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  • 99
    New England Biolabs t5 exonuclease digestion
    Base Editing of HBV cccDNA (A) Southern blot analysis of intracellular HBV replicative intermediates of BE/gRNA-transduced HepG2-NTCP-C4 cells infected by 5 × 10 5 genome equivalents (GEs) of HBV at 9 days post-infection. Lane 1, mock infection; lane 2, HBV infection without enzymatic treatment; lane 3, HBV infection with Eco RI treatment; lane 4, HBV infection with <t>T5</t> exonuclease treatment. RC, HBV RC-DNA; DSL, double-stranded linear DNA; CCC, cccDNA. (B) Sanger sequencing of the base-edited sites in cccDNA targeted by individual gRNAs gP9 and gS8. The labels are the same as those in Figure 1 B. (C) The fold change of secreted HBsAg levels, measured by the quantitative HBsAg assay, in the supernatant of HepG2-NTCP-C4 cells at day 6 and day 8 after HBV infection. (D) Fold change of supernatant HBV DNA in HepG2-NTCP-C4 cells transduced by individual gRNAs gP9 and gS8 in comparison to those transduced by the control glacZ. HepG2-NTCP-C4 cells were initially transduced by individual gRNAs, control glacZ, gP9, or gS8, along with SpCas9-BE and subsequently infected by HBV. (E) Individual percentages of C-to-T conversion at the target sites of cccDNA measured by NGS. (F) Individual percentages of indels at the gRNA-targeting sites of cccDNA measured by NGS. The results of (C) and (D)–(F) are combined from three independent experiments and shown in bar graphs with means plus standard error (SE). ∗∗p
    T5 Exonuclease Digestion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t5 exonuclease digestion/product/New England Biolabs
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    t5 exonuclease digestion - by Bioz Stars, 2020-08
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    99
    Millipore plasmid dna
    Base Editing of HBV cccDNA (A) Southern blot analysis of intracellular HBV replicative intermediates of BE/gRNA-transduced HepG2-NTCP-C4 cells infected by 5 × 10 5 genome equivalents (GEs) of HBV at 9 days post-infection. Lane 1, mock infection; lane 2, HBV infection without enzymatic treatment; lane 3, HBV infection with Eco RI treatment; lane 4, HBV infection with <t>T5</t> exonuclease treatment. RC, HBV RC-DNA; DSL, double-stranded linear DNA; CCC, cccDNA. (B) Sanger sequencing of the base-edited sites in cccDNA targeted by individual gRNAs gP9 and gS8. The labels are the same as those in Figure 1 B. (C) The fold change of secreted HBsAg levels, measured by the quantitative HBsAg assay, in the supernatant of HepG2-NTCP-C4 cells at day 6 and day 8 after HBV infection. (D) Fold change of supernatant HBV DNA in HepG2-NTCP-C4 cells transduced by individual gRNAs gP9 and gS8 in comparison to those transduced by the control glacZ. HepG2-NTCP-C4 cells were initially transduced by individual gRNAs, control glacZ, gP9, or gS8, along with SpCas9-BE and subsequently infected by HBV. (E) Individual percentages of C-to-T conversion at the target sites of cccDNA measured by NGS. (F) Individual percentages of indels at the gRNA-targeting sites of cccDNA measured by NGS. The results of (C) and (D)–(F) are combined from three independent experiments and shown in bar graphs with means plus standard error (SE). ∗∗p
    Plasmid Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7013 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid dna/product/Millipore
    Average 99 stars, based on 7013 article reviews
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    plasmid dna - by Bioz Stars, 2020-08
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    99
    New England Biolabs exonuclease t5
    Base Editing of HBV cccDNA (A) Southern blot analysis of intracellular HBV replicative intermediates of BE/gRNA-transduced HepG2-NTCP-C4 cells infected by 5 × 10 5 genome equivalents (GEs) of HBV at 9 days post-infection. Lane 1, mock infection; lane 2, HBV infection without enzymatic treatment; lane 3, HBV infection with Eco RI treatment; lane 4, HBV infection with <t>T5</t> exonuclease treatment. RC, HBV RC-DNA; DSL, double-stranded linear DNA; CCC, cccDNA. (B) Sanger sequencing of the base-edited sites in cccDNA targeted by individual gRNAs gP9 and gS8. The labels are the same as those in Figure 1 B. (C) The fold change of secreted HBsAg levels, measured by the quantitative HBsAg assay, in the supernatant of HepG2-NTCP-C4 cells at day 6 and day 8 after HBV infection. (D) Fold change of supernatant HBV DNA in HepG2-NTCP-C4 cells transduced by individual gRNAs gP9 and gS8 in comparison to those transduced by the control glacZ. HepG2-NTCP-C4 cells were initially transduced by individual gRNAs, control glacZ, gP9, or gS8, along with SpCas9-BE and subsequently infected by HBV. (E) Individual percentages of C-to-T conversion at the target sites of cccDNA measured by NGS. (F) Individual percentages of indels at the gRNA-targeting sites of cccDNA measured by NGS. The results of (C) and (D)–(F) are combined from three independent experiments and shown in bar graphs with means plus standard error (SE). ∗∗p
    Exonuclease T5, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease t5/product/New England Biolabs
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    exonuclease t5 - by Bioz Stars, 2020-08
    99/100 stars
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    Image Search Results


