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  • 95
    New England Biolabs m13mp18 rf i dna
    M13mp18 Rf I Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher phusion high fidelity dna polymerase
    Phusion High Fidelity Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 14136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs double stranded dna dsdna
    Double Stranded Dna Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    New England Biolabs double stranded dna dsdna fragmentase
    Double Stranded Dna Dsdna Fragmentase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 87/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    New England Biolabs m13ke double stranded dna dsdna
    Standard curves of <t>M13KE</t> (Panel A) and T7 (Panel B) phage <t>DNA</t> from TaqMan qPCR Log (gc/μL) was calculated from standard M13KE DNA from 0.1 fg/μL - 10 6 fg/μL and T7 packaging control DNA at concentrations ranging from 1 fg/μL - 10 7 fg/μL. At each concentration, every sample had a Ct value from TaqMan qPCR. Linear regression was plotted with Ct versus log (genome copies (gc)/μL). Linear equation and correlation coefficient are in the inset.
    M13ke Double Stranded Dna Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    New England Biolabs rfii circular double stranded dna dsdna substrates
    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no <t>DNA</t> (filled diamond), <t>dsDNA</t> (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).
    Rfii Circular Double Stranded Dna Dsdna Substrates, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    New England Biolabs λ phage double strand dna dsdna
    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no <t>DNA</t> (filled diamond), <t>dsDNA</t> (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).
    λ Phage Double Strand Dna Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    New England Biolabs ii dna dsdna
    <t>DNA-binding</t> activity of MlaA. ( A ) Electrophoretic mobility shift assays show that MlaA binds to <t>dsDNA</t> but only weakly to ssDNA. In all, 0, 0.25, 0.5, 1 or 2 μg MlaA is incubated with 50 fmol ssDNA or dsDNA 20-mer and analysed on 6% native acrylamide gels. ( B ) Competition experiments using labelled dsDNA oligonucleotides and unlabelled circular dsDNA. The fraction of labelled dsDNA that is shifted by MlaA versus the competitor:probe ratio is plotted. The highly efficient competition of dsDNA oligonucleotide binding by circular plasmid dsDNA (approximately 50% at a 1:1 ratio) indicates that MlaA does not need DNA ends for efficient binding. ( C ). The fraction of MlaA:DNA complexes (retained at nitrocellulose filters) relative to total DNA (sum of DNA retained at both nitrocellulose and hybond) versus MlaA concentration is shown. Data were fitted to f = p n /( K d + p n ), where f is the fraction of DNA bound to MlaA relative to total DNA, K d is the dissociation constant, p is the protein concentration and n is the Hill coefficient. MlaA poorly binds ssDNA (filled triangles), but more strongly binds a dsDNA Y-substrate (open circles). The tightest binding is observed for a Holliday-junction substrate (filled circles), suggesting a preference for DNA secondary structures. dsDNA ( n =2.5–4) but not ssDNA ( n ≈1) substrates are evidently bound cooperatively.
    Ii Dna Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs e coli dna ligase
    <t>DNA-binding</t> activity of MlaA. ( A ) Electrophoretic mobility shift assays show that MlaA binds to <t>dsDNA</t> but only weakly to ssDNA. In all, 0, 0.25, 0.5, 1 or 2 μg MlaA is incubated with 50 fmol ssDNA or dsDNA 20-mer and analysed on 6% native acrylamide gels. ( B ) Competition experiments using labelled dsDNA oligonucleotides and unlabelled circular dsDNA. The fraction of labelled dsDNA that is shifted by MlaA versus the competitor:probe ratio is plotted. The highly efficient competition of dsDNA oligonucleotide binding by circular plasmid dsDNA (approximately 50% at a 1:1 ratio) indicates that MlaA does not need DNA ends for efficient binding. ( C ). The fraction of MlaA:DNA complexes (retained at nitrocellulose filters) relative to total DNA (sum of DNA retained at both nitrocellulose and hybond) versus MlaA concentration is shown. Data were fitted to f = p n /( K d + p n ), where f is the fraction of DNA bound to MlaA relative to total DNA, K d is the dissociation constant, p is the protein concentration and n is the Hill coefficient. MlaA poorly binds ssDNA (filled triangles), but more strongly binds a dsDNA Y-substrate (open circles). The tightest binding is observed for a Holliday-junction substrate (filled circles), suggesting a preference for DNA secondary structures. dsDNA ( n =2.5–4) but not ssDNA ( n ≈1) substrates are evidently bound cooperatively.
    E Coli Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 392 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs nebnext dsdna fragmentase
