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    New England Biolabs taq dna ligase
    FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified <t>NC-DNA</t> (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and <t>Taq</t> DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P
    Taq Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6723 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Article Snippet: NC-DNA (108 copies) was incubated with 32 units (U) of Thermostable FEN1 in ThermoPol Buffer (New England Biolabs) at 65°C for 10 min, followed by incubation with 8 U of Bst DNA polymerase, 40 U of Taq DNA ligase, 100 μM dNTPs, and NAD+ (all from New England Biolabs).

    Techniques: In Vitro, Tube Formation Assay, Purification, Incubation, Recombinant, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing

    Synthesis of a functional chloramphenicol acetyltransferase gene with changed codon composition. The ratio r of ‘active clones’ to ‘analyzed clones’ as described in the text is shown for different gene synthesis methods with or without an EMC step. A significant increase of r can be observed only in the cases where EMC is combined with an exonuclease activity present in the reaction or in the later amplification reaction. Prolonged incubation with E.coli endonuclease V results in no detectable product after the amplification steps (ss, single-stranded synthesis, ds, double-stranded synthesis; VII, T4 endonuclease VII; V, E.coli endonuclease V; T, Taq DNA polymerase; and Vn, Vent DNA polymerase).

    Journal: Nucleic Acids Research

    Article Title: Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage

    doi: 10.1093/nar/gni058

    Figure Lengend Snippet: Synthesis of a functional chloramphenicol acetyltransferase gene with changed codon composition. The ratio r of ‘active clones’ to ‘analyzed clones’ as described in the text is shown for different gene synthesis methods with or without an EMC step. A significant increase of r can be observed only in the cases where EMC is combined with an exonuclease activity present in the reaction or in the later amplification reaction. Prolonged incubation with E.coli endonuclease V results in no detectable product after the amplification steps (ss, single-stranded synthesis, ds, double-stranded synthesis; VII, T4 endonuclease VII; V, E.coli endonuclease V; T, Taq DNA polymerase; and Vn, Vent DNA polymerase).

    Article Snippet: Synthesis of double-stranded DNA was performed by thermal cycling of 10 μl phosphorylated sense oligonucleotides, 10 μl phosphorylated antisense oligonucleotides, 3 μl 10× Taq DNA ligase buffer (NEB) in a total volume of 30 μl.

    Techniques: Functional Assay, Clone Assay, Activity Assay, Amplification, Incubation

    Principle of OLA-based SNP genotyping. (a) For each polymorphism, a set of three genotyping oligos are allowed to anneal to denatured PCR product (blue) in the presence of Taq DNA ligase. Ligation of up- and downstream oligos occurs only if there is a perfect match to template. Upstream oligos are color-coded gray (M13 forward amplification primer sequence), red/green (a pair of barcode sequences), and black (assay-specific sequence flanking the query SNP). The downstream oligo is 5'-phosphorylated, and color-coded gray (reverse complemented sequence of the M13 reverse amplification primer), and black (assay-specific flanking sequence). (b) Addition of common M13 primers (gray) allows amplification of all ligated products. (c) After arraying amplified OLA products, membranes are hybridized with probes complementary to the barcode sequences. Probes can be fluorescently labeled with infrared (IR) fluors and both alleles hybridized simultaneously, or radiolabeled and hybridized sequentially.

    Journal: Genome Biology

    Article Title: A low-cost open-source SNP genotyping platform for association mapping applications

    doi: 10.1186/gb-2005-6-12-r105

    Figure Lengend Snippet: Principle of OLA-based SNP genotyping. (a) For each polymorphism, a set of three genotyping oligos are allowed to anneal to denatured PCR product (blue) in the presence of Taq DNA ligase. Ligation of up- and downstream oligos occurs only if there is a perfect match to template. Upstream oligos are color-coded gray (M13 forward amplification primer sequence), red/green (a pair of barcode sequences), and black (assay-specific sequence flanking the query SNP). The downstream oligo is 5'-phosphorylated, and color-coded gray (reverse complemented sequence of the M13 reverse amplification primer), and black (assay-specific flanking sequence). (b) Addition of common M13 primers (gray) allows amplification of all ligated products. (c) After arraying amplified OLA products, membranes are hybridized with probes complementary to the barcode sequences. Probes can be fluorescently labeled with infrared (IR) fluors and both alleles hybridized simultaneously, or radiolabeled and hybridized sequentially.

    Article Snippet: OLA and OLA amplification reaction conditions The OLA reactions are just 3 μl in volume, and contain 1 × OLA buffer (50 mM Tris-HCl pH 8.5, 50 mM KCl, 7.5 mM MgCl2 , 1 mM NAD), 2.5 mM dithiothreitol, 1.6 units Taq (Thermus aquaticus ) DNA ligase (NEB), and 0.03 pmol of each genotyping oligo.