    Base Editing of HBV cccDNA (A) Southern blot analysis of intracellular HBV replicative intermediates of BE/gRNA-transduced HepG2-NTCP-C4 cells infected by 5 × 10 5 genome equivalents (GEs) of HBV at 9 days post-infection. Lane 1, mock infection; lane 2, HBV infection without enzymatic treatment; lane 3, HBV infection with Eco RI treatment; lane 4, HBV infection with T5 exonuclease treatment. RC, HBV RC-DNA; DSL, double-stranded linear DNA; CCC, cccDNA. (B) Sanger sequencing of the base-edited sites in cccDNA targeted by individual gRNAs gP9 and gS8. The labels are the same as those in Figure 1 B. (C) The fold change of secreted HBsAg levels, measured by the quantitative HBsAg assay, in the supernatant of HepG2-NTCP-C4 cells at day 6 and day 8 after HBV infection. (D) Fold change of supernatant HBV DNA in HepG2-NTCP-C4 cells transduced by individual gRNAs gP9 and gS8 in comparison to those transduced by the control glacZ. HepG2-NTCP-C4 cells were initially transduced by individual gRNAs, control glacZ, gP9, or gS8, along with SpCas9-BE and subsequently infected by HBV. (E) Individual percentages of C-to-T conversion at the target sites of cccDNA measured by NGS. (F) Individual percentages of indels at the gRNA-targeting sites of cccDNA measured by NGS. The results of (C) and (D)–(F) are combined from three independent experiments and shown in bar graphs with means plus standard error (SE). ∗∗p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing

    doi: 10.1016/j.omtn.2020.03.005

    Figure Lengend Snippet: Base Editing of HBV cccDNA (A) Southern blot analysis of intracellular HBV replicative intermediates of BE/gRNA-transduced HepG2-NTCP-C4 cells infected by 5 × 10 5 genome equivalents (GEs) of HBV at 9 days post-infection. Lane 1, mock infection; lane 2, HBV infection without enzymatic treatment; lane 3, HBV infection with Eco RI treatment; lane 4, HBV infection with T5 exonuclease treatment. RC, HBV RC-DNA; DSL, double-stranded linear DNA; CCC, cccDNA. (B) Sanger sequencing of the base-edited sites in cccDNA targeted by individual gRNAs gP9 and gS8. The labels are the same as those in Figure 1 B. (C) The fold change of secreted HBsAg levels, measured by the quantitative HBsAg assay, in the supernatant of HepG2-NTCP-C4 cells at day 6 and day 8 after HBV infection. (D) Fold change of supernatant HBV DNA in HepG2-NTCP-C4 cells transduced by individual gRNAs gP9 and gS8 in comparison to those transduced by the control glacZ. HepG2-NTCP-C4 cells were initially transduced by individual gRNAs, control glacZ, gP9, or gS8, along with SpCas9-BE and subsequently infected by HBV. (E) Individual percentages of C-to-T conversion at the target sites of cccDNA measured by NGS. (F) Individual percentages of indels at the gRNA-targeting sites of cccDNA measured by NGS. The results of (C) and (D)–(F) are combined from three independent experiments and shown in bar graphs with means plus standard error (SE). ∗∗p

    Article Snippet: To remove linear and RC-form HBV DNAs for Sanger and MiSeq sequencing of cccDNA, genomic DNAs were extracted and digested with T5 exonuclease (New England Biolabs) in the reaction mixture of 50 μL containing 500 ng of DNA, 5 μL of ,10× reaction buffer and 1 μL of T5 Exo at 37°C for 1 h, and afterward 11 mM EDTA was added to stop reaction.

    Techniques: Southern Blot, Infection, Countercurrent Chromatography, Sequencing, HBsAg Assay, Next-Generation Sequencing