taq dna ligase buffer  (New England Biolabs)


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    Name:
    Taq DNA Ligase
    Description:
    Taq DNA Ligase 10 000 units
    Catalog Number:
    m0208l
    Price:
    320
    Size:
    10 000 units
    Category:
    DNA Ligases
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    New England Biolabs taq dna ligase buffer
    Taq DNA Ligase
    Taq DNA Ligase 10 000 units
    https://www.bioz.com/result/taq dna ligase buffer/product/New England Biolabs
    Average 99 stars, based on 1387 article reviews
    Price from $9.99 to $1999.99
    taq dna ligase buffer - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "SNPWaveTM: a flexible multiplexed SNP genotyping technology"

    Article Title: SNPWaveTM: a flexible multiplexed SNP genotyping technology

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh045

    Principle of the SNPWave method. Allele-specific ligation probes are hybridized to denatured genomic DNA. SNP allele discrimination is based on the specificity of the Taq ( Thermus aquaticus ) DNA ligase. ( A and B ) Closed circular probes are formed only
    Figure Legend Snippet: Principle of the SNPWave method. Allele-specific ligation probes are hybridized to denatured genomic DNA. SNP allele discrimination is based on the specificity of the Taq ( Thermus aquaticus ) DNA ligase. ( A and B ) Closed circular probes are formed only

    Techniques Used: Ligation

    2) Product Images from "Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni058

    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).
    Figure Legend 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).

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

    3) Product Images from "Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus"

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007124

    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
    Figure Legend 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

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

    4) Product Images from "A low-cost open-source SNP genotyping platform for association mapping applications"

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

    Journal: Genome Biology

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

    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.
    Figure Legend 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.

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

    5) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    6) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    7) Product Images from "Bleomycin-induced genome structural variations in normal, non-tumor cells"

    Article Title: Bleomycin-induced genome structural variations in normal, non-tumor cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34580-8

    Ligation-mediated Chimera-Free (LCF) protocol ensures preparation of sequencing libraries virtually free from artificial chimeras. ( A ) LCF protocol outline. LCF is based on assignment of sequencing adapters as single-stranded oligonucleotides at elevated temperature using thermostable Taq DNA ligase. The ligation is facilitated by hybridization of adapter carrying thymine residuals on 3′-end and DNA fragment with A-overhangs at 3′-ends of both strands. At the final step sequencing library is completed by treatment with T4 DNA polymerase in the presence of dNTPs. ( B ) Frequency of artificial chimeras in sequencing libraries prepared with different approaches. ( C ) Spectra of artificial chimeras in sequencing libraries prepared with different approaches. Data in ( B ) shown as the average ± s.d.; n = 3 for each, ligation-based and MuPlus libraries and n = 8 for LCF libraries; statistically significant differences determined by two-tailed t-test.
    Figure Legend Snippet: Ligation-mediated Chimera-Free (LCF) protocol ensures preparation of sequencing libraries virtually free from artificial chimeras. ( A ) LCF protocol outline. LCF is based on assignment of sequencing adapters as single-stranded oligonucleotides at elevated temperature using thermostable Taq DNA ligase. The ligation is facilitated by hybridization of adapter carrying thymine residuals on 3′-end and DNA fragment with A-overhangs at 3′-ends of both strands. At the final step sequencing library is completed by treatment with T4 DNA polymerase in the presence of dNTPs. ( B ) Frequency of artificial chimeras in sequencing libraries prepared with different approaches. ( C ) Spectra of artificial chimeras in sequencing libraries prepared with different approaches. Data in ( B ) shown as the average ± s.d.; n = 3 for each, ligation-based and MuPlus libraries and n = 8 for LCF libraries; statistically significant differences determined by two-tailed t-test.