    Example fragmentation profiles and coverage plots. Example fragmentation profiles and coverage plots are shown for two mitochondrial genomes (examples 62 and 80). A . These Bioanalyzer profiles (from a DNA 7500 chip) show pooled long-range PCR products after digestion with <t>NEBNext</t> <t>dsDNA</t> <t>Fragmentase</t> (10 min at 37°C). The x -axis shows the inferred size of the DNA fragments based on the two internal markers of known size (the peaks at 50 and 10,380 bp). The y -axis shows the amount of DNA present based on fluorescence units. Both example digestion profiles show fragments between distributed between ~300 bp and ~5 kb in length, with the distribution skewed towards smaller fragments. These profiles show fragments in the ideal size range for 454 sequencing. The difference in yields between the two samples is probably due to different recovery efficiencies in the preceding AMPure XP purification step. Screen captures are taken from the 2100 Expert software (Agilent). B . These coverage plots for two mitochondrial genomes were generated using the software described in this paper. The x -axis shows the nucleotide positions based on the revised Cambridge Reference Sequence (rCRS). The y -axis shows coverage depth. The horizontal dashed line indicates mean coverage for that genome. On the left of each heading line is the individual name (e.g. 62.sff or 80.sff); the following number (here, 16569) is the number of positions that were covered by at least 1 read, and the final number (here, also 16569) is the length of the reference sequence. Note the large peak from 8,000–9,000 bp, which is discussed in the main text. The blue lines represent the corresponding long-range PCR products and the associated numbers the positions of the ends of those products (see Table 1 ). The data used to generated these coverage plots is available in Additional file 6 .
    Nebnext Dsdna Fragmentase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 616 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs ϕx174 virion ssdna
    Three Strand Exchange Recombinase Assay A Active RAD51 nucleoprotein filament is formed on <t>ϕX174</t> Virion circular <t>ssDNA</t> in reaction conditions permitting ATP hydrolysis followed by addition of RPA which helps remove secondary structures in the ssDNA allowing stable nucleoprotein filament formation. The RAD51 nucleoprotein then invades ϕX174 RFI linear dsDNA to form nicked circular dsDNA products through several joint-molecule intermediates showing various stages of branch migration. The RPA also helps sequester any free displaced ssDNA following strand exchange thereby preventing a reverse reaction. B . Typical agarose gel image from a strand exchange assay used to resolve the substrates, intermediates and the products of the reaction over a time course of 0, 30, 60, 120 and 180 minutes. In reactions containing the wild type protein, nicked circular products can be observed as early as 30 minutes. C. Quantitation of the strand exchange assay gel showing appearance of nicked circular products (black) and joint molecules (blue) on the left Y axis. Disappearance of the linear dsDNA substrate (red) is quantitated on the right Y axis. Time in minutes is indicated on the X axis.
    ϕx174 Virion Ssdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs 100 bp dsdna ladder
    RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained <t>100</t> nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of <t>dsDNA</t> (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.
    100 Bp Dsdna Ladder, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs lambda dsdna
    RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained <t>100</t> nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of <t>dsDNA</t> (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.
    Lambda Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ultra ii dna library prep kit
    A general overview of the <t>dsDNA</t> Fragmentation Through Polymerization (FTP) method. The FTP method is based on two enzymatic reactions: a <t>DNA</t> nicking reaction with DNase I and a strand-displacement DNA polymerization with SD DNA polymerase. As a result, multiple double-stranded DNA fragments with overlapping sequences are generated. De novo synthesized DNA is indicated in grey, and SD polymerase is indicated in red.
    Ultra Ii Dna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1985 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    New England Biolabs m13mp18 dna sequencing standard m13mp18 dsdna
