double stranded dna dsdna  (New England Biolabs)


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    Structured Review

    New England Biolabs double stranded dna dsdna
    CRISPR adaptation to HHPV-2 infection. ( A ) Depiction of the single CRISPR structure and the preceding cas operon carried by the H. hispanica ATCC 33960 genome. Primers used to examine CRISPR expansion (in panel B) are shown as black arrows and listed in Supplementary Table S2 . ( B ) PCR assay to detect CRISPR expansion at the leader end (L1–L2), the inner part (I1–I2) or the distal end (D1–D2). <t>DNA</t> sampled from infected (+) or uninfected (−) cells was used as PCR templates. Lane M, <t>dsDNA</t> size marker. ( C ) The sequence logo showing the conserved PAM of TTC. The 20 nt upstream of each protospacer observed during HHPV-2 infection were collected and analyzed with WebLogo ( http://weblogo.berkeley.edu/logo.cgi ).
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    Images

    1) Product Images from "Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process"

    Article Title: Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1154

    CRISPR adaptation to HHPV-2 infection. ( A ) Depiction of the single CRISPR structure and the preceding cas operon carried by the H. hispanica ATCC 33960 genome. Primers used to examine CRISPR expansion (in panel B) are shown as black arrows and listed in Supplementary Table S2 . ( B ) PCR assay to detect CRISPR expansion at the leader end (L1–L2), the inner part (I1–I2) or the distal end (D1–D2). DNA sampled from infected (+) or uninfected (−) cells was used as PCR templates. Lane M, dsDNA size marker. ( C ) The sequence logo showing the conserved PAM of TTC. The 20 nt upstream of each protospacer observed during HHPV-2 infection were collected and analyzed with WebLogo ( http://weblogo.berkeley.edu/logo.cgi ).
    Figure Legend Snippet: CRISPR adaptation to HHPV-2 infection. ( A ) Depiction of the single CRISPR structure and the preceding cas operon carried by the H. hispanica ATCC 33960 genome. Primers used to examine CRISPR expansion (in panel B) are shown as black arrows and listed in Supplementary Table S2 . ( B ) PCR assay to detect CRISPR expansion at the leader end (L1–L2), the inner part (I1–I2) or the distal end (D1–D2). DNA sampled from infected (+) or uninfected (−) cells was used as PCR templates. Lane M, dsDNA size marker. ( C ) The sequence logo showing the conserved PAM of TTC. The 20 nt upstream of each protospacer observed during HHPV-2 infection were collected and analyzed with WebLogo ( http://weblogo.berkeley.edu/logo.cgi ).

    Techniques Used: CRISPR, Infection, Polymerase Chain Reaction, Marker, Sequencing

    Adaptation to HHPV-2 infection under different cas genetic backgrounds. ( A ) Cas requirement for adaptation. For each cas mutant, DNA was sampled from cells transformed with an empty plasmid (−) or the plasmid carrying the deleted cas gene(s) (+). The plasmid-carried cas gene(s) was/were under the control of the cas operon promoter. ( B ) Requirements for the nuclease and helicase activities of Cas3. Alanine replacement was performed for the putative key residues in the HD nuclease domain (H20A, H55A, D56A and D229A) and the DExD/H helicase domain (K315A, D439A and E440A). Another two conserved residues (His6 and Lys113) were also mutated. The empty plasmid (−) and the plasmid carrying a wild-type Cas3 (Cas3 WT ) were used, respectively, as negative and positive controls. Lane Ms, dsDNA size markers.
    Figure Legend Snippet: Adaptation to HHPV-2 infection under different cas genetic backgrounds. ( A ) Cas requirement for adaptation. For each cas mutant, DNA was sampled from cells transformed with an empty plasmid (−) or the plasmid carrying the deleted cas gene(s) (+). The plasmid-carried cas gene(s) was/were under the control of the cas operon promoter. ( B ) Requirements for the nuclease and helicase activities of Cas3. Alanine replacement was performed for the putative key residues in the HD nuclease domain (H20A, H55A, D56A and D229A) and the DExD/H helicase domain (K315A, D439A and E440A). Another two conserved residues (His6 and Lys113) were also mutated. The empty plasmid (−) and the plasmid carrying a wild-type Cas3 (Cas3 WT ) were used, respectively, as negative and positive controls. Lane Ms, dsDNA size markers.