    Techniques Used: Ligation, Sequencing, Hybridization, Two Tailed Test

    8) Product Images from "Plant Enzymes but Not Agrobacterium VirD2 Mediate T-DNA Ligation In Vitro"

    Article Title: Plant Enzymes but Not Agrobacterium VirD2 Mediate T-DNA Ligation In Vitro

    Journal: Molecular and Cellular Biology

    doi:

    T-DNA ligation in vitro is performed by plant enzymes, likely by a DNA ligase. In vitro ligation was performed with nuclear extract from tobacco BY2 cells (A), with extract from pea shoot apices (B), and with the following purified prokaryotic ligases: E. coli DNA ligase (10 U), Taq DNA ligase (40 U), T4 DNA ligase (40 U), and T4 RNA ligase (20 U) (C). (D) Effect of inhibitors (150 μM aphidicolin, 5 μM ddTTP, and 1 mM dTTP) on T-DNA in vitro ligation. Inhibition values represent comparisons of the ligation efficiencies in the presence and absence of the inhibitor. 8-mer–VirD2 (8 pmol) and 8-mer-P (4 pmol) were used as ligation substrates.
    Figure Legend Snippet: T-DNA ligation in vitro is performed by plant enzymes, likely by a DNA ligase. In vitro ligation was performed with nuclear extract from tobacco BY2 cells (A), with extract from pea shoot apices (B), and with the following purified prokaryotic ligases: E. coli DNA ligase (10 U), Taq DNA ligase (40 U), T4 DNA ligase (40 U), and T4 RNA ligase (20 U) (C). (D) Effect of inhibitors (150 μM aphidicolin, 5 μM ddTTP, and 1 mM dTTP) on T-DNA in vitro ligation. Inhibition values represent comparisons of the ligation efficiencies in the presence and absence of the inhibitor. 8-mer–VirD2 (8 pmol) and 8-mer-P (4 pmol) were used as ligation substrates.

    Techniques Used: DNA Ligation, In Vitro, Ligation, Purification, Inhibition

    9) Product Images from "Cellular reagents for diagnostics and synthetic biology"

    Article Title: Cellular reagents for diagnostics and synthetic biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201681

    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
    Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay

    RNA detection by two-step reverse transcription TaqMan qPCR using cellular reagents for MMLV RT and Taq DNA polymerase. Indicated copies of synthetic RNA template derived from Zika virus genomic sequence were tested using 2 x 10 7 ).
    Figure Legend Snippet: RNA detection by two-step reverse transcription TaqMan qPCR using cellular reagents for MMLV RT and Taq DNA polymerase. Indicated copies of synthetic RNA template derived from Zika virus genomic sequence were tested using 2 x 10 7 ).

    Techniques Used: RNA Detection, Real-time Polymerase Chain Reaction, Derivative Assay, Sequencing

    TaqMan qPCR analysis using lyophilized Taq DNA polymerase cellular reagents. Indicated copies of synthetic DNA templates derived from Zika virus genomic sequence were amplified using 2.5 units of pure commercial Taq DNA polymerase (panels a and b) or 2 x 10 7 cells of rehydrated cellular reagents expressing Taq DNA polymerase (panels c and d). Amplification was assessed in real-time by measuring increase in TaqMan probe fluorescence over time. Representative amplification curves generated using the “Abs quant” analysis in the LightCycler 96 software are depicted in panels a and c. Amplification curve colors distinguish starting template copies–yellow: 60,000 template copies; green: 6000 template copies; blue: 600 template copies; red: 60 template copies; and gray: no template control. These curves depict the real-time kinetics of PCR amplification mediated by pure versus cellular reagents. The corresponding standard curve analyses performed using the “Abs quant” protocol in the LightCycler 96 software are depicted in panels b and d, respectively. Standard curve analyses data for comparing amplification efficiency, linearity, and error are tabulated as insets.
    Figure Legend Snippet: TaqMan qPCR analysis using lyophilized Taq DNA polymerase cellular reagents. Indicated copies of synthetic DNA templates derived from Zika virus genomic sequence were amplified using 2.5 units of pure commercial Taq DNA polymerase (panels a and b) or 2 x 10 7 cells of rehydrated cellular reagents expressing Taq DNA polymerase (panels c and d). Amplification was assessed in real-time by measuring increase in TaqMan probe fluorescence over time. Representative amplification curves generated using the “Abs quant” analysis in the LightCycler 96 software are depicted in panels a and c. Amplification curve colors distinguish starting template copies–yellow: 60,000 template copies; green: 6000 template copies; blue: 600 template copies; red: 60 template copies; and gray: no template control. These curves depict the real-time kinetics of PCR amplification mediated by pure versus cellular reagents. The corresponding standard curve analyses performed using the “Abs quant” protocol in the LightCycler 96 software are depicted in panels b and d, respectively. Standard curve analyses data for comparing amplification efficiency, linearity, and error are tabulated as insets.