    Molecular events and ionic current trace for a 2D read of a 7.25 kb M13 phage <t>dsDNA</t> molecule. (a) Schematic for the steps in <t>DNA</t> translocation through the nanopore. (i) Open channel; (ii) dsDNA with a ligated lead adaptor (blue), with a molecular motor bound to it (orange), and a hairpin adaptor (red), is captured by the nanopore. DNA translocation through the nanopore begins through the effect of an applied voltage across the membrane and the action of a molecular motor; (iii) Translocation of the lead adaptor (blue); (iv) Translocation of the template strand (gold); (v) Translocation of the hairpin adaptor (red); (vi) Translocation of the complement strand (dark blue); (vii) Translocation of the trailing adaptor (brown); (viii) Return to open channel. (b) Raw current trace for the passage of the M13 dsDNA construct through the nanopore. Regions of the ionic current trace corresponding to steps i-viii are labeled. (c) Expanded time and current scale for raw current traces corresponding to steps i–viii. Each adaptor generates a unique current signal used to aid base calling.
    M13mp18 Dna Sequencing Standard M13mp18 Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    New England Biolabs dsdna fragmentase buffer
    Molecular events and ionic current trace for a 2D read of a 7.25 kb M13 phage <t>dsDNA</t> molecule. (a) Schematic for the steps in <t>DNA</t> translocation through the nanopore. (i) Open channel; (ii) dsDNA with a ligated lead adaptor (blue), with a molecular motor bound to it (orange), and a hairpin adaptor (red), is captured by the nanopore. DNA translocation through the nanopore begins through the effect of an applied voltage across the membrane and the action of a molecular motor; (iii) Translocation of the lead adaptor (blue); (iv) Translocation of the template strand (gold); (v) Translocation of the hairpin adaptor (red); (vi) Translocation of the complement strand (dark blue); (vii) Translocation of the trailing adaptor (brown); (viii) Return to open channel. (b) Raw current trace for the passage of the M13 dsDNA construct through the nanopore. Regions of the ionic current trace corresponding to steps i-viii are labeled. (c) Expanded time and current scale for raw current traces corresponding to steps i–viii. Each adaptor generates a unique current signal used to aid base calling.
    Dsdna Fragmentase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs library quant kit
    Molecular events and ionic current trace for a 2D read of a 7.25 kb M13 phage <t>dsDNA</t> molecule. (a) Schematic for the steps in <t>DNA</t> translocation through the nanopore. (i) Open channel; (ii) dsDNA with a ligated lead adaptor (blue), with a molecular motor bound to it (orange), and a hairpin adaptor (red), is captured by the nanopore. DNA translocation through the nanopore begins through the effect of an applied voltage across the membrane and the action of a molecular motor; (iii) Translocation of the lead adaptor (blue); (iv) Translocation of the template strand (gold); (v) Translocation of the hairpin adaptor (red); (vi) Translocation of the complement strand (dark blue); (vii) Translocation of the trailing adaptor (brown); (viii) Return to open channel. (b) Raw current trace for the passage of the M13 dsDNA construct through the nanopore. Regions of the ionic current trace corresponding to steps i-viii are labeled. (c) Expanded time and current scale for raw current traces corresponding to steps i–viii. Each adaptor generates a unique current signal used to aid base calling.
    Library Quant Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 709 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs phusion high fidelity pcr master mix
    Molecular events and ionic current trace for a 2D read of a 7.25 kb M13 phage <t>dsDNA</t> molecule. (a) Schematic for the steps in <t>DNA</t> translocation through the nanopore. (i) Open channel; (ii) dsDNA with a ligated lead adaptor (blue), with a molecular motor bound to it (orange), and a hairpin adaptor (red), is captured by the nanopore. DNA translocation through the nanopore begins through the effect of an applied voltage across the membrane and the action of a molecular motor; (iii) Translocation of the lead adaptor (blue); (iv) Translocation of the template strand (gold); (v) Translocation of the hairpin adaptor (red); (vi) Translocation of the complement strand (dark blue); (vii) Translocation of the trailing adaptor (brown); (viii) Return to open channel. (b) Raw current trace for the passage of the M13 dsDNA construct through the nanopore. Regions of the ionic current trace corresponding to steps i-viii are labeled. (c) Expanded time and current scale for raw current traces corresponding to steps i–viii. Each adaptor generates a unique current signal used to aid base calling.
    Phusion High Fidelity Pcr Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 4138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Standard curves of M13KE (Panel A) and T7 (Panel B) phage DNA from TaqMan qPCR Log (gc/μL) was calculated from standard M13KE DNA from 0.1 fg/μL - 10 6 fg/μL and T7 packaging control DNA at concentrations ranging from 1 fg/μL - 10 7 fg/μL. At each concentration, every sample had a Ct value from TaqMan qPCR. Linear regression was plotted with Ct versus log (genome copies (gc)/μL). Linear equation and correlation coefficient are in the inset.