    Techniques Used: Infection, Mutagenesis, Transformation Assay, Plasmid Preparation, Mass Spectrometry

    2) Product Images from "The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing"

    Article Title: The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw877

    BCCIPβ binds DNA. ( A ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) incubated with ϕX174 (+) ssDNA (ss; 30 μM nucleotides). ( B ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) was incubated with ϕX174 RF (I) dsDNA (ds; 30 μM base pairs). The reaction products were separated on a 1.0% agarose gel, and were stained with ethidium bromide. Lane 1 contained no protein, and lane 8 was deproteinized with SDS and Proteinase K (S/P) prior to loading.
    Figure Legend Snippet: BCCIPβ binds DNA. ( A ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) incubated with ϕX174 (+) ssDNA (ss; 30 μM nucleotides). ( B ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) was incubated with ϕX174 RF (I) dsDNA (ds; 30 μM base pairs). The reaction products were separated on a 1.0% agarose gel, and were stained with ethidium bromide. Lane 1 contained no protein, and lane 8 was deproteinized with SDS and Proteinase K (S/P) prior to loading.

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining

    3) Product Images from "RecA Protein from the Extremely Radioresistant Bacterium Deinococcus radiodurans: Expression, Purification, and Characterization"

    Article Title: RecA Protein from the Extremely Radioresistant Bacterium Deinococcus radiodurans: Expression, Purification, and Characterization

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.184.6.1649-1660.2002

    Electron microscopy of the RecA* Dr protein bound to DNA derived from bacteriophage φX174. (A) RecA Dr filamented linear dsDNA. (B) RecA Dr filamented circular ssDNA. This view was chosen because it includes in the background, at lower contrast, several collapsed DNA circles bound with SSB.
    Figure Legend Snippet: Electron microscopy of the RecA* Dr protein bound to DNA derived from bacteriophage φX174. (A) RecA Dr filamented linear dsDNA. (B) RecA Dr filamented circular ssDNA. This view was chosen because it includes in the background, at lower contrast, several collapsed DNA circles bound with SSB.

    Techniques Used: Electron Microscopy, Derivative Assay

    Effects of pH on the hydrolysis of ATP and dATP by the RecA Dr protein. Circular single-stranded (css or SS), linear single-stranded (lss), and linear double-stranded DNA from φX174 linearized with Pst I restriction enzyme (DS) were used as DNA cofactors. (A) (d)ATP hydrolysis reactions of the RecA* Dr protein. Closed and open symbols represent dATP and ATP hydrolysis, respectively. Circles and squares represent reactions with circular and linear ssDNA, respectively. Reactions were carried out as described in Materials and Methods, and reaction mixtures contained 5 μM ssDNA, 3 μM RecA* Dr protein, 0.5 μM E. coli SSB protein, and a 3 mM concentration of either ATP or dATP. (B) Effect of pH on the DNA-dependent (d)ATP hydrolytic activities of the native RecA Dr protein. Reactions were carried out as described in Materials and Methods, and reaction mixtures contained 2 mM ATP (or dATP), 2 μM RecA Dr , 0.5 μM E. coli SSB protein, and 5 μM ssDNA (or 10 μM dsDNA). The E. coli SSB protein was omitted in the dsDNA-dependent reactions. (C) Reactions were the same as the ssDNA reactions in panel B, but poly(dT) was substituted for the ssDNA.
    Figure Legend Snippet: Effects of pH on the hydrolysis of ATP and dATP by the RecA Dr protein. Circular single-stranded (css or SS), linear single-stranded (lss), and linear double-stranded DNA from φX174 linearized with Pst I restriction enzyme (DS) were used as DNA cofactors. (A) (d)ATP hydrolysis reactions of the RecA* Dr protein. Closed and open symbols represent dATP and ATP hydrolysis, respectively. Circles and squares represent reactions with circular and linear ssDNA, respectively. Reactions were carried out as described in Materials and Methods, and reaction mixtures contained 5 μM ssDNA, 3 μM RecA* Dr protein, 0.5 μM E. coli SSB protein, and a 3 mM concentration of either ATP or dATP. (B) Effect of pH on the DNA-dependent (d)ATP hydrolytic activities of the native RecA Dr protein. Reactions were carried out as described in Materials and Methods, and reaction mixtures contained 2 mM ATP (or dATP), 2 μM RecA Dr , 0.5 μM E. coli SSB protein, and 5 μM ssDNA (or 10 μM dsDNA). The E. coli SSB protein was omitted in the dsDNA-dependent reactions. (C) Reactions were the same as the ssDNA reactions in panel B, but poly(dT) was substituted for the ssDNA.

