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  • 99
    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 .
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    99
    New England Biolabs e coli dna ligase
    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 .
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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: 99/100, based on 2463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.
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    New England Biolabs double stranded dna fragments
    Gka RloC’s ATPase activates its ACNase. Gka RloC’s ACNase of the IMAC fraction was assayed in vitro in panels ( A )–( C ) and ( E ) essentially as described in Materials and Methods but in the absence of added <t>DNA.</t> ( A ) Dependence of Gka RloC’s ACNase activity on <t>ATP’s</t> level. ( B ) Gka RloC’s ACNase activity was assayed in the presence of 500 µM of the indicated nucleotides. ( C ) Time courses of Gka RloC’s ACNase activity in the presence of 0.5 mM ATP and indicated amounts of AMPPNP. ( D ) In vivo ACNase activity of the indicated Gka RloC alleles. Left panel—RNA extracted from cells expressing these alleles was 5′-end labelled using T4 Pnk and separated by denaturing PAGE. Right panel—the expression of the indicated Gka RloC alleles were monitored by Western using an anti-His tag monoclonal antibody ( 4 ). ( E ) Nucleotide specificity of Gka RloC’s ACNase activation. The activation reaction was performed in the presence of the indicated nucleotides (GTP and ATP at 0.5 mM each, dTTP at 5 µM).
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    New England Biolabs dsdna ladder
    Gka RloC’s ATPase activates its ACNase. Gka RloC’s ACNase of the IMAC fraction was assayed in vitro in panels ( A )–( C ) and ( E ) essentially as described in Materials and Methods but in the absence of added <t>DNA.</t> ( A ) Dependence of Gka RloC’s ACNase activity on <t>ATP’s</t> level. ( B ) Gka RloC’s ACNase activity was assayed in the presence of 500 µM of the indicated nucleotides. ( C ) Time courses of Gka RloC’s ACNase activity in the presence of 0.5 mM ATP and indicated amounts of AMPPNP. ( D ) In vivo ACNase activity of the indicated Gka RloC alleles. Left panel—RNA extracted from cells expressing these alleles was 5′-end labelled using T4 Pnk and separated by denaturing PAGE. Right panel—the expression of the indicated Gka RloC alleles were monitored by Western using an anti-His tag monoclonal antibody ( 4 ). ( E ) Nucleotide specificity of Gka RloC’s ACNase activation. The activation reaction was performed in the presence of the indicated nucleotides (GTP and ATP at 0.5 mM each, dTTP at 5 µM).
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    New England Biolabs dsdna
    Nucleic acid-binding activity of <t>AtUSP</t> in vitro. Indicated amounts of purified recombinant AtUSP protein were incubated with either ( A ) M13mp8 ssDNA, ( B ) M13mp8 <t>dsDNA,</t> or ( C ) in vitro transcribed luciferase ( luc ) mRNA. To analyze the effect of AtUSP in RNA mobility and the AtUSP-RNA complexes, 0.8% agarose gels were used for gel-shift assays. Bovine serum albumin (BSA) protein (100 μg/μL) was used as a negative control. SM presents size marker from Thermo Scientific Company.
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    New England Biolabs large klenow fragment
    Nucleic acid-binding activity of <t>AtUSP</t> in vitro. Indicated amounts of purified recombinant AtUSP protein were incubated with either ( A ) M13mp8 ssDNA, ( B ) M13mp8 <t>dsDNA,</t> or ( C ) in vitro transcribed luciferase ( luc ) mRNA. To analyze the effect of AtUSP in RNA mobility and the AtUSP-RNA complexes, 0.8% agarose gels were used for gel-shift assays. Bovine serum albumin (BSA) protein (100 μg/μL) was used as a negative control. SM presents size marker from Thermo Scientific Company.
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    New England Biolabs nebnext dna fragmentation reaction buffer
    Nucleic acid-binding activity of <t>AtUSP</t> in vitro. Indicated amounts of purified recombinant AtUSP protein were incubated with either ( A ) M13mp8 ssDNA, ( B ) M13mp8 <t>dsDNA,</t> or ( C ) in vitro transcribed luciferase ( luc ) mRNA. To analyze the effect of AtUSP in RNA mobility and the AtUSP-RNA complexes, 0.8% agarose gels were used for gel-shift assays. Bovine serum albumin (BSA) protein (100 μg/μL) was used as a negative control. SM presents size marker from Thermo Scientific Company.
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    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: PDF file of the protocol for fragmenting LR-PCR products with NEBNext® dsDNA Fragmentase™.

    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: PDF file of the protocol for fragmenting LR-PCR products with NEBNext® dsDNA Fragmentase™.

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

    DSB s formation at suboptimal pH e. (A) Left panel: PFGE in neutral conditions to resolve DSB s. Arrows show maximal density (apex) for each track. Right panel: corresponding densitometric scan of the PFGE tracks shown in A. Arrows indicate the corresponding position of apex shown on the gel. (B) qTUNEL quantification of DNA double‐stranded breaks for the highest and lowest values of the suboptimal pH e range. Frag indicates fragmentase‐digested DNA from fibroblasts used as a positive control. Values are mean ± SEM of three determinations.