    Journal: Journal of virological methods

    Article Title: Quantification of M13 and T7 bacteriophages by TaqMan and SYBR Green qPCR

    doi: 10.1016/j.jviromet.2017.11.012

    Figure Lengend Snippet: Standard curves of M13KE (Panel A) and T7 (Panel B) phage DNA from TaqMan qPCR Log (gc/μL) was calculated from standard M13KE DNA from 0.1 fg/μL - 10 6 fg/μL and T7 packaging control DNA at concentrations ranging from 1 fg/μL - 10 7 fg/μL. At each concentration, every sample had a Ct value from TaqMan qPCR. Linear regression was plotted with Ct versus log (genome copies (gc)/μL). Linear equation and correlation coefficient are in the inset.

    Article Snippet: M13KE double stranded DNA (dsDNA) (NEB, 7222 bp, concentration 1μg/μL) was used as a standard for the calibration curve to quantify M13KE genomic DNA at 10-fold dilutions from 0.01 fg/μL - 106 fg/μL.

    Techniques: Real-time Polymerase Chain Reaction, Concentration Assay

    The workflow of TaqMan and SYBR Green qPCR of genomic DNA from M13KE and T7 wild type (WT) phage and C(X) 7 C clones, as compared to double-layer agar plaque assay.

    Journal: Journal of virological methods

    Article Title: Quantification of M13 and T7 bacteriophages by TaqMan and SYBR Green qPCR

    doi: 10.1016/j.jviromet.2017.11.012

    Figure Lengend Snippet: The workflow of TaqMan and SYBR Green qPCR of genomic DNA from M13KE and T7 wild type (WT) phage and C(X) 7 C clones, as compared to double-layer agar plaque assay.

    Article Snippet: M13KE double stranded DNA (dsDNA) (NEB, 7222 bp, concentration 1μg/μL) was used as a standard for the calibration curve to quantify M13KE genomic DNA at 10-fold dilutions from 0.01 fg/μL - 106 fg/μL.

    Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction, Clone Assay, Plaque Assay

    Standard curves of M13KE (Panel A) and T7 (Panel B) phage DNA from SYBR Green qPCR Log (gc/μL) was calculated from serial concentrations of M13KE DNA from 10 −2 – 10 5 fg/μL and of T7 DNA from 10 0 – 10 6 fg/μL. Ct (threshold cycle) values were the SYBR Green qPCR results at the respective concentration. Linear regression was plotted with Ct versus log (gc/μL). Linear equation and correlation coefficient are in the inset.

    Journal: Journal of virological methods

    Article Title: Quantification of M13 and T7 bacteriophages by TaqMan and SYBR Green qPCR

    doi: 10.1016/j.jviromet.2017.11.012

    Figure Lengend Snippet: Standard curves of M13KE (Panel A) and T7 (Panel B) phage DNA from SYBR Green qPCR Log (gc/μL) was calculated from serial concentrations of M13KE DNA from 10 −2 – 10 5 fg/μL and of T7 DNA from 10 0 – 10 6 fg/μL. Ct (threshold cycle) values were the SYBR Green qPCR results at the respective concentration. Linear regression was plotted with Ct versus log (gc/μL). Linear equation and correlation coefficient are in the inset.

    Article Snippet: M13KE double stranded DNA (dsDNA) (NEB, 7222 bp, concentration 1μg/μL) was used as a standard for the calibration curve to quantify M13KE genomic DNA at 10-fold dilutions from 0.01 fg/μL - 106 fg/μL.

    Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction, Concentration Assay

    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).

    Journal: Nucleic Acids Research

    Article Title: Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands

    doi: 10.1093/nar/gki685

    Figure Lengend Snippet: Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).

    Article Snippet: ATPase assays Hydrolysis of ATP by Hel308a was measured spectroscopically using malachite green assays ( ). φX174 circular ssDNA and RFII circular double-stranded DNA (dsDNA) substrates were from New England Biolabs.

    Techniques: Sequencing, SDS Page, Purification, Recombinant, Marker, Activity Assay, Derivative Assay

    DNA-binding activity of MlaA. ( A ) Electrophoretic mobility shift assays show that MlaA binds to dsDNA but only weakly to ssDNA. In all, 0, 0.25, 0.5, 1 or 2 μg MlaA is incubated with 50 fmol ssDNA or dsDNA 20-mer and analysed on 6% native acrylamide gels. ( B ) Competition experiments using labelled dsDNA oligonucleotides and unlabelled circular dsDNA. The fraction of labelled dsDNA that is shifted by MlaA versus the competitor:probe ratio is plotted. The highly efficient competition of dsDNA oligonucleotide binding by circular plasmid dsDNA (approximately 50% at a 1:1 ratio) indicates that MlaA does not need DNA ends for efficient binding. ( C ). The fraction of MlaA:DNA complexes (retained at nitrocellulose filters) relative to total DNA (sum of DNA retained at both nitrocellulose and hybond) versus MlaA concentration is shown. Data were fitted to f = p n /( K d + p n ), where f is the fraction of DNA bound to MlaA relative to total DNA, K d is the dissociation constant, p is the protein concentration and n is the Hill coefficient. MlaA poorly binds ssDNA (filled triangles), but more strongly binds a dsDNA Y-substrate (open circles). The tightest binding is observed for a Holliday-junction substrate (filled circles), suggesting a preference for DNA secondary structures. dsDNA ( n =2.5–4) but not ssDNA ( n ≈1) substrates are evidently bound cooperatively.

    Journal: EMBO Reports

    Article Title: MlaA, a hexameric ATPase linked to the Mre11 complex in archaeal genomes

    doi: 10.1038/sj.embor.7400037

    Figure Lengend Snippet: DNA-binding activity of MlaA. ( A ) Electrophoretic mobility shift assays show that MlaA binds to dsDNA but only weakly to ssDNA. In all, 0, 0.25, 0.5, 1 or 2 μg MlaA is incubated with 50 fmol ssDNA or dsDNA 20-mer and analysed on 6% native acrylamide gels. ( B ) Competition experiments using labelled dsDNA oligonucleotides and unlabelled circular dsDNA. The fraction of labelled dsDNA that is shifted by MlaA versus the competitor:probe ratio is plotted. The highly efficient competition of dsDNA oligonucleotide binding by circular plasmid dsDNA (approximately 50% at a 1:1 ratio) indicates that MlaA does not need DNA ends for efficient binding. ( C ). The fraction of MlaA:DNA complexes (retained at nitrocellulose filters) relative to total DNA (sum of DNA retained at both nitrocellulose and hybond) versus MlaA concentration is shown. Data were fitted to f = p n /( K d + p n ), where f is the fraction of DNA bound to MlaA relative to total DNA, K d is the dissociation constant, p is the protein concentration and n is the Hill coefficient. MlaA poorly binds ssDNA (filled triangles), but more strongly binds a dsDNA Y-substrate (open circles). The tightest binding is observed for a Holliday-junction substrate (filled circles), suggesting a preference for DNA secondary structures. dsDNA ( n =2.5–4) but not ssDNA ( n ≈1) substrates are evidently bound cooperatively.

    Article Snippet: We performed competition assays with phiX174 form II DNA (dsDNA) (New England BioLabs) by incubating 1 μg of MlaA with 50 fmol labelled ds oligonucleotide substrate as described above.