    Techniques Used: Concentration Assay

    The RecA Dr protein binds to dsDNA in preference to ssDNA. (A) Reactions were carried out at pH 7.5 and contained 1.4 μM RecA Dr protein, DNA as indicated (5 μM css φX174 DNA (ssDNA), 10 μM lds φX174 (Hm dsDNA or dsDNA), and/or 10 μM lds M13mp8 DNA (Ht dsDNA), 0.4 μM E. coli SSB, 2 mM ATP, and an ATP regenerating system. (B) Reactions were carried out at pH 8.1, and reaction mixtures contained 1.0 μM RecA Dr protein, DNA as indicated [4 μM poly(dT) and/or 8 μM lds M13mp8 DNA], no E. coli SSB, 2 mM dATP, and a dATP regenerating system.
    Figure Legend Snippet: The RecA Dr protein binds to dsDNA in preference to ssDNA. (A) Reactions were carried out at pH 7.5 and contained 1.4 μM RecA Dr protein, DNA as indicated (5 μM css φX174 DNA (ssDNA), 10 μM lds φX174 (Hm dsDNA or dsDNA), and/or 10 μM lds M13mp8 DNA (Ht dsDNA), 0.4 μM E. coli SSB, 2 mM ATP, and an ATP regenerating system. (B) Reactions were carried out at pH 8.1, and reaction mixtures contained 1.0 μM RecA Dr protein, DNA as indicated [4 μM poly(dT) and/or 8 μM lds M13mp8 DNA], no E. coli SSB, 2 mM dATP, and a dATP regenerating system.

    Techniques Used:

    4) Product Images from "Heterodimeric complexes of Hop2 and Mnd1 function with Dmc1 to promote meiotic homolog juxtaposition and strand assimilation"

    Article Title: Heterodimeric complexes of Hop2 and Mnd1 function with Dmc1 to promote meiotic homolog juxtaposition and strand assimilation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0404195101

    H2M1 complexes associate with dsDNA. ( A ) φX174 dsDNA (15 μMin bp) and ssDNA (30 μM in nt) were incubated with H2M1 protein (0–1.5 μM). Nucleoprotein supercomplexes were separated on a 0.8% agarose gel, stained with ethidium bromide, and visualized by UV transillumination. ( B ). ( C ) AFM images of H2M1 protein ( 1 ), 872-bp dsDNA ( 2 ), and H2M1–dsDNA complexes ( 3 – 6 ).
    Figure Legend Snippet: H2M1 complexes associate with dsDNA. ( A ) φX174 dsDNA (15 μMin bp) and ssDNA (30 μM in nt) were incubated with H2M1 protein (0–1.5 μM). Nucleoprotein supercomplexes were separated on a 0.8% agarose gel, stained with ethidium bromide, and visualized by UV transillumination. ( B ). ( C ) AFM images of H2M1 protein ( 1 ), 872-bp dsDNA ( 2 ), and H2M1–dsDNA complexes ( 3 – 6 ).

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining

    5) Product Images from "Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations"

    Article Title: Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations

    Journal: eLife

    doi: 10.7554/eLife.51605

    Purification and activity of NEIL2. ( A ) Purified NEIL2-His 6 (50 ng) from E. coli was subjected to NuPAGE and stained with Coomassie blue. ( B ) Activity of purified NEIL2-His 6 on 5’-[ 32 P]-labeled oligonucleotides (51 nt) containing: hydroxyuracil (OHU), OHU/G, U, or U/G. ssDNA, single-stranded DNA; dsDNA, double-stranded DNA. S, substrate; P, product. ( C ) Validation of UDG-generated AP sites from 5’-[ 32 P]-labeled single-stranded and double-stranded oligonucleotides (35 nt) by treatment with NaOH. AP sites are lysed by alkali treatment. ( D ) NEIL2 cleaves Fluorescein (Fluor)-labeled U-containing single strand oligonucleotide (39 nt) in the presence of UDG. S, substrate; P, product.
    Figure Legend Snippet: Purification and activity of NEIL2. ( A ) Purified NEIL2-His 6 (50 ng) from E. coli was subjected to NuPAGE and stained with Coomassie blue. ( B ) Activity of purified NEIL2-His 6 on 5’-[ 32 P]-labeled oligonucleotides (51 nt) containing: hydroxyuracil (OHU), OHU/G, U, or U/G. ssDNA, single-stranded DNA; dsDNA, double-stranded DNA. S, substrate; P, product. ( C ) Validation of UDG-generated AP sites from 5’-[ 32 P]-labeled single-stranded and double-stranded oligonucleotides (35 nt) by treatment with NaOH. AP sites are lysed by alkali treatment. ( D ) NEIL2 cleaves Fluorescein (Fluor)-labeled U-containing single strand oligonucleotide (39 nt) in the presence of UDG. S, substrate; P, product.