    Journal: FEBS Open Bio

    Article Title: Suboptimal extracellular pH values alter DNA damage response to induced double‐strand breaks

    doi: 10.1002/2211-5463.12384

    Figure Lengend Snippet: DSB s formation at suboptimal pH e. (A) Left panel: PFGE in neutral conditions to resolve DSB s. Arrows show maximal density (apex) for each track. Right panel: corresponding densitometric scan of the PFGE tracks shown in A. Arrows indicate the corresponding position of apex shown on the gel. (B) qTUNEL quantification of DNA double‐stranded breaks for the highest and lowest values of the suboptimal pH e range. Frag indicates fragmentase‐digested DNA from fibroblasts used as a positive control. Values are mean ± SEM of three determinations.

    Article Snippet: As a positive control, digestion of the DNA was performed with 2 μL of fragmentase (M0348, NEB, Ipswich, MA, USA).

    Techniques: Positive Control

    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

    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

    Gka RloC’s ATPase activates its ACNase. Gka RloC’s ACNase of the IMAC fraction was assayed in vitro in panels ( A )–( C ) and ( E ) essentially as described in Materials and Methods but in the absence of added DNA. ( A ) Dependence of Gka RloC’s ACNase activity on ATP’s level. ( B ) Gka RloC’s ACNase activity was assayed in the presence of 500 µM of the indicated nucleotides. ( C ) Time courses of Gka RloC’s ACNase activity in the presence of 0.5 mM ATP and indicated amounts of AMPPNP. ( D ) In vivo ACNase activity of the indicated Gka RloC alleles. Left panel—RNA extracted from cells expressing these alleles was 5′-end labelled using T4 Pnk and separated by denaturing PAGE. Right panel—the expression of the indicated Gka RloC alleles were monitored by Western using an anti-His tag monoclonal antibody ( 4 ). ( E ) Nucleotide specificity of Gka RloC’s ACNase activation. The activation reaction was performed in the presence of the indicated nucleotides (GTP and ATP at 0.5 mM each, dTTP at 5 µM).

    Journal: Nucleic Acids Research

    Article Title: The wobble nucleotide-excising anticodon nuclease RloC is governed by the zinc-hook and DNA-dependent ATPase of its Rad50-like region

    doi: 10.1093/nar/gks593

    Figure Lengend Snippet: Gka RloC’s ATPase activates its ACNase. Gka RloC’s ACNase of the IMAC fraction was assayed in vitro in panels ( A )–( C ) and ( E ) essentially as described in Materials and Methods but in the absence of added DNA. ( A ) Dependence of Gka RloC’s ACNase activity on ATP’s level. ( B ) Gka RloC’s ACNase activity was assayed in the presence of 500 µM of the indicated nucleotides. ( C ) Time courses of Gka RloC’s ACNase activity in the presence of 0.5 mM ATP and indicated amounts of AMPPNP. ( D ) In vivo ACNase activity of the indicated Gka RloC alleles. Left panel—RNA extracted from cells expressing these alleles was 5′-end labelled using T4 Pnk and separated by denaturing PAGE. Right panel—the expression of the indicated Gka RloC alleles were monitored by Western using an anti-His tag monoclonal antibody ( 4 ). ( E ) Nucleotide specificity of Gka RloC’s ACNase activation. The activation reaction was performed in the presence of the indicated nucleotides (GTP and ATP at 0.5 mM each, dTTP at 5 µM).

    Article Snippet: Gka RloC ACNase assays The standard in vitro ACNase assay mixtures (10 µl) contained 100–500 ng Gka RloC protein, 70 mM Tris–HCl, pH 7.5, 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP, 10 ng/µl double-stranded DNA fragments (BstEII-digested λ DNA, NEB) and 0.05–0.1 pmol of [5′-32 P]Lys3-ASL or 0.01–0.02 pmol of the internally labelled sc-tRNAGlu(UUC) .

    Techniques: In Vitro, Activity Assay, In Vivo, Expressing, Polyacrylamide Gel Electrophoresis, Western Blot, Activation Assay

    Plot of fluorescence intensity for a λ-DNA stock solution, free enzyme digestion, and the effluent from an IMERs digestion. The emission spectra were taken from 490 to 700 nm with 480 nm excitation. The spectrum labeled in black depicts the intensity of the λ-DNA stock. The blue line represents the spectrum of the IMERS digestion and the red line was that for the free solution digestion. For the IMERs digestion, the amount of immobilized enzyme was 4.96 pmol. For the λ-DNA stock, the IMERs was free of immobilized enzyme. In all cases, the solutions were incubated with PicoGreen following the reaction.