    Techniques: Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Incubation, Plasmid Preparation, Concentration Assay, Protein Concentration

    Example fragmentation profiles and coverage plots. Example fragmentation profiles and coverage plots are shown for two mitochondrial genomes (examples 62 and 80). A . These Bioanalyzer profiles (from a DNA 7500 chip) show pooled long-range PCR products after digestion with NEBNext dsDNA Fragmentase (10 min at 37°C). The x -axis shows the inferred size of the DNA fragments based on the two internal markers of known size (the peaks at 50 and 10,380 bp). The y -axis shows the amount of DNA present based on fluorescence units. Both example digestion profiles show fragments between distributed between ~300 bp and ~5 kb in length, with the distribution skewed towards smaller fragments. These profiles show fragments in the ideal size range for 454 sequencing. The difference in yields between the two samples is probably due to different recovery efficiencies in the preceding AMPure XP purification step. Screen captures are taken from the 2100 Expert software (Agilent). B . These coverage plots for two mitochondrial genomes were generated using the software described in this paper. The x -axis shows the nucleotide positions based on the revised Cambridge Reference Sequence (rCRS). The y -axis shows coverage depth. The horizontal dashed line indicates mean coverage for that genome. On the left of each heading line is the individual name (e.g. 62.sff or 80.sff); the following number (here, 16569) is the number of positions that were covered by at least 1 read, and the final number (here, also 16569) is the length of the reference sequence. Note the large peak from 8,000–9,000 bp, which is discussed in the main text. The blue lines represent the corresponding long-range PCR products and the associated numbers the positions of the ends of those products (see Table 1 ). The data used to generated these coverage plots is available in Additional file 6 .

    Journal: BMC Genomics

    Article Title: From cheek swabs to consensus sequences: an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes

    doi: 10.1186/1471-2164-15-68

    Figure Lengend Snippet: Example fragmentation profiles and coverage plots. Example fragmentation profiles and coverage plots are shown for two mitochondrial genomes (examples 62 and 80). A . These Bioanalyzer profiles (from a DNA 7500 chip) show pooled long-range PCR products after digestion with NEBNext dsDNA Fragmentase (10 min at 37°C). The x -axis shows the inferred size of the DNA fragments based on the two internal markers of known size (the peaks at 50 and 10,380 bp). The y -axis shows the amount of DNA present based on fluorescence units. Both example digestion profiles show fragments between distributed between ~300 bp and ~5 kb in length, with the distribution skewed towards smaller fragments. These profiles show fragments in the ideal size range for 454 sequencing. The difference in yields between the two samples is probably due to different recovery efficiencies in the preceding AMPure XP purification step. Screen captures are taken from the 2100 Expert software (Agilent). B . These coverage plots for two mitochondrial genomes were generated using the software described in this paper. The x -axis shows the nucleotide positions based on the revised Cambridge Reference Sequence (rCRS). The y -axis shows coverage depth. The horizontal dashed line indicates mean coverage for that genome. On the left of each heading line is the individual name (e.g. 62.sff or 80.sff); the following number (here, 16569) is the number of positions that were covered by at least 1 read, and the final number (here, also 16569) is the length of the reference sequence. Note the large peak from 8,000–9,000 bp, which is discussed in the main text. The blue lines represent the corresponding long-range PCR products and the associated numbers the positions of the ends of those products (see Table 1 ). The data used to generated these coverage plots is available in Additional file 6 .

    Article Snippet: If samples of different length are pooled for library construction then the mass of DNA used for each sample should be adjusted accordingly to ensure coverage levels are the same across all samples (see ‘Fragmentation of PCR products using NEBNext dsDNA fragmentase’ above).

    Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Fluorescence, Sequencing, Purification, Software, Generated

    Overview of the A to Z method for high-throughput DNA sequencing of complete human mitochondrial genomes. DNA is collected using cheek swabs and then extracted using a phenol–chloroform method. Long-range PCR is used to amplify each mitochondrial genome in two overlapping amplicons. The two amplicons from each genome are then pooled and fragmented using NEBNext dsDNA Fragmentase. Barcoding of the fragments is then achieved using Parallel Tagged Sequencing (PTS) [ 12 ]. Barcoded fragments are then pooled for library preparation, emulsion PCR (emPCR) and pyrosequencing on the 454 GS FLX platform. Using a number of bioinformatics tools, the resulting sequence data are de-multiplexed and barcodes and primers are removed. Reference-based mapping (to a circular reference) is carried out, followed by the output of coverage plots, consensus sequences and SNP calling for each individual.