    Techniques Used: Purification, Activity Assay, Staining, Labeling, Generated

    6) Product Images from "Quantification of M13 and T7 bacteriophages by TaqMan and SYBR Green qPCR"

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

    Journal: Journal of virological methods

    doi: 10.1016/j.jviromet.2017.11.012

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    7) Product Images from "Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki685

    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).
    Figure Legend 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).

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

    Related Articles

    Electroporation:

    Article Title: Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone
    Article Snippet: RNA was electroporated into Vero cells (170 V, 0 resistance, and 950 μF; 1 pulse with the BTX ECM 630 electroporation system), and cells were subsequently monitored for cytopathic effect (CPE) daily. .. DNA was then fragmented with double-stranded DNA (dsDNA) fragmentase (NEB) for 15 min at 37°C.

    Transfection:

    Article Title: Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone
    Article Snippet: Paragraph title: In vitro transcription and transfection. ... DNA was then fragmented with double-stranded DNA (dsDNA) fragmentase (NEB) for 15 min at 37°C.

    Amplification:

    Article Title: Genome-Wide Identification of Fitness Factors in Mastitis-Associated Escherichia coli
    Article Snippet: Genomic DNA (150 ng) was digested using double-stranded DNA (dsDNA) Fragmentase (NEB) for exactly 12 min to create fragments that were between 500 bp and 3,000 bp. .. To facilitate the PCR amplification of transposon insertion junctions, cytosine was added to the ends of the fragments using terminal transferase (NEB).

    Article Title: Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone
    Article Snippet: Recovered infectious clone virus was sequenced using amplicon-based RNA sequencing with next-generation sequencing (NGS). .. DNA was then fragmented with double-stranded DNA (dsDNA) fragmentase (NEB) for 15 min at 37°C.

    RNA Sequencing Assay:

    Article Title: Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone
    Article Snippet: Recovered infectious clone virus was sequenced using amplicon-based RNA sequencing with next-generation sequencing (NGS). .. DNA was then fragmented with double-stranded DNA (dsDNA) fragmentase (NEB) for 15 min at 37°C.

    In Vitro:

    Article Title: Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone
    Article Snippet: Paragraph title: In vitro transcription and transfection. ... DNA was then fragmented with double-stranded DNA (dsDNA) fragmentase (NEB) for 15 min at 37°C.

    Isolation:

    Article Title: Genome-Wide Identification of Fitness Factors in Mastitis-Associated Escherichia coli
    Article Snippet: Genomic DNA (150 ng) was digested using double-stranded DNA (dsDNA) Fragmentase (NEB) for exactly 12 min to create fragments that were between 500 bp and 3,000 bp. .. Genomic DNA fragments were isolated and cleaned using a QIAprep Spin Miniprep kit (Qiagen).

    Article Title: Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone
    Article Snippet: For NGS, RNA was isolated from recovered virus, cDNA was produced, and PCR was performed to amplify amplicons for sequencing. .. DNA was then fragmented with double-stranded DNA (dsDNA) fragmentase (NEB) for 15 min at 37°C.

    Next-Generation Sequencing:

    Article Title: Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone
    Article Snippet: For NGS, RNA was isolated from recovered virus, cDNA was produced, and PCR was performed to amplify amplicons for sequencing. .. DNA was then fragmented with double-stranded DNA (dsDNA) fragmentase (NEB) for 15 min at 37°C.

    Sequencing:

    Article Title: Genome-Wide Identification of Fitness Factors in Mastitis-Associated Escherichia coli
    Article Snippet: Paragraph title: DNA preparation and Illumina sequencing. ... Genomic DNA (150 ng) was digested using double-stranded DNA (dsDNA) Fragmentase (NEB) for exactly 12 min to create fragments that were between 500 bp and 3,000 bp.

    Article Title: Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone
    Article Snippet: For NGS, RNA was isolated from recovered virus, cDNA was produced, and PCR was performed to amplify amplicons for sequencing. .. DNA was then fragmented with double-stranded DNA (dsDNA) fragmentase (NEB) for 15 min at 37°C.

