bss hii  (New England Biolabs)


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    Name:
    BssHII
    Description:
    BssHII 2 500 units
    Catalog Number:
    r0199l
    Price:
    282
    Size:
    2 500 units
    Category:
    Restriction Enzymes
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    New England Biolabs bss hii
    BssHII
    BssHII 2 500 units
    https://www.bioz.com/result/bss hii/product/New England Biolabs
    Average 95 stars, based on 1254 article reviews
    Price from $9.99 to $1999.99
    bss hii - by Bioz Stars, 2020-07
    95/100 stars

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    Clone Assay:

    Article Title: Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements
    Article Snippet: .. The HPyV NCCRs were chemically synthesized in pUC57 (Eurogentec S.A, Belgium) , excised using the restriction enzymes BssHII and MluI (New England BioLabs, England), and cloned into the corresponding restriction sites of pRG13D12. .. HPyV NCCR constructs were verified by Sanger sequencing for correct NCCR sequences and orientations using the 3130 genetic analyzer (Applied Biosystems, Switzerland).

    Synthesized:

    Article Title: Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements
    Article Snippet: .. The HPyV NCCRs were chemically synthesized in pUC57 (Eurogentec S.A, Belgium) , excised using the restriction enzymes BssHII and MluI (New England BioLabs, England), and cloned into the corresponding restriction sites of pRG13D12. .. HPyV NCCR constructs were verified by Sanger sequencing for correct NCCR sequences and orientations using the 3130 genetic analyzer (Applied Biosystems, Switzerland).

    Isolation:

    Article Title: Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes
    Article Snippet: .. Probes corresponding to five unique E.cuniculi genes isolated in our laboratory ( , ; C.Biderre, personal communication), were hybridised to Bss HII- and Mlu I-KARD-PFGE gels. ..

    Methylation:

    Article Title: Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes
    Article Snippet: .. This low frequency might also be explained by a DNA modification to which both Bss HII and Mlu I are sensitive, e.g. cytosine methylation as in higher eukaryotes. .. If DNA methylation occurs in E.cuniculi , differences in the methylation pattern of two homologous chromosomes should lead to differentially migrated fragments in KARD-PFGE gels.

    Labeling:

    Article Title: Murine Cytomegalovirus CC Chemokine Homolog MCK-2 (m131-129) Is a Determinant of Dissemination That Increases Inflammation at Initial Sites of Infection
    Article Snippet: .. Virion DNA (0.5 to 1.0 μg) was digested with Hin dIII, Bss HII, Afl II, or Spe I (New England Biolabs, Beverly, Mass.) and end labeled in the presence of 2.5 μCi of [α-32 P]dCTP (Amersham), 125 μM each dATP, dGTP, and dTTP, and 0.5 U of Klenow polymerase (Roche, Indianapolis, Ind.) for 15 min at room temperature in 20 μl of restriction enzyme buffer. .. Restriction fragments were separated on a 0.6% agarose gel, which was fixed in 95% ethanol and vacuum dried at 80°C, followed by autoradiography.

    other:

    Article Title: Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes
    Article Snippet: In order to place the Bss HII and Mlu I restriction sites on each chromosome, we developed a mapping procedure applied to individual chromosomes (DDIC-PFGE) that can be viewed as the counterpart of 2D-PFGE in bacterial genomics ( ).

    Article Title: Genetic relationships between Candida albicans strains isolated from dental plaque, trachea, and bronchoalveolar lavage fluid from mechanically ventilated intensive care unit patients
    Article Snippet: In comparison, restriction endonuclease analysis of the genome (REAG) from the 49 isolates of 22 patients yielded 24 patterns after digestion with Sfi I ( ) and 25 patterns after digestion with Bss HII ( ).

    Modification:

    Article Title: Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes
    Article Snippet: .. This low frequency might also be explained by a DNA modification to which both Bss HII and Mlu I are sensitive, e.g. cytosine methylation as in higher eukaryotes. .. If DNA methylation occurs in E.cuniculi , differences in the methylation pattern of two homologous chromosomes should lead to differentially migrated fragments in KARD-PFGE gels.

    Plasmid Preparation:

    Article Title: Gene cloning, sequence analysis, and expression of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
    Article Snippet: .. According to the pBluescript map, we would expect to see the DNA fragment next to the pBluescript vector at the T3 promoter site in pBluMH to be about 0.1 kb smaller when double-digested with Bss HII + Pst I than when digested with Bss HII alone. .. The Southern hybridization results show that the 1.1-kb fragment from Bss HII digestion corresponds to a 1-kb fragment in the Bss HII + Pst I double-digestion.

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    New England Biolabs vl pcr
    One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and <t>PCR</t> amplicons contain or are flanked by <t>Bsa</t> I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping
    Vl Pcr, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    New England Biolabs a13 vl
    Competition ELISA for comparison of relative affinities of anti-EGFR IgGs. ER414 is a fully human IgG1 originated from the mAb <t>A13</t> by guided panning. Binding inhibition of anti-EGFR IgGs to EGFR was analyzed on EGFR-coated plates with different concentrations
    A13 Vl, 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
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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
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    One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

    Journal: Microbial Cell Factories

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries

    doi: 10.1186/s12934-017-0853-z

    Figure Lengend Snippet: One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

    Article Snippet: 160 ng of VH PCR product and 160 ng VL PCR product as well as 200 U Bsa I (New England Biolabs), 800 U T4 DNA ligase (New England Biolabs) and 10 µL 10× T4 Ligase buffer (New England Biolabs).