    Journal: BMC Genomics

    Article Title: From cheek swabs to consensus sequences: an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes

    doi: 10.1186/1471-2164-15-68

    Figure Lengend Snippet: Overview of the A to Z method for high-throughput DNA sequencing of complete human mitochondrial genomes. DNA is collected using cheek swabs and then extracted using a phenol–chloroform method. Long-range PCR is used to amplify each mitochondrial genome in two overlapping amplicons. The two amplicons from each genome are then pooled and fragmented using NEBNext dsDNA Fragmentase. Barcoding of the fragments is then achieved using Parallel Tagged Sequencing (PTS) [ 12 ]. Barcoded fragments are then pooled for library preparation, emulsion PCR (emPCR) and pyrosequencing on the 454 GS FLX platform. Using a number of bioinformatics tools, the resulting sequence data are de-multiplexed and barcodes and primers are removed. Reference-based mapping (to a circular reference) is carried out, followed by the output of coverage plots, consensus sequences and SNP calling for each individual.

    Article Snippet: If samples of different length are pooled for library construction then the mass of DNA used for each sample should be adjusted accordingly to ensure coverage levels are the same across all samples (see ‘Fragmentation of PCR products using NEBNext dsDNA fragmentase’ above).

    Techniques: High Throughput Screening Assay, DNA Sequencing, Polymerase Chain Reaction, Sequencing

    Three Strand Exchange Recombinase Assay A Active RAD51 nucleoprotein filament is formed on ϕX174 Virion circular ssDNA in reaction conditions permitting ATP hydrolysis followed by addition of RPA which helps remove secondary structures in the ssDNA allowing stable nucleoprotein filament formation. The RAD51 nucleoprotein then invades ϕX174 RFI linear dsDNA to form nicked circular dsDNA products through several joint-molecule intermediates showing various stages of branch migration. The RPA also helps sequester any free displaced ssDNA following strand exchange thereby preventing a reverse reaction. B . Typical agarose gel image from a strand exchange assay used to resolve the substrates, intermediates and the products of the reaction over a time course of 0, 30, 60, 120 and 180 minutes. In reactions containing the wild type protein, nicked circular products can be observed as early as 30 minutes. C. Quantitation of the strand exchange assay gel showing appearance of nicked circular products (black) and joint molecules (blue) on the left Y axis. Disappearance of the linear dsDNA substrate (red) is quantitated on the right Y axis. Time in minutes is indicated on the X axis.

    Journal: Methods in enzymology

    Article Title: Expression, Purification and Biochemical Evaluation of Human RAD51 Protein

    doi: 10.1016/bs.mie.2017.11.011

    Figure Lengend Snippet: Three Strand Exchange Recombinase Assay A Active RAD51 nucleoprotein filament is formed on ϕX174 Virion circular ssDNA in reaction conditions permitting ATP hydrolysis followed by addition of RPA which helps remove secondary structures in the ssDNA allowing stable nucleoprotein filament formation. The RAD51 nucleoprotein then invades ϕX174 RFI linear dsDNA to form nicked circular dsDNA products through several joint-molecule intermediates showing various stages of branch migration. The RPA also helps sequester any free displaced ssDNA following strand exchange thereby preventing a reverse reaction. B . Typical agarose gel image from a strand exchange assay used to resolve the substrates, intermediates and the products of the reaction over a time course of 0, 30, 60, 120 and 180 minutes. In reactions containing the wild type protein, nicked circular products can be observed as early as 30 minutes. C. Quantitation of the strand exchange assay gel showing appearance of nicked circular products (black) and joint molecules (blue) on the left Y axis. Disappearance of the linear dsDNA substrate (red) is quantitated on the right Y axis. Time in minutes is indicated on the X axis.

    Article Snippet: The oligonucleotides are resuspended in 10mM Tris (pH8.0) and stored at −20°C. ϕX174 Virion ssDNA (NEB# N3023) ϕX174 RFI dsDNA (NEB# N3021)

    Techniques: Recombinase Polymerase Amplification, Migration, Agarose Gel Electrophoresis, Quantitation Assay

    RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.