    Purification:

    Article Title: Inhibitors of RecA Activity Discovered by High-Throughput Screening: Cell-Permeable Small Molecules Attenuate the SOS Response in Escherichia coli
    Article Snippet: RecA was purified and stored as previously described. .. Crystalline L-ascorbic acid and sulfuric acid were purchased from Fisher Scientific. ϕχ174 circular single-stranded DNA (cssDNA) and double stranded DNA (dsDNA) were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone
    Article Snippet: DNA was then fragmented with double-stranded DNA (dsDNA) fragmentase (NEB) for 15 min at 37°C. .. Fragmented DNA was then purified with AMPure XP beads (1.2 bead/DNA ratio) before library preparation with the NEBNext Ultra II DNA library prep kit for Illumina sequencing in accordance with the manufacturer's protocol.

    Produced:

    Article Title: Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone
    Article Snippet: For NGS, RNA was isolated from recovered virus, cDNA was produced, and PCR was performed to amplify amplicons for sequencing. .. DNA was then fragmented with double-stranded DNA (dsDNA) fragmentase (NEB) for 15 min at 37°C.

    Concentration Assay:

    Article Title: Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone
    Article Snippet: On day 8 postelectroporation, significant CPE was observed and supernatant was harvested, centrifuged to remove cellular debris, and supplemented to a final concentration of 20% fetal bovine serum (FBS) and 10 mM HEPES before being frozen in single-use aliquots. .. DNA was then fragmented with double-stranded DNA (dsDNA) fragmentase (NEB) for 15 min at 37°C.

    Article Title: Quantification of M13 and T7 bacteriophages by TaqMan and SYBR Green qPCR
    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. .. T7 packaging control dsDNA (Millipore Sigma, 37314 bp, 0.1μg/μL) was diluted into a serial concentration of 1 fg/μL - 107 fg/μL, which was used to prepare the standard curve for titers of genome copies of T7 phages.

    Polymerase Chain Reaction:

    Article Title: Genome-Wide Identification of Fitness Factors in Mastitis-Associated Escherichia coli
    Article Snippet: Genomic DNA (150 ng) was digested using double-stranded DNA (dsDNA) Fragmentase (NEB) for exactly 12 min to create fragments that were between 500 bp and 3,000 bp. .. To facilitate the PCR amplification of transposon insertion junctions, cytosine was added to the ends of the fragments using terminal transferase (NEB).

    Article Title: Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone
    Article Snippet: For NGS, RNA was isolated from recovered virus, cDNA was produced, and PCR was performed to amplify amplicons for sequencing. .. DNA was then fragmented with double-stranded DNA (dsDNA) fragmentase (NEB) for 15 min at 37°C.

    Sample Prep:

    Article Title: Quantification of M13 and T7 bacteriophages by TaqMan and SYBR Green qPCR
    Article Snippet: Paragraph title: 2.2.1 DNA sample preparation ... 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.

    Variant Assay:

    Article Title: Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands
    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. .. Reactions contained 100 nM Hel308a or its variant K51L, 100 ng DNA or RNA and were at 45°C for the time specified in the figures.

    Plaque Assay:

    Article Title: Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone
    Article Snippet: The titer was measured using the plaque assay on Vero cells. .. DNA was then fragmented with double-stranded DNA (dsDNA) fragmentase (NEB) for 15 min at 37°C.

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    New England Biolabs double stranded dna dsdna
    CRISPR adaptation to HHPV-2 infection. ( A ) Depiction of the single CRISPR structure and the preceding cas operon carried by the H. hispanica ATCC 33960 genome. Primers used to examine CRISPR expansion (in panel B) are shown as black arrows and listed in Supplementary Table S2 . ( B ) PCR assay to detect CRISPR expansion at the leader end (L1–L2), the inner part (I1–I2) or the distal end (D1–D2). <t>DNA</t> sampled from infected (+) or uninfected (−) cells was used as PCR templates. Lane M, <t>dsDNA</t> size marker. ( C ) The sequence logo showing the conserved PAM of TTC. The 20 nt upstream of each protospacer observed during HHPV-2 infection were collected and analyzed with WebLogo ( http://weblogo.berkeley.edu/logo.cgi ).
    Double Stranded Dna Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 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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    New England Biolabs rna dependent dna recombinational repair rad52 dependent rna bridging reactions
    <t>RAD52</t> promotes <t>RNA-dependent</t> <t>DNA</t> recombination. a Schematic of assay (left). Non-denaturing gels showing RAD52 RNA−DNA recombination (RNA-bridging of homologous DNA) in the presence of the indicated substrates (right). b Schematic of assay (left). Non-denaturing gel showing RNase H digestion of a RAD52-mediated RNA−DNA recombination intermediate (RNA−DNA recombinant bridge) (right). c Graph showing a time course of RNA–DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking ssDNA without RPA and in the presence and absence of RAD52. Data shown as average ± SD, n = 3. d Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination in the presence of the indicated RPA-coated substrates (right). e Graph showing a time course of RNA−DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking RPA-bound ssDNA in the presence and absence of RAD52. Data shown as average ± SD, n = 3. f Schematic of assay (left). Non-denaturing gel showing RAD51 RNA−DNA recombination (bridging) in the presence of RPA pre-coated substrates (right). g Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated pssDNA substrates (right). h Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated RPA-coated pssDNA substrates (right). * = 32 P label. % bridging indicated
    Rna Dependent Dna Recombinational Repair Rad52 Dependent Rna Bridging Reactions, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CRISPR adaptation to HHPV-2 infection. ( A ) Depiction of the single CRISPR structure and the preceding cas operon carried by the H. hispanica ATCC 33960 genome. Primers used to examine CRISPR expansion (in panel B) are shown as black arrows and listed in Supplementary Table S2 . ( B ) PCR assay to detect CRISPR expansion at the leader end (L1–L2), the inner part (I1–I2) or the distal end (D1–D2). DNA sampled from infected (+) or uninfected (−) cells was used as PCR templates. Lane M, dsDNA size marker. ( C ) The sequence logo showing the conserved PAM of TTC. The 20 nt upstream of each protospacer observed during HHPV-2 infection were collected and analyzed with WebLogo ( http://weblogo.berkeley.edu/logo.cgi ).

