation buffer [1.5 M Tris (pH 7.4), 1.5 M NaCl] were performed after which DNA was transferred to a neutral nylon membrane (Hybond N, Amersham) by upward capillary transfer of 10× SSC.

    Agarose Gel Electrophoresis:

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine
    Article Snippet: .. For endonuclease treatment of MB + VL-damaged DNA, the EcoRI-digested samples were divided in half and incubated with or without 16 units of Fpg (1× NEB buffer 1, 100 µg/ml BSA) for 5 h. Following incubation, the samples were loaded directly into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V). .. Ad DNA was separated on denaturing 8% alkaline agarose gels run at 10 V for 20–24 h. Following gel electrophoresis, two 30-min washes in neutralisation buffer [1.5 M Tris (pH 7.4), 1.5 M NaCl] were performed after which DNA was transferred to a neutral nylon membrane (Hybond N, Amersham) by upward capillary transfer of 10× SSC.

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    New England Biolabs vl damaged dna
    Repair of UVC-induced CPDs from the 3-kb <t>EcoRI</t> fragment of the Ad-encoded lacZ gene in human and rodent cells measured by loss of T4pdg-sensitive sites. ( A ) Southern blot analysis of the repair of UVC-induced CPDs in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad <t>DNA,</t> while lanes 3 and 4 contain Ad DNA exposed to 240 J/m 2 UVC. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with T4pdg (lanes 1 and 3) represent ssDNA breaks generated by other sources. Following UVC exposure, a large number of T4pdg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, NER removes CPDs resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as CPDs persist in the lacZ DNA, T4pdg will induce ssDNA breaks resulting in less full-length signal compared to the control. ( B ) Quantification of the percent removal of CPDs from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents an arithmetic mean ± SE of the percent removal of UVC-induced T4pdg-sensitive sites from three independent experiments. A significant increase in the percent removal of UVC-induced T4pdg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of T4pdg-sensitive sites was observed between GM637F and CHO-AA8 24 h (indicated by a cross/plus sign).
    Vl Damaged Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vl damaged dna/product/New England Biolabs
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vl damaged dna - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    88
    New England Biolabs vl plasmid dna
    Repair of UVC-induced CPDs from the 3-kb <t>EcoRI</t> fragment of the Ad-encoded lacZ gene in human and rodent cells measured by loss of T4pdg-sensitive sites. ( A ) Southern blot analysis of the repair of UVC-induced CPDs in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad <t>DNA,</t> while lanes 3 and 4 contain Ad DNA exposed to 240 J/m 2 UVC. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with T4pdg (lanes 1 and 3) represent ssDNA breaks generated by other sources. Following UVC exposure, a large number of T4pdg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, NER removes CPDs resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as CPDs persist in the lacZ DNA, T4pdg will induce ssDNA breaks resulting in less full-length signal compared to the control. ( B ) Quantification of the percent removal of CPDs from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents an arithmetic mean ± SE of the percent removal of UVC-induced T4pdg-sensitive sites from three independent experiments. A significant increase in the percent removal of UVC-induced T4pdg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of T4pdg-sensitive sites was observed between GM637F and CHO-AA8 24 h (indicated by a cross/plus sign).
    Vl Plasmid Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vl plasmid dna/product/New England Biolabs
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vl plasmid dna - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    Image Search Results


    Repair of UVC-induced CPDs from the 3-kb EcoRI fragment of the Ad-encoded lacZ gene in human and rodent cells measured by loss of T4pdg-sensitive sites. ( A ) Southern blot analysis of the repair of UVC-induced CPDs in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 240 J/m 2 UVC. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with T4pdg (lanes 1 and 3) represent ssDNA breaks generated by other sources. Following UVC exposure, a large number of T4pdg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, NER removes CPDs resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as CPDs persist in the lacZ DNA, T4pdg will induce ssDNA breaks resulting in less full-length signal compared to the control. ( B ) Quantification of the percent removal of CPDs from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents an arithmetic mean ± SE of the percent removal of UVC-induced T4pdg-sensitive sites from three independent experiments. A significant increase in the percent removal of UVC-induced T4pdg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of T4pdg-sensitive sites was observed between GM637F and CHO-AA8 24 h (indicated by a cross/plus sign).

    Journal: Mutagenesis

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine

    doi: 10.1093/mutage/get027

    Figure Lengend Snippet: Repair of UVC-induced CPDs from the 3-kb EcoRI fragment of the Ad-encoded lacZ gene in human and rodent cells measured by loss of T4pdg-sensitive sites. ( A ) Southern blot analysis of the repair of UVC-induced CPDs in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 240 J/m 2 UVC. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with T4pdg (lanes 1 and 3) represent ssDNA breaks generated by other sources. Following UVC exposure, a large number of T4pdg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, NER removes CPDs resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as CPDs persist in the lacZ DNA, T4pdg will induce ssDNA breaks resulting in less full-length signal compared to the control. ( B ) Quantification of the percent removal of CPDs from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents an arithmetic mean ± SE of the percent removal of UVC-induced T4pdg-sensitive sites from three independent experiments. A significant increase in the percent removal of UVC-induced T4pdg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of T4pdg-sensitive sites was observed between GM637F and CHO-AA8 24 h (indicated by a cross/plus sign).

    Article Snippet: For endonuclease treatment of MB + VL-damaged DNA, the EcoRI-digested samples were divided in half and incubated with or without 16 units of Fpg (1× NEB buffer 1, 100 µg/ml BSA) for 5 h. Following incubation, the samples were loaded directly into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V).

    Techniques: Southern Blot, Generated, Incubation

    Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Journal: Mutagenesis

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine

    doi: 10.1093/mutage/get027

    Figure Lengend Snippet: Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Article Snippet: For endonuclease treatment of MB + VL-damaged DNA, the EcoRI-digested samples were divided in half and incubated with or without 16 units of Fpg (1× NEB buffer 1, 100 µg/ml BSA) for 5 h. Following incubation, the samples were loaded directly into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V).

    Techniques: Southern Blot, Incubation, Generated