    Journal: Analytical Chemistry

    Article Title: Immobilization of Lambda Exonuclease onto Polymer Micropillar Arrays for the Solid-Phase Digestion of dsDNAs

    doi: 10.1021/ac5002965

    Figure Lengend Snippet: Plot of fluorescence intensity for a λ-DNA stock solution, free enzyme digestion, and the effluent from an IMERs digestion. The emission spectra were taken from 490 to 700 nm with 480 nm excitation. The spectrum labeled in black depicts the intensity of the λ-DNA stock. The blue line represents the spectrum of the IMERS digestion and the red line was that for the free solution digestion. For the IMERs digestion, the amount of immobilized enzyme was 4.96 pmol. For the λ-DNA stock, the IMERs was free of immobilized enzyme. In all cases, the solutions were incubated with PicoGreen following the reaction.

    Article Snippet: Digestion Studies of dsDNA Duplexed λ-DNA (48 502 bp), purchased from New England Biolabs (Ipswich, MA), was incubated in the enzyme-modified IMERs for various reaction times.

    Techniques: Fluorescence, Labeling, Incubation

    (A–D) Fluorescence still images for the real-time digestion of dsDNA using λ-Exo covalently immobilized to a PMMA substrate configured in the IMER device. (E–H) The corresponding fluorescence intensity line plots taken from the still images shown in parts A–D. In these cases, the line plot was secured from a horizontal line that crossed the section in the still image containing the stained DNA molecule. (I) Graphical depiction of the relative fluorescence intensity of a single dsDNA that was digested by an immobilized λ-Exo molecule as a function of reaction time, where possible pausing events were seen in each inset. Immobilization of λ-Exo was accomplished using EDC/NHS onto a PMMA substrate. The λ-DNA was labeled in a 1:50 dye/bp ratio with YOYO-1. The fluorescence intensity was measured in the presence (black) or absence (red) of the enzyme cofactor, Mg 2+ . The dotted line for the intensity profile in the presence of Mg 2+ indicates the time at which the cofactor was infused into the IMER.

    Journal: Analytical Chemistry

    Article Title: Immobilization of Lambda Exonuclease onto Polymer Micropillar Arrays for the Solid-Phase Digestion of dsDNAs

    doi: 10.1021/ac5002965

    Figure Lengend Snippet: (A–D) Fluorescence still images for the real-time digestion of dsDNA using λ-Exo covalently immobilized to a PMMA substrate configured in the IMER device. (E–H) The corresponding fluorescence intensity line plots taken from the still images shown in parts A–D. In these cases, the line plot was secured from a horizontal line that crossed the section in the still image containing the stained DNA molecule. (I) Graphical depiction of the relative fluorescence intensity of a single dsDNA that was digested by an immobilized λ-Exo molecule as a function of reaction time, where possible pausing events were seen in each inset. Immobilization of λ-Exo was accomplished using EDC/NHS onto a PMMA substrate. The λ-DNA was labeled in a 1:50 dye/bp ratio with YOYO-1. The fluorescence intensity was measured in the presence (black) or absence (red) of the enzyme cofactor, Mg 2+ . The dotted line for the intensity profile in the presence of Mg 2+ indicates the time at which the cofactor was infused into the IMER.

    Article Snippet: Digestion Studies of dsDNA Duplexed λ-DNA (48 502 bp), purchased from New England Biolabs (Ipswich, MA), was incubated in the enzyme-modified IMERs for various reaction times.

    Techniques: Fluorescence, Staining, Labeling

    Nucleic acid-binding activity of AtUSP in vitro. Indicated amounts of purified recombinant AtUSP protein were incubated with either ( A ) M13mp8 ssDNA, ( B ) M13mp8 dsDNA, or ( C ) in vitro transcribed luciferase ( luc ) mRNA. To analyze the effect of AtUSP in RNA mobility and the AtUSP-RNA complexes, 0.8% agarose gels were used for gel-shift assays. Bovine serum albumin (BSA) protein (100 μg/μL) was used as a negative control. SM presents size marker from Thermo Scientific Company.

    Journal: International Journal of Molecular Sciences

    Article Title: RNA Chaperone Function of a Universal Stress Protein in Arabidopsis Confers Enhanced Cold Stress Tolerance in Plants

    doi: 10.3390/ijms18122546

    Figure Lengend Snippet: Nucleic acid-binding activity of AtUSP in vitro. Indicated amounts of purified recombinant AtUSP protein were incubated with either ( A ) M13mp8 ssDNA, ( B ) M13mp8 dsDNA, or ( C ) in vitro transcribed luciferase ( luc ) mRNA. To analyze the effect of AtUSP in RNA mobility and the AtUSP-RNA complexes, 0.8% agarose gels were used for gel-shift assays. Bovine serum albumin (BSA) protein (100 μg/μL) was used as a negative control. SM presents size marker from Thermo Scientific Company.

    Article Snippet: Recombinant AtUSP or BSA was incubated with ssDNA (M13mp18, NEB), dsDNA (M13mp18 RF I DNA, NEB), and luc mRNA (TriLink Biotechnologies Co., San Diego, CA, USA) in 15 μL binding buffer (10 mM Tris-HCl, pH 7.5) on ice for 30 min.

    Techniques: Binding Assay, Activity Assay, In Vitro, Purification, Recombinant, Incubation, Luciferase, Electrophoretic Mobility Shift Assay, Negative Control, Marker