    Journal: Nucleic Acids Research

    Article Title: Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process

    doi: 10.1093/nar/gkt1154

    Figure Lengend Snippet: CRISPR adaptation to HHPV-2 infection. ( A ) Depiction of the single CRISPR structure and the preceding cas operon carried by the H. hispanica ATCC 33960 genome. Primers used to examine CRISPR expansion (in panel B) are shown as black arrows and listed in Supplementary Table S2 . ( B ) PCR assay to detect CRISPR expansion at the leader end (L1–L2), the inner part (I1–I2) or the distal end (D1–D2). DNA sampled from infected (+) or uninfected (−) cells was used as PCR templates. Lane M, dsDNA size marker. ( C ) The sequence logo showing the conserved PAM of TTC. The 20 nt upstream of each protospacer observed during HHPV-2 infection were collected and analyzed with WebLogo ( http://weblogo.berkeley.edu/logo.cgi ).

    Article Snippet: The single-stranded DNA (ssDNA) (ФX174ss) and double-stranded DNA (dsDNA) (ФX174ds) from phiX174 phage (purchased from New England Biolabs) were used as controls.

    Techniques: CRISPR, Infection, Polymerase Chain Reaction, Marker, Sequencing

    Adaptation to HHPV-2 infection under different cas genetic backgrounds. ( A ) Cas requirement for adaptation. For each cas mutant, DNA was sampled from cells transformed with an empty plasmid (−) or the plasmid carrying the deleted cas gene(s) (+). The plasmid-carried cas gene(s) was/were under the control of the cas operon promoter. ( B ) Requirements for the nuclease and helicase activities of Cas3. Alanine replacement was performed for the putative key residues in the HD nuclease domain (H20A, H55A, D56A and D229A) and the DExD/H helicase domain (K315A, D439A and E440A). Another two conserved residues (His6 and Lys113) were also mutated. The empty plasmid (−) and the plasmid carrying a wild-type Cas3 (Cas3 WT ) were used, respectively, as negative and positive controls. Lane Ms, dsDNA size markers.

    Journal: Nucleic Acids Research

    Article Title: Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process

    doi: 10.1093/nar/gkt1154

    Figure Lengend Snippet: Adaptation to HHPV-2 infection under different cas genetic backgrounds. ( A ) Cas requirement for adaptation. For each cas mutant, DNA was sampled from cells transformed with an empty plasmid (−) or the plasmid carrying the deleted cas gene(s) (+). The plasmid-carried cas gene(s) was/were under the control of the cas operon promoter. ( B ) Requirements for the nuclease and helicase activities of Cas3. Alanine replacement was performed for the putative key residues in the HD nuclease domain (H20A, H55A, D56A and D229A) and the DExD/H helicase domain (K315A, D439A and E440A). Another two conserved residues (His6 and Lys113) were also mutated. The empty plasmid (−) and the plasmid carrying a wild-type Cas3 (Cas3 WT ) were used, respectively, as negative and positive controls. Lane Ms, dsDNA size markers.