    Techniques Used: Real-time Polymerase Chain Reaction, Derivative Assay, Sequencing, Amplification, Expressing, Fluorescence, Generated, Software, Polymerase Chain Reaction

    10) Product Images from "Bleomycin-induced genome structural variations in normal, non-tumor cells"

    Article Title: Bleomycin-induced genome structural variations in normal, non-tumor cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34580-8

    Ligation-mediated Chimera-Free (LCF) protocol ensures preparation of sequencing libraries virtually free from artificial chimeras. ( A ) LCF protocol outline. LCF is based on assignment of sequencing adapters as single-stranded oligonucleotides at elevated temperature using thermostable Taq DNA ligase. The ligation is facilitated by hybridization of adapter carrying thymine residuals on 3′-end and DNA fragment with A-overhangs at 3′-ends of both strands. At the final step sequencing library is completed by treatment with T4 DNA polymerase in the presence of dNTPs. ( B ) Frequency of artificial chimeras in sequencing libraries prepared with different approaches. ( C ) Spectra of artificial chimeras in sequencing libraries prepared with different approaches. Data in ( B ) shown as the average ± s.d.; n = 3 for each, ligation-based and MuPlus libraries and n = 8 for LCF libraries; statistically significant differences determined by two-tailed t-test.
    Figure Legend Snippet: Ligation-mediated Chimera-Free (LCF) protocol ensures preparation of sequencing libraries virtually free from artificial chimeras. ( A ) LCF protocol outline. LCF is based on assignment of sequencing adapters as single-stranded oligonucleotides at elevated temperature using thermostable Taq DNA ligase. The ligation is facilitated by hybridization of adapter carrying thymine residuals on 3′-end and DNA fragment with A-overhangs at 3′-ends of both strands. At the final step sequencing library is completed by treatment with T4 DNA polymerase in the presence of dNTPs. ( B ) Frequency of artificial chimeras in sequencing libraries prepared with different approaches. ( C ) Spectra of artificial chimeras in sequencing libraries prepared with different approaches. Data in ( B ) shown as the average ± s.d.; n = 3 for each, ligation-based and MuPlus libraries and n = 8 for LCF libraries; statistically significant differences determined by two-tailed t-test.

    Techniques Used: Ligation, Sequencing, Hybridization, Two Tailed Test

    11) Product Images from "Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost"

