dsdna  (New England Biolabs)


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    Structured Review

    New England Biolabs dsdna
    The C-terminal domain of hRAD52 but not of yRad52 is dispensable for <t>ssDNA</t> annealing. (A) Illustration of ssDNA annealing assay. The reaction contained DAPI, fluorescence of which increased upon binding to the <t>dsDNA</t> product. (B and D) Sixteen nanomolar of yRad52, yRad52NM, hRAD52, or hRAD52NM was added to complementary 70-nt ssDNA (2.86 nM each) that were pre-complexed with their cognate RPA (36 nM). (C and E). The same reactions as in B and D were repeated with various concentrations of the Rad52 derivatives and the initial rates of the reactions were plotted with the concentrations of Rad52 derivatives. Error bases are STD (n = 3 to 4).
    Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing"

    Article Title: Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158436

    The C-terminal domain of hRAD52 but not of yRad52 is dispensable for ssDNA annealing. (A) Illustration of ssDNA annealing assay. The reaction contained DAPI, fluorescence of which increased upon binding to the dsDNA product. (B and D) Sixteen nanomolar of yRad52, yRad52NM, hRAD52, or hRAD52NM was added to complementary 70-nt ssDNA (2.86 nM each) that were pre-complexed with their cognate RPA (36 nM). (C and E). The same reactions as in B and D were repeated with various concentrations of the Rad52 derivatives and the initial rates of the reactions were plotted with the concentrations of Rad52 derivatives. Error bases are STD (n = 3 to 4).
    Figure Legend Snippet: The C-terminal domain of hRAD52 but not of yRad52 is dispensable for ssDNA annealing. (A) Illustration of ssDNA annealing assay. The reaction contained DAPI, fluorescence of which increased upon binding to the dsDNA product. (B and D) Sixteen nanomolar of yRad52, yRad52NM, hRAD52, or hRAD52NM was added to complementary 70-nt ssDNA (2.86 nM each) that were pre-complexed with their cognate RPA (36 nM). (C and E). The same reactions as in B and D were repeated with various concentrations of the Rad52 derivatives and the initial rates of the reactions were plotted with the concentrations of Rad52 derivatives. Error bases are STD (n = 3 to 4).

    Techniques Used: Fluorescence, Binding Assay, Recombinase Polymerase Amplification