    Article Snippet: The single-stranded DNA (ssDNA) (ФX174ss) and double-stranded DNA (dsDNA) (ФX174ds) from phiX174 phage (purchased from New England Biolabs) were used as controls.

    Techniques: Infection, Mutagenesis, Transformation Assay, Plasmid Preparation, Mass Spectrometry

    BCCIPβ binds DNA. ( A ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) incubated with ϕX174 (+) ssDNA (ss; 30 μM nucleotides). ( B ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) was incubated with ϕX174 RF (I) dsDNA (ds; 30 μM base pairs). The reaction products were separated on a 1.0% agarose gel, and were stained with ethidium bromide. Lane 1 contained no protein, and lane 8 was deproteinized with SDS and Proteinase K (S/P) prior to loading.

    Journal: Nucleic Acids Research

    Article Title: The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing

    doi: 10.1093/nar/gkw877

    Figure Lengend Snippet: BCCIPβ binds DNA. ( A ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) incubated with ϕX174 (+) ssDNA (ss; 30 μM nucleotides). ( B ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) was incubated with ϕX174 RF (I) dsDNA (ds; 30 μM base pairs). The reaction products were separated on a 1.0% agarose gel, and were stained with ethidium bromide. Lane 1 contained no protein, and lane 8 was deproteinized with SDS and Proteinase K (S/P) prior to loading.

    Article Snippet: All oligonucleotides were purchased from Integrated DNA Technologies. pBluescript was purified from E. coli using a Giga Kit (Qiagen). ϕX174 (+) virion ssDNA and ϕX174 replicative form I double-stranded DNA (dsDNA) were purchased from New England BioLabs—ϕX174 dsDNA was linearized with ApaLI (New England BioLabs).

    Techniques: Incubation, Agarose Gel Electrophoresis, Staining

    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

    RAD52 promotes RNA-dependent DNA recombination. a Schematic of assay (left). Non-denaturing gels showing RAD52 RNA−DNA recombination (RNA-bridging of homologous DNA) in the presence of the indicated substrates (right). b Schematic of assay (left). Non-denaturing gel showing RNase H digestion of a RAD52-mediated RNA−DNA recombination intermediate (RNA−DNA recombinant bridge) (right). c Graph showing a time course of RNA–DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking ssDNA without RPA and in the presence and absence of RAD52. Data shown as average ± SD, n = 3. d Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination in the presence of the indicated RPA-coated substrates (right). e Graph showing a time course of RNA−DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking RPA-bound ssDNA in the presence and absence of RAD52. Data shown as average ± SD, n = 3. f Schematic of assay (left). Non-denaturing gel showing RAD51 RNA−DNA recombination (bridging) in the presence of RPA pre-coated substrates (right). g Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated pssDNA substrates (right). h Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated RPA-coated pssDNA substrates (right). * = 32 P label. % bridging indicated

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: RAD52 promotes RNA-dependent DNA recombination. a Schematic of assay (left). Non-denaturing gels showing RAD52 RNA−DNA recombination (RNA-bridging of homologous DNA) in the presence of the indicated substrates (right). b Schematic of assay (left). Non-denaturing gel showing RNase H digestion of a RAD52-mediated RNA−DNA recombination intermediate (RNA−DNA recombinant bridge) (right). c Graph showing a time course of RNA–DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking ssDNA without RPA and in the presence and absence of RAD52. Data shown as average ± SD, n = 3. d Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination in the presence of the indicated RPA-coated substrates (right). e Graph showing a time course of RNA−DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking RPA-bound ssDNA in the presence and absence of RAD52. Data shown as average ± SD, n = 3. f Schematic of assay (left). Non-denaturing gel showing RAD51 RNA−DNA recombination (bridging) in the presence of RPA pre-coated substrates (right). g Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated pssDNA substrates (right). h Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated RPA-coated pssDNA substrates (right). * = 32 P label. % bridging indicated

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Recombinant, Recombinase Polymerase Amplification

    RAD52 promotes RNA-dependent recombinational repair of DSBs. a Schematic of assay (left). Non-denaturing gel showing a time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (middle). Plot showing time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (right). Data shown as average ± SEM, n = 3. b Schematic of assay (left). Non-denaturing gels showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence (left) and absence (right) of RPA. c Schematic of assays showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA employing either RAD52-dsDNA pre-incubation (right schematic) or RAD52-RNA (left schematic) pre-incubation steps, and performed either with and without RPA. Graph showing quantification of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA utilizing the indicated pre-incubation steps and with and without RPA (right). Data shown as average ± SD, n = 3. d Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA recombinational repair (bridging followed by ligation) of blunt-ended DNA in the presence of the indicated proteins and substrates (middle). Graph showing percent of RAD52-dependent RNA-mediated recombinational repair of blunt-ended DNA (% ligation) (right). Data shown as average ± SD, n = 3. ***, p = 0.0008 (unpaired Student’s t- test). * = 32 P label