    Article Title: Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0153158

    “Dissection” of the Gibson assembly into its component reactions. In the Gibson assembly, single-stranded 3’-overhangs are produced using T5 exonuclease. After insert-to-plasmid annealing at complementary single-stranded DNA ends, gaps are filled-in by Phusion DNA polymerase, and finally, Taq DNA ligase ligates the nicks. Here, we compared the efficiencies at which insert-plasmid mixtures transformed chemically competent E . coli cells. We used untreated insert-plasmid mixtures (co-transformation cloning), insert-plasmid mixtures treated with T5 exonuclease (sequence- and ligation-independent cloning), insert-plasmid mixtures treated with T5 exonuclease and Phusion DNA polymerase (sequence- and ligation-independent cloning plus gap filling), and insert-plasmid mixtures treated with T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase (Gibson assembly). A) Data points represent the number of positive (blue) colonies averaged over three experiments ±SD. B) Data points represent the percentage of positive colonies averaged over three experiments ±SD.
    Figure Legend Snippet: “Dissection” of the Gibson assembly into its component reactions. In the Gibson assembly, single-stranded 3’-overhangs are produced using T5 exonuclease. After insert-to-plasmid annealing at complementary single-stranded DNA ends, gaps are filled-in by Phusion DNA polymerase, and finally, Taq DNA ligase ligates the nicks. Here, we compared the efficiencies at which insert-plasmid mixtures transformed chemically competent E . coli cells. We used untreated insert-plasmid mixtures (co-transformation cloning), insert-plasmid mixtures treated with T5 exonuclease (sequence- and ligation-independent cloning), insert-plasmid mixtures treated with T5 exonuclease and Phusion DNA polymerase (sequence- and ligation-independent cloning plus gap filling), and insert-plasmid mixtures treated with T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase (Gibson assembly). A) Data points represent the number of positive (blue) colonies averaged over three experiments ±SD. B) Data points represent the percentage of positive colonies averaged over three experiments ±SD.

    Techniques Used: Produced, Plasmid Preparation, Transformation Assay, Clone Assay, Sequencing, Ligation

    12) Product Images from "Bleomycin-induced genome structural variations in normal, non-tumor cells"

    Article Title: Bleomycin-induced genome structural variations in normal, non-tumor cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34580-8

    Ligation-mediated Chimera-Free (LCF) protocol ensures preparation of sequencing libraries virtually free from artificial chimeras. ( A ) LCF protocol outline. LCF is based on assignment of sequencing adapters as single-stranded oligonucleotides at elevated temperature using thermostable Taq DNA ligase. The ligation is facilitated by hybridization of adapter carrying thymine residuals on 3′-end and DNA fragment with A-overhangs at 3′-ends of both strands. At the final step sequencing library is completed by treatment with T4 DNA polymerase in the presence of dNTPs. ( B ) Frequency of artificial chimeras in sequencing libraries prepared with different approaches. ( C ) Spectra of artificial chimeras in sequencing libraries prepared with different approaches. Data in ( B ) shown as the average ± s.d.; n = 3 for each, ligation-based and MuPlus libraries and n = 8 for LCF libraries; statistically significant differences determined by two-tailed t-test.
    Figure Legend Snippet: Ligation-mediated Chimera-Free (LCF) protocol ensures preparation of sequencing libraries virtually free from artificial chimeras. ( A ) LCF protocol outline. LCF is based on assignment of sequencing adapters as single-stranded oligonucleotides at elevated temperature using thermostable Taq DNA ligase. The ligation is facilitated by hybridization of adapter carrying thymine residuals on 3′-end and DNA fragment with A-overhangs at 3′-ends of both strands. At the final step sequencing library is completed by treatment with T4 DNA polymerase in the presence of dNTPs. ( B ) Frequency of artificial chimeras in sequencing libraries prepared with different approaches. ( C ) Spectra of artificial chimeras in sequencing libraries prepared with different approaches. Data in ( B ) shown as the average ± s.d.; n = 3 for each, ligation-based and MuPlus libraries and n = 8 for LCF libraries; statistically significant differences determined by two-tailed t-test.

    Techniques Used: Ligation, Sequencing, Hybridization, Two Tailed Test

    13) Product Images from "SNPWaveTM: a flexible multiplexed SNP genotyping technology"

    Article Title: SNPWaveTM: a flexible multiplexed SNP genotyping technology

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh045

    Principle of the SNPWave method. Allele-specific ligation probes are hybridized to denatured genomic DNA. SNP allele discrimination is based on the specificity of the Taq ( Thermus aquaticus ) DNA ligase. ( A and B ) Closed circular probes are formed only
    Figure Legend Snippet: Principle of the SNPWave method. Allele-specific ligation probes are hybridized to denatured genomic DNA. SNP allele discrimination is based on the specificity of the Taq ( Thermus aquaticus ) DNA ligase. ( A and B ) Closed circular probes are formed only

    Techniques Used: Ligation

    14) Product Images from "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."