    Techniques: Clone Assay, Polymerase Chain Reaction, Expressing, Sequencing, Generated, Plasmid Preparation

    One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

    Journal: Microbial Cell Factories

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries

    doi: 10.1186/s12934-017-0853-z

    Figure Lengend Snippet: One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

    Article Snippet: 160 ng of VH PCR product and 160 ng VL PCR product as well as 200 U Bsa I (New England Biolabs), 800 U T4 DNA ligase (New England Biolabs) and 10 µL 10× T4 Ligase buffer (New England Biolabs).

    Techniques: Clone Assay, Polymerase Chain Reaction, Expressing, Sequencing, Generated, Plasmid Preparation

    Competition ELISA for comparison of relative affinities of anti-EGFR IgGs. ER414 is a fully human IgG1 originated from the mAb A13 by guided panning. Binding inhibition of anti-EGFR IgGs to EGFR was analyzed on EGFR-coated plates with different concentrations

    Journal: Experimental & Molecular Medicine

    Article Title: Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

    doi: 10.3858/emm.2012.44.1.005

    Figure Lengend Snippet: Competition ELISA for comparison of relative affinities of anti-EGFR IgGs. ER414 is a fully human IgG1 originated from the mAb A13 by guided panning. Binding inhibition of anti-EGFR IgGs to EGFR was analyzed on EGFR-coated plates with different concentrations

    Article Snippet: Again the PCR DNAs of A13 VH and A13 VL were amplified with primers H-extension(F)/H-extension(R) and K-extension(F)/K-extension(R) ( ) and inserted to pSC73His vector ( ) by digestion with Sfi I/BstE II (New England Bio Labs, Beverly, MA) for VH and BstE II/Not I (New England Bio Lab) for VL , which was designated as pSC73His-13VH -13VL .

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Inhibition

    Competition ELISA to analyze whether the ER414 has the same binding site with the murine counterpart A13 mAb. The competition was performed on the EGFR-coated plates with 1 µg of biotinylated ER414 and different concentrations of free ER414, A13

    Journal: Experimental & Molecular Medicine

    Article Title: Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

    doi: 10.3858/emm.2012.44.1.005

    Figure Lengend Snippet: Competition ELISA to analyze whether the ER414 has the same binding site with the murine counterpart A13 mAb. The competition was performed on the EGFR-coated plates with 1 µg of biotinylated ER414 and different concentrations of free ER414, A13

    Article Snippet: Again the PCR DNAs of A13 VH and A13 VL were amplified with primers H-extension(F)/H-extension(R) and K-extension(F)/K-extension(R) ( ) and inserted to pSC73His vector ( ) by digestion with Sfi I/BstE II (New England Bio Labs, Beverly, MA) for VH and BstE II/Not I (New England Bio Lab) for VL , which was designated as pSC73His-13VH -13VL .

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

    Competition ELISA for measuring relative affinities of anti-EGFR human scFvs which were isolated by guided panning of mAb A13. The inhibition of binding of anti-EGFR human scFvs to EGFR was analyzed on EGFR-coated plates with different concentrations

    Journal: Experimental & Molecular Medicine

    Article Title: Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

    doi: 10.3858/emm.2012.44.1.005

    Figure Lengend Snippet: Competition ELISA for measuring relative affinities of anti-EGFR human scFvs which were isolated by guided panning of mAb A13. The inhibition of binding of anti-EGFR human scFvs to EGFR was analyzed on EGFR-coated plates with different concentrations

    Article Snippet: Again the PCR DNAs of A13 VH and A13 VL were amplified with primers H-extension(F)/H-extension(R) and K-extension(F)/K-extension(R) ( ) and inserted to pSC73His vector ( ) by digestion with Sfi I/BstE II (New England Bio Labs, Beverly, MA) for VH and BstE II/Not I (New England Bio Lab) for VL , which was designated as pSC73His-13VH -13VL .

    Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Inhibition, Binding Assay

    FACS analysis for binding activity of anti-EGFR human scFvs to cell surface-expressed EGFR, in which the scFvs were isolated by guided panning of mAb A13. Differential binding of scFvs to EGFR-positive A431 tumor cells and EGFR-negative HL60 cells (indicated

    Journal: Experimental & Molecular Medicine

    Article Title: Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

    doi: 10.3858/emm.2012.44.1.005

    Figure Lengend Snippet: FACS analysis for binding activity of anti-EGFR human scFvs to cell surface-expressed EGFR, in which the scFvs were isolated by guided panning of mAb A13. Differential binding of scFvs to EGFR-positive A431 tumor cells and EGFR-negative HL60 cells (indicated

    Article Snippet: Again the PCR DNAs of A13 VH and A13 VL were amplified with primers H-extension(F)/H-extension(R) and K-extension(F)/K-extension(R) ( ) and inserted to pSC73His vector ( ) by digestion with Sfi I/BstE II (New England Bio Labs, Beverly, MA) for VH and BstE II/Not I (New England Bio Lab) for VL , which was designated as pSC73His-13VH -13VL .

    Techniques: FACS, Binding Assay, Activity Assay, Isolation

    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