    Journal: Journal of molecular biology

    Article Title: TRIM25 binds RNA to modulate cellular anti-viral defense

    doi: 10.1016/j.jmb.2018.10.003

    Figure Lengend Snippet: RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.

    Article Snippet: Experiments in used RNase A (Qiagen), DNase I (Sigma), dsRNA ladder (NEB), and 100-bp dsDNA ladder (NEB).

    Techniques: Activity Assay, In Vitro, Purification, Incubation

    A general overview of the dsDNA Fragmentation Through Polymerization (FTP) method. The FTP method is based on two enzymatic reactions: a DNA nicking reaction with DNase I and a strand-displacement DNA polymerization with SD DNA polymerase. As a result, multiple double-stranded DNA fragments with overlapping sequences are generated. De novo synthesized DNA is indicated in grey, and SD polymerase is indicated in red.

    Journal: PLoS ONE

    Article Title: Fragmentation Through Polymerization (FTP): A new method to fragment DNA for next-generation sequencing

    doi: 10.1371/journal.pone.0210374

    Figure Lengend Snippet: A general overview of the dsDNA Fragmentation Through Polymerization (FTP) method. The FTP method is based on two enzymatic reactions: a DNA nicking reaction with DNase I and a strand-displacement DNA polymerization with SD DNA polymerase. As a result, multiple double-stranded DNA fragments with overlapping sequences are generated. De novo synthesized DNA is indicated in grey, and SD polymerase is indicated in red.

    Article Snippet: NEBNext dsDNA Fragmentase and the NEBNext Ultra II DNA Library Prep kit were supplied by New England Biolabs, Inc. (Ipswich, MA, USA).

    Techniques: Generated, Synthesized

    Molecular events and ionic current trace for a 2D read of a 7.25 kb M13 phage dsDNA molecule. (a) Schematic for the steps in DNA translocation through the nanopore. (i) Open channel; (ii) dsDNA with a ligated lead adaptor (blue), with a molecular motor bound to it (orange), and a hairpin adaptor (red), is captured by the nanopore. DNA translocation through the nanopore begins through the effect of an applied voltage across the membrane and the action of a molecular motor; (iii) Translocation of the lead adaptor (blue); (iv) Translocation of the template strand (gold); (v) Translocation of the hairpin adaptor (red); (vi) Translocation of the complement strand (dark blue); (vii) Translocation of the trailing adaptor (brown); (viii) Return to open channel. (b) Raw current trace for the passage of the M13 dsDNA construct through the nanopore. Regions of the ionic current trace corresponding to steps i-viii are labeled. (c) Expanded time and current scale for raw current traces corresponding to steps i–viii. Each adaptor generates a unique current signal used to aid base calling.

    Journal: Nature methods

    Article Title: Improved data analysis for the MinION nanopore sequencer

    doi: 10.1038/nmeth.3290

    Figure Lengend Snippet: Molecular events and ionic current trace for a 2D read of a 7.25 kb M13 phage dsDNA molecule. (a) Schematic for the steps in DNA translocation through the nanopore. (i) Open channel; (ii) dsDNA with a ligated lead adaptor (blue), with a molecular motor bound to it (orange), and a hairpin adaptor (red), is captured by the nanopore. DNA translocation through the nanopore begins through the effect of an applied voltage across the membrane and the action of a molecular motor; (iii) Translocation of the lead adaptor (blue); (iv) Translocation of the template strand (gold); (v) Translocation of the hairpin adaptor (red); (vi) Translocation of the complement strand (dark blue); (vii) Translocation of the trailing adaptor (brown); (viii) Return to open channel. (b) Raw current trace for the passage of the M13 dsDNA construct through the nanopore. Regions of the ionic current trace corresponding to steps i-viii are labeled. (c) Expanded time and current scale for raw current traces corresponding to steps i–viii. Each adaptor generates a unique current signal used to aid base calling.

    Article Snippet: M13mp18 DNA sequencing standard M13mp18 dsDNA was obtained from New England Biolabs (Cat No. N4018S).

    Techniques: Translocation Assay, Construct, Labeling