    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.

    Journal: Nanoscale

    doi: 10.1039/c3nr01010d

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Fluorescence

    15) Product Images from "Cellular reagents for diagnostics and synthetic biology"

    Article Title: Cellular reagents for diagnostics and synthetic biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201681

    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
    Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay

    RNA detection by two-step reverse transcription TaqMan qPCR using cellular reagents for MMLV RT and Taq DNA polymerase. Indicated copies of synthetic RNA template derived from Zika virus genomic sequence were tested using 2 x 10 7 cells each of MMLV RT and Taq DNA polymerase lyophilized cellular reagents. Amplification was assessed in real-time by measuring increase in TaqMan probe fluorescence over time. Representative amplification curves generated using the “Abs quant” analysis in the LightCycler 96 software are presented. Color of the traces indicate presence (black traces) or absence (red traces) of MMLV RT cellular reagents, or the absence of templates (blue traces). The corresponding derivation of template copies from Cq analyses are tabulated. Cq values were converted to template copies using standard curve analyses of the same RNA samples with commercial qRT-PCR master mix ( S8 Fig ).
    Figure Legend Snippet: RNA detection by two-step reverse transcription TaqMan qPCR using cellular reagents for MMLV RT and Taq DNA polymerase. Indicated copies of synthetic RNA template derived from Zika virus genomic sequence were tested using 2 x 10 7 cells each of MMLV RT and Taq DNA polymerase lyophilized cellular reagents. Amplification was assessed in real-time by measuring increase in TaqMan probe fluorescence over time. Representative amplification curves generated using the “Abs quant” analysis in the LightCycler 96 software are presented. Color of the traces indicate presence (black traces) or absence (red traces) of MMLV RT cellular reagents, or the absence of templates (blue traces). The corresponding derivation of template copies from Cq analyses are tabulated. Cq values were converted to template copies using standard curve analyses of the same RNA samples with commercial qRT-PCR master mix ( S8 Fig ).

    Techniques Used: RNA Detection, Real-time Polymerase Chain Reaction, Derivative Assay, Sequencing, Amplification, Fluorescence, Generated, Software, Quantitative RT-PCR

    TaqMan qPCR analysis using lyophilized Taq DNA polymerase cellular reagents. Indicated copies of synthetic DNA templates derived from Zika virus genomic sequence were amplified using 2.5 units of pure commercial Taq DNA polymerase (panels a and b) or 2 x 10 7 cells of rehydrated cellular reagents expressing Taq DNA polymerase (panels c and d). Amplification was assessed in real-time by measuring increase in TaqMan probe fluorescence over time. Representative amplification curves generated using the “Abs quant” analysis in the LightCycler 96 software are depicted in panels a and c. Amplification curve colors distinguish starting template copies–yellow: 60,000 template copies; green: 6000 template copies; blue: 600 template copies; red: 60 template copies; and gray: no template control. These curves depict the real-time kinetics of PCR amplification mediated by pure versus cellular reagents. The corresponding standard curve analyses performed using the “Abs quant” protocol in the LightCycler 96 software are depicted in panels b and d, respectively. Standard curve analyses data for comparing amplification efficiency, linearity, and error are tabulated as insets.
    Figure Legend Snippet: TaqMan qPCR analysis using lyophilized Taq DNA polymerase cellular reagents. Indicated copies of synthetic DNA templates derived from Zika virus genomic sequence were amplified using 2.5 units of pure commercial Taq DNA polymerase (panels a and b) or 2 x 10 7 cells of rehydrated cellular reagents expressing Taq DNA polymerase (panels c and d). Amplification was assessed in real-time by measuring increase in TaqMan probe fluorescence over time. Representative amplification curves generated using the “Abs quant” analysis in the LightCycler 96 software are depicted in panels a and c. Amplification curve colors distinguish starting template copies–yellow: 60,000 template copies; green: 6000 template copies; blue: 600 template copies; red: 60 template copies; and gray: no template control. These curves depict the real-time kinetics of PCR amplification mediated by pure versus cellular reagents. The corresponding standard curve analyses performed using the “Abs quant” protocol in the LightCycler 96 software are depicted in panels b and d, respectively. Standard curve analyses data for comparing amplification efficiency, linearity, and error are tabulated as insets.