    Techniques: Polymerase Chain Reaction, Ligation, Amplification, Sequencing, Labeling

    Schematic illustration of our strategy for specific SNP DNA detection. A biotin-probe (blue) and a phosphate-probe (green) are hybridized to each half of a complementary DNA target, which are then ligated by the Taq DNA ligase if the sequences between the probes and target are fully complementary, but not for those having a single-base mismatch ( ca. SNP) at the nicking site. The system is then subjected to multiple cycles of denaturation, annealing and ligation, where each full-match template produces a ligated product in each cycle. A capture-DNA modified magnetic nanoparticle (note that hundreds of capture-DNA strands are linked to each MNP, only one is shown here for simplicity) was added to capture the ligated products, and followed by magnetic separation. Finally, a neutravidin conjugated horseradish peroxidase (NAV-HRP) is bound to the MNP, allowing for sensitive detection of the full-match DNA target via the HRP catalysed enzymatic assay.

    Journal: Nanoscale

    Article Title: Ultrasensitive single-nucleotide polymorphism detection using target-recycled ligation, strand displacement and enzymatic amplification single-nucleotide polymorphism detection using target-recycled ligation, strand displacement and enzymatic amplification †Electronic supplementary information (ESI) available: The preparation, modification and magnetic retrieval of MNPs, optimisation of the Cap-MNP probe and experimental procedures and the SNP discrimination at low target abundance (5 fmol). See DOI: 10.1039/c3nr01010dClick here for additional data file.

    doi: 10.1039/c3nr01010d

    Figure Lengend Snippet: Schematic illustration of our strategy for specific SNP DNA detection. A biotin-probe (blue) and a phosphate-probe (green) are hybridized to each half of a complementary DNA target, which are then ligated by the Taq DNA ligase if the sequences between the probes and target are fully complementary, but not for those having a single-base mismatch ( ca. SNP) at the nicking site. The system is then subjected to multiple cycles of denaturation, annealing and ligation, where each full-match template produces a ligated product in each cycle. A capture-DNA modified magnetic nanoparticle (note that hundreds of capture-DNA strands are linked to each MNP, only one is shown here for simplicity) was added to capture the ligated products, and followed by magnetic separation. Finally, a neutravidin conjugated horseradish peroxidase (NAV-HRP) is bound to the MNP, allowing for sensitive detection of the full-match DNA target via the HRP catalysed enzymatic assay.

    Article Snippet: Materials and reagents Taq DNA ligase and 10× ligation buffer (200 mM Tris–HCl, 250 mM potassium acetate, 100 mM magnesium acetate, 10 mM NAD, 100 mM dithiothreitol and 1.0% Triton X-100, pH 7.6) were purchased from New England Biolabs (UK).

    Techniques: Ligation, Modification, Enzymatic Assay

    Fluorescence response (rate of increase, cps min –1 ) for different samples after identical thermal cycle treatments. All samples contain the same amount of Amplex red (2 μM) and H 2 O 2 (2 μM). The samples are (a) PBS, (b) Cap-MNP in PBS, (c) Cap-MNP + ligase but no T2, (d) Cap-MNP + T2 but no Taq ligase, and (e) Cap-MNP + T2 + Taq DNA ligase.

    Journal: Nanoscale

    Article Title: Ultrasensitive single-nucleotide polymorphism detection using target-recycled ligation, strand displacement and enzymatic amplification single-nucleotide polymorphism detection using target-recycled ligation, strand displacement and enzymatic amplification †Electronic supplementary information (ESI) available: The preparation, modification and magnetic retrieval of MNPs, optimisation of the Cap-MNP probe and experimental procedures and the SNP discrimination at low target abundance (5 fmol). See DOI: 10.1039/c3nr01010dClick here for additional data file.

    doi: 10.1039/c3nr01010d

    Figure Lengend Snippet: Fluorescence response (rate of increase, cps min –1 ) for different samples after identical thermal cycle treatments. All samples contain the same amount of Amplex red (2 μM) and H 2 O 2 (2 μM). The samples are (a) PBS, (b) Cap-MNP in PBS, (c) Cap-MNP + ligase but no T2, (d) Cap-MNP + T2 but no Taq ligase, and (e) Cap-MNP + T2 + Taq DNA ligase.

    Article Snippet: Materials and reagents Taq DNA ligase and 10× ligation buffer (200 mM Tris–HCl, 250 mM potassium acetate, 100 mM magnesium acetate, 10 mM NAD, 100 mM dithiothreitol and 1.0% Triton X-100, pH 7.6) were purchased from New England Biolabs (UK).

    Techniques: Fluorescence