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: RAD52 promotes RNA-dependent recombinational repair of DSBs. a Schematic of assay (left). Non-denaturing gel showing a time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (middle). Plot showing time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (right). Data shown as average ± SEM, n = 3. b Schematic of assay (left). Non-denaturing gels showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence (left) and absence (right) of RPA. c Schematic of assays showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA employing either RAD52-dsDNA pre-incubation (right schematic) or RAD52-RNA (left schematic) pre-incubation steps, and performed either with and without RPA. Graph showing quantification of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA utilizing the indicated pre-incubation steps and with and without RPA (right). Data shown as average ± SD, n = 3. d Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA recombinational repair (bridging followed by ligation) of blunt-ended DNA in the presence of the indicated proteins and substrates (middle). Graph showing percent of RAD52-dependent RNA-mediated recombinational repair of blunt-ended DNA (% ligation) (right). Data shown as average ± SD, n = 3. ***, p = 0.0008 (unpaired Student’s t- test). * = 32 P label

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Recombinase Polymerase Amplification, Incubation, Ligation, DNA Ligation

    RAD52 promotes RNA transcript-dependent DNA recombinational repair. a Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of left and right ssDNA flanks and the indicated proteins (right). b Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (right). c Schematic of assay (left). Denaturing gel showing RAD52-mediated RNA transcript-dependent DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (middle). Graph showing percent of RNA transcript-dependent DNA recombinational repair (right). Data shown as average ± SD, n = 3. *, p = 0.016 (unpaired Student’s t -test). Sequencing chromatogram of RNA transcript-dependent DNA recombinational repair product (bottom). * = 32 P label

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: RAD52 promotes RNA transcript-dependent DNA recombinational repair. a Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of left and right ssDNA flanks and the indicated proteins (right). b Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (right). c Schematic of assay (left). Denaturing gel showing RAD52-mediated RNA transcript-dependent DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (middle). Graph showing percent of RNA transcript-dependent DNA recombinational repair (right). Data shown as average ± SD, n = 3. *, p = 0.016 (unpaired Student’s t -test). Sequencing chromatogram of RNA transcript-dependent DNA recombinational repair product (bottom). * = 32 P label

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Recombinase Polymerase Amplification, Sequencing

    Models of RAD52-mediated RNA−DNA repair. a RNA-bridging DSB repair model. RAD52 utilizes RNA to tether both ends of a homologous DSB which forms a DNA synapse for ligation. RNA degradation by RNase H may also occur. b RNA-templated DSB repair model. RAD52 forms an RNA−DNA hybrid along the 3′ overhang of a DSB. The RNA is then used as a template for DNA repair synthesis by RT. The RNA is then degraded by RNase H and RAD52 promotes SSA of the opposing homologous ssDNA overhangs. Final processing of the DSB involves gap filling and ligation

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: Models of RAD52-mediated RNA−DNA repair. a RNA-bridging DSB repair model. RAD52 utilizes RNA to tether both ends of a homologous DSB which forms a DNA synapse for ligation. RNA degradation by RNase H may also occur. b RNA-templated DSB repair model. RAD52 forms an RNA−DNA hybrid along the 3′ overhang of a DSB. The RNA is then used as a template for DNA repair synthesis by RT. The RNA is then degraded by RNase H and RAD52 promotes SSA of the opposing homologous ssDNA overhangs. Final processing of the DSB involves gap filling and ligation

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Ligation

    RAD52 promotes RNA transcript-templated DNA recombination. a Schematic of assay (left). Denaturing gel showing reverse transcription of a RNA−DNA recombinant half-bridge in the presence of the indicated proteins and RNA (middle). Graph showing percent extension of a RNA−DNA recombinant half-bridge by RT in the presence and absence of RAD52 (right). Data shown as average ± SD, n = 4, ***, p

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: RAD52 promotes RNA transcript-templated DNA recombination. a Schematic of assay (left). Denaturing gel showing reverse transcription of a RNA−DNA recombinant half-bridge in the presence of the indicated proteins and RNA (middle). Graph showing percent extension of a RNA−DNA recombinant half-bridge by RT in the presence and absence of RAD52 (right). Data shown as average ± SD, n = 4, ***, p

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Recombinant