    Techniques Used: Real-time Polymerase Chain Reaction, Derivative Assay, Sequencing, Amplification, Expressing, Fluorescence, Generated, Software, Polymerase Chain Reaction

    16) Product Images from "Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus"

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007124

    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
    Figure Legend 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

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

    17) Product Images from "Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni058

    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).
    Figure Legend 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).

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

    18) Product Images from "A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli"

    Article Title: A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-11-19

    Construction outline of the MCP tac s promoter clusters . Fragment 5CP tac s with the flanking sequence was amplified by PCR with p5TG as the template. Fragment 1 was generated by digesting fragment 5CP tac s with BamH I. Fragment 2 was digested from fragment 5CP tac s with BamH I and Hind III. Fragment 3 was linearized from the plasmid p5TG with Hind III. Then, the three fragments were assembled together under the action of T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase in the isothermal process.
    Figure Legend Snippet: Construction outline of the MCP tac s promoter clusters . Fragment 5CP tac s with the flanking sequence was amplified by PCR with p5TG as the template. Fragment 1 was generated by digesting fragment 5CP tac s with BamH I. Fragment 2 was digested from fragment 5CP tac s with BamH I and Hind III. Fragment 3 was linearized from the plasmid p5TG with Hind III. Then, the three fragments were assembled together under the action of T5 exonuclease, Phusion DNA polymerase and Taq DNA ligase in the isothermal process.

    Techniques Used: Sequencing, Amplification, Polymerase Chain Reaction, Generated, Plasmid Preparation

    19) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    20) Product Images from "T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis"

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1169

    Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Enzymes and buffer components required for TEDA. ( A ) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. ( B ) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD + were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; ( C ) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl 2 and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Clone Assay

    Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.
    Figure Legend Snippet: Comparison of different assembly methods. ( A ) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle- lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. ( B ) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. ( C ) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A ), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.

    Techniques Used: Plasmid Preparation, Clone Assay, Transformation Assay

    Related Articles

    Sequencing:

    Article Title: Bleomycin-induced genome structural variations in normal, non-tumor cells
    Article Snippet: .. Ligation-mediated chimera-free (LCF) libraries were prepared as follows: Fragmentation using NEBNext dsDNA Fragmentase (NEB); End-repair I using S1 nuclease (Thermo Fisher Scientific, Waltham, MA USA); A-tailing using Terminal Transferase (TdT) polymerase (NEB) and dATP (NEB); Ligation of single-stranded sequencing adaptors using Taq DNA Ligase (NEB). .. Ligation-mediated chimera-free (LCF) libraries were prepared as follows: Fragmentation using NEBNext dsDNA Fragmentase (NEB); End-repair I using S1 nuclease (Thermo Fisher Scientific, Waltham, MA USA); A-tailing using Terminal Transferase (TdT) polymerase (NEB) and dATP (NEB); Ligation of single-stranded sequencing adaptors using Taq DNA Ligase (NEB).

    Clone Assay:

    Article Title: T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis
    Article Snippet: .. 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 ).

    Incubation:

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus
    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). .. After further incubation at 37°C for 20 min, DNA was purified by phenol/chloroform extraction and ethanol precipitation, and subjected to cccDNA-selective qPCR or RCA, as described above.

