t5 exonuclease digestion  (New England Biolabs)


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    Name:
    T5 Exonuclease
    Description:

    Catalog Number:
    M0663
    Price:
    270
    Category:
    DNA Modifying Enzymes
    Applications:
    DNA Manipulation
    Size:
    5000 units
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    Structured Review

    New England Biolabs t5 exonuclease digestion
    T5 Exonuclease

    https://www.bioz.com/result/t5 exonuclease digestion/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t5 exonuclease digestion - by Bioz Stars, 2021-07
    97/100 stars

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    other:

    Article Title: A Simple Enhancement for Gibson Isothermal Assembly
    Article Snippet: To create a 1.33x Gibson Assembly mix, 40 units Phusion High-Fidelity DNA Polymerase (NEB M0530), 12 units T5 exonuclease (NEB M0363), and 6,400 units Tth DNA Ligase (homemade, supplementary protocol 1) were added to 320 ul of 5x Isothermal Reaction Mix.

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: TEDA uses T5 exonuclease that generates 3′-overhangs.

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: TEDA uses only T5 exonuclease to create 3′-overhangs of linear DNA fragments, which anneal to form circle DNA with gaps.

    Clone Assay:

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: The PCR and sequencing primers for the plasmids used in TEDA optimization and testing were listed in . .. T5 exonuclease in a Tris buffer with PEG 8000 works well for cloning The Gibson method applies three enzymes, and it can be simplified by removing Taq DNA ligase without reducing the cloning efficiency ( , ). .. To test whether the method could be further simplified, we checked the requirement of the enzymes and other components in the Gibson system for cloning Pkat-eGFP into pBluescript SK- (Figure ).

    Sequencing:

    Article Title: Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing
    Article Snippet: The PCR products were purified by the Illustra GFX PCR DNA and gel band purification kits (GE Healthcare), and were subjected to Sanger sequencing. .. 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. .. Immunoblotting Assay Cells were washed with phosphate-buffered saline (PBS) and lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 [NP-40], 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail [Roche]).

    Amplification:

    Article Title: Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques
    Article Snippet: Quantification was assessed separately for each primer/probe target set against an absolute standard curve using the plasmid pcDNA_HBV1.3. .. To control for low-level amplification of rcDNA with the primer/probe set targeting the nick and gap of the HBV genome, cccDNA quantification from rhesus macaque PH was normalized by digesting HBV DNA, extracted from HBV stocks and thus containing only rcDNA, with T5 exonuclease and measuring quantification of this product with both primer sets. .. hNTCP DNA and RNA quantificationTo quantify human NTCP DNA and RNA in macaque liver samples, total DNA and RNA were extracted using the AllPrep DNA/RNA kit according to manufacturer’s instructions (Qiagen).

    Incubation:

    Article Title: The gp44 Ejection Protein of Staphylococcus aureus Bacteriophage 80α Binds to the Ends of the Genome and Protects It from Degradation
    Article Snippet: The mixture was then run on either a Novex 6% polyacrylamide DNA retardation gel (Thermo Fisher) or on 0.7% agarose gel in 0.5× or 1× TBE buffer, respectively, and stained with ethidium bromide for DNA detection. .. For nuclease assays, the DNA–protein mixture was incubated with 0.025 U T5 exonuclease or 0.5 µg DNAse I and incubated for 2 hours at 30 °C. .. The reaction was halted by addition of EDTA (final concentration 14 mM) and proteinase K (final concentration 0.3 mg/mL) and incubated at 55 °C for 1 hour.

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  • 97
    New England Biolabs t5 exonuclease
    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, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t5 exonuclease/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t5 exonuclease - by Bioz Stars, 2021-07
    97/100 stars
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    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

    In vitro HBV infection of rhesus macaque PH.  a  Predicted schematic of NTCP showing amino acid differences between human and macaque NTCP. Differences in the sequences were labeled with lighter red for amino acid exchanges with similar physiochemical properties and darker red for exchanges with different physiochemical properties. Gray box represents cellular membrane. N-linked glycosylation sites represented by black brackets. macNTCP = macaque NTCP.  b  Rhesus macaque PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and stained 3 days later with Myrcludex B-atto488.  c  Rhesus macaque and baboon PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later. Productive infection was monitored by quantification of HBsAg and HBeAg in the supernatant by ELISA. Each condition represents a single-biological sample ( N  = 1). Figure is representative data of two separate experiments.  d  HBV DNA qPCR on the same supernatants shown in  c . Each condition represents a single-biological sample ( N  = 1).  e  Total intracellular DNA from 1 × 10 6  rhesus macaque PH and HepG2-hNTCP cells was used in a cccDNA-specific qPCR. Rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later showed formation of cccDNA, while the non-transduced, HBV challenged PH did not. Bars represent standard error of measurement from two qPCR replicates.  f  Southern blot shows presence of cccDNA in rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100). DNA was purified after Hirt extraction to remove protein-bound DNA forms. SM = size marker; T5 Exo = T5 exonuclease; PF-rcDNA = polymerase-free relaxed circular DNA; PF-dlDNA = polymerase-free duplex linear DNA. Figure is representative data of two separate experiments.  g  Neonate rhesus macaque PH were transduced with AAV-hNTCP (MOI = 1 × 10 4  or 5 × 10 2 ) and infected with HBV (MOI = 100) 3 days later. HBV infection was then monitored longitudinally by HBsAg ELISA. Each condition represents a single-biological sample ( N  = 1)