    Amplification:

    Article Title: A low-cost open-source SNP genotyping platform for association mapping applications
    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. .. Each OLA reaction mix is spiked with 0.2 μl of PCR product using a HydraII 96-syringe pipetting unit (Matrix Technologies Corporation, Hudson, NH, USA).

    Ligation:

    Article Title: Bleomycin-induced genome structural variations in normal, non-tumor cells
    Article Snippet: .. Ligation-mediated chimera-free (LCF) libraries were prepared as follows: Fragmentation using NEBNext dsDNA Fragmentase (NEB); End-repair I using S1 nuclease (Thermo Fisher Scientific, Waltham, MA USA); A-tailing using Terminal Transferase (TdT) polymerase (NEB) and dATP (NEB); Ligation of single-stranded sequencing adaptors using Taq DNA Ligase (NEB). .. Ligation-mediated chimera-free (LCF) libraries were prepared as follows: Fragmentation using NEBNext dsDNA Fragmentase (NEB); End-repair I using S1 nuclease (Thermo Fisher Scientific, Waltham, MA USA); A-tailing using Terminal Transferase (TdT) polymerase (NEB) and dATP (NEB); Ligation of single-stranded sequencing adaptors using Taq DNA Ligase (NEB).

    Article Title: SNPWaveTM: a flexible multiplexed SNP genotyping technology
    Article Snippet: .. Ligation reactions for Arabidopsis polymorphisms were performed in a 25 µl volume containing 625 ng of Arabidopsis DNA, 1× Taq DNA ligase buffer [20 mM Tris–HCl, 25 mM KAc, 10 mM MgAc2 , 10 mM dithiothreitol (DTT), 1 mM NAD, 0.1% Triton X-100; pH 7.6 at 25°C; New England Biolabs Inc., Beverly, MA], 0.2 U/µl Taq DNA ligase (NEB) and 0.05 fmol/µl of each of 200 ligation probes. .. Next, 10 cycles of repeated denaturation, probe hybridization and ligation were performed in a Perkin Elmer 9700 thermal cycler (Applied Biosystems, Foster City, CA) using the following profile: initial denaturation for 2 min at 94°C, followed by 10 cycles of 15 s at 94°C and 60 min at 60°C, followed by storage at 4°C.

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    New England Biolabs taq dna ligase buffer
    Principle of the SNPWave method. Allele-specific ligation probes are hybridized to denatured genomic <t>DNA.</t> SNP allele discrimination is based on the specificity of the <t>Taq</t> ( Thermus aquaticus ) DNA ligase. ( A and B ) Closed circular probes are formed only
    Taq Dna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 392 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Principle of the SNPWave method. Allele-specific ligation probes are hybridized to denatured genomic DNA. SNP allele discrimination is based on the specificity of the Taq ( Thermus aquaticus ) DNA ligase. ( A and B ) Closed circular probes are formed only

    Journal: Nucleic Acids Research

    Article Title: SNPWaveTM: a flexible multiplexed SNP genotyping technology

    doi: 10.1093/nar/gnh045

    Figure Lengend Snippet: Principle of the SNPWave method. Allele-specific ligation probes are hybridized to denatured genomic DNA. SNP allele discrimination is based on the specificity of the Taq ( Thermus aquaticus ) DNA ligase. ( A and B ) Closed circular probes are formed only

    Article Snippet: Ligation reactions for Arabidopsis polymorphisms were performed in a 25 µl volume containing 625 ng of Arabidopsis DNA, 1× Taq DNA ligase buffer [20 mM Tris–HCl, 25 mM KAc, 10 mM MgAc2 , 10 mM dithiothreitol (DTT), 1 mM NAD, 0.1% Triton X-100; pH 7.6 at 25°C; New England Biolabs Inc., Beverly, MA], 0.2 U/µl Taq DNA ligase (NEB) and 0.05 fmol/µl of each of 200 ligation probes.

    Techniques: Ligation

    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

    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

    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