    Journal: Nature Communications

    Article Title: Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques

    doi: 10.1038/s41467-017-01953-y

    Figure Lengend Snippet: In vitro HBV infection of rhesus macaque PH. a Predicted schematic of NTCP showing amino acid differences between human and macaque NTCP. Differences in the sequences were labeled with lighter red for amino acid exchanges with similar physiochemical properties and darker red for exchanges with different physiochemical properties. Gray box represents cellular membrane. N-linked glycosylation sites represented by black brackets. macNTCP = macaque NTCP. b Rhesus macaque PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and stained 3 days later with Myrcludex B-atto488. c Rhesus macaque and baboon PH were transduced with either HDAd-hNTCP (MOI = 2) or AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later. Productive infection was monitored by quantification of HBsAg and HBeAg in the supernatant by ELISA. Each condition represents a single-biological sample ( N  = 1). Figure is representative data of two separate experiments. d HBV DNA qPCR on the same supernatants shown in c . Each condition represents a single-biological sample ( N  = 1). e Total intracellular DNA from 1 × 10 6 rhesus macaque PH and HepG2-hNTCP cells was used in a cccDNA-specific qPCR. Rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100) 3 days later showed formation of cccDNA, while the non-transduced, HBV challenged PH did not. Bars represent standard error of measurement from two qPCR replicates. f Southern blot shows presence of cccDNA in rhesus macaque PH transduced with AAV-hNTCP (MOI = 1 × 10 4 ) and infected with HBV (MOI = 100). DNA was purified after Hirt extraction to remove protein-bound DNA forms. SM = size marker; T5 Exo = T5 exonuclease; PF-rcDNA = polymerase-free relaxed circular DNA; PF-dlDNA = polymerase-free duplex linear DNA. Figure is representative data of two separate experiments. g Neonate rhesus macaque PH were transduced with AAV-hNTCP (MOI = 1 × 10 4 or 5 × 10 2 ) and infected with HBV (MOI = 100) 3 days later. HBV infection was then monitored longitudinally by HBsAg ELISA. Each condition represents a single-biological sample ( N  = 1)

    Article Snippet: To confirm the presence of cccDNA, Hirt extracted DNA was digested with T5 exonuclease (New England Biolabs) at 37 °C for 30 min.

    Techniques: In Vitro, Infection, Labeling, Transduction, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Southern Blot, Purification, Marker

    Susceptibility of rcDNA and DP-rcDNA to exonuclease digestion. (A) HBV cytoplasmic core DNA was left untreated (mock) or treated with 5′ exonuclease T5 at the indicated doses at 37°C for 30 min, followed by Southern blotting with HBV (−)- or (+)strand-specific riboprobe. (B and C) Similar amounts of HBV cytoplasmic core DNA and DP-rcDNA were left untreated or treated with 5′ exonuclease T5 (0.8 U) or the 3′ exonuclease combination ExoI/III (10/50 U) at 37°C for 30 min and subjected to Southern blotting and hybridization with HBV DNA strand-specific riboprobes.

    Journal: Journal of Virology

    Article Title: Characterization of the Termini of Cytoplasmic Hepatitis B Virus Deproteinated Relaxed Circular DNA

    doi: 10.1128/JVI.00922-20

    Figure Lengend Snippet: Susceptibility of rcDNA and DP-rcDNA to exonuclease digestion. (A) HBV cytoplasmic core DNA was left untreated (mock) or treated with 5′ exonuclease T5 at the indicated doses at 37°C for 30 min, followed by Southern blotting with HBV (−)- or (+)strand-specific riboprobe. (B and C) Similar amounts of HBV cytoplasmic core DNA and DP-rcDNA were left untreated or treated with 5′ exonuclease T5 (0.8 U) or the 3′ exonuclease combination ExoI/III (10/50 U) at 37°C for 30 min and subjected to Southern blotting and hybridization with HBV DNA strand-specific riboprobes.

    Article Snippet: HBV cytoplasmic core DNA and DP-rcDNA extracted from HepDE19 cells were digested with 5′ exonuclease T5 (NEB) or 3′ exonuclease ExoI/III (NEB) at the indicated doses in a total volume of 30 μl per reaction mixture at 37°C for 30 min.

    Techniques: Southern Blot, Hybridization