    Mediator assay of yRad52 with hRAD51. (A) Illustration of the DNA strand exchange experiment to analyze the mediator activity. ΦX-174 ssDNA was incubated with yRPA, yRad52, hRAD51, and then with ΦX-174 dsDNA in the indicated order. The reaction produced joint molecules (jm) and a nicked circular (nc) dsDNA. (B) Derivatives of yRad52. N-terminal (N), middle (M), and C-terminal (C) region have been described in previous paper [ 18 ]. For bottom three constructs, yRad52NM was fused with a human BRC4 (NM-BRC4), BRC3-BRC4 (NM-BRC3-4), or three repeats of BRC4 (NM-BRC4 x3 ). (C) Same amount (2 μg) of purified yRad52 and its derivatives were separated by SDS-PAGE and stained with coomassie brilliant blue. Asterisk indicates a contaminating protein present in all preparations. (D and E) DNA strand exchange was performed in the absence (lane 2) or presence of 1, 2, 3, 4 μM (lane 3 to 6) of yRad52 (D) or yRad52NM (E). DNA products were separated through agarose gel and visualized with ethidium bromide staining. Lane 1 shows a control reaction without any protein. (F) The products (nicked circles (nc) and joint molecules (jm)) were quantified from D (yRad52) and E (yRad52NM) and repeated experiments and relative product formation was plotted against the mediator concentration. Product formation in the absence of the mediator was 31.2%, which was defined as 1.0. Error bars are standard deviations (n = 3). (G) hRAD51 and His-tagged yRad52 were mixed as indicated and precipitated with Ni-beads. Proteins on the beads were then eluted and analyzed by SDS-PAGE.
    Figure Legend Snippet: Mediator assay of yRad52 with hRAD51. (A) Illustration of the DNA strand exchange experiment to analyze the mediator activity. ΦX-174 ssDNA was incubated with yRPA, yRad52, hRAD51, and then with ΦX-174 dsDNA in the indicated order. The reaction produced joint molecules (jm) and a nicked circular (nc) dsDNA. (B) Derivatives of yRad52. N-terminal (N), middle (M), and C-terminal (C) region have been described in previous paper [ 18 ]. For bottom three constructs, yRad52NM was fused with a human BRC4 (NM-BRC4), BRC3-BRC4 (NM-BRC3-4), or three repeats of BRC4 (NM-BRC4 x3 ). (C) Same amount (2 μg) of purified yRad52 and its derivatives were separated by SDS-PAGE and stained with coomassie brilliant blue. Asterisk indicates a contaminating protein present in all preparations. (D and E) DNA strand exchange was performed in the absence (lane 2) or presence of 1, 2, 3, 4 μM (lane 3 to 6) of yRad52 (D) or yRad52NM (E). DNA products were separated through agarose gel and visualized with ethidium bromide staining. Lane 1 shows a control reaction without any protein. (F) The products (nicked circles (nc) and joint molecules (jm)) were quantified from D (yRad52) and E (yRad52NM) and repeated experiments and relative product formation was plotted against the mediator concentration. Product formation in the absence of the mediator was 31.2%, which was defined as 1.0. Error bars are standard deviations (n = 3). (G) hRAD51 and His-tagged yRad52 were mixed as indicated and precipitated with Ni-beads. Proteins on the beads were then eluted and analyzed by SDS-PAGE.

    Techniques Used: Activity Assay, Incubation, Produced, Construct, Purification, SDS Page, Staining, Agarose Gel Electrophoresis, Concentration Assay

    2) Product Images from "Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing"

    Article Title: Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158436

    The C-terminal domain of hRAD52 but not of yRad52 is dispensable for ssDNA annealing. (A) Illustration of ssDNA annealing assay. The reaction contained DAPI, fluorescence of which increased upon binding to the dsDNA product. (B and D) Sixteen nanomolar of yRad52, yRad52NM, hRAD52, or hRAD52NM was added to complementary 70-nt ssDNA (2.86 nM each) that were pre-complexed with their cognate RPA (36 nM). (C and E). The same reactions as in B and D were repeated with various concentrations of the Rad52 derivatives and the initial rates of the reactions were plotted with the concentrations of Rad52 derivatives. Error bases are STD (n = 3 to 4).
    Figure Legend Snippet: The C-terminal domain of hRAD52 but not of yRad52 is dispensable for ssDNA annealing. (A) Illustration of ssDNA annealing assay. The reaction contained DAPI, fluorescence of which increased upon binding to the dsDNA product. (B and D) Sixteen nanomolar of yRad52, yRad52NM, hRAD52, or hRAD52NM was added to complementary 70-nt ssDNA (2.86 nM each) that were pre-complexed with their cognate RPA (36 nM). (C and E). The same reactions as in B and D were repeated with various concentrations of the Rad52 derivatives and the initial rates of the reactions were plotted with the concentrations of Rad52 derivatives. Error bases are STD (n = 3 to 4).

    Techniques Used: Fluorescence, Binding Assay, Recombinase Polymerase Amplification

    Mediator assay of yRad52 with hRAD51. (A) Illustration of the DNA strand exchange experiment to analyze the mediator activity. ΦX-174 ssDNA was incubated with yRPA, yRad52, hRAD51, and then with ΦX-174 dsDNA in the indicated order. The reaction produced joint molecules (jm) and a nicked circular (nc) dsDNA. (B) Derivatives of yRad52. N-terminal (N), middle (M), and C-terminal (C) region have been described in previous paper [ 18 ]. For bottom three constructs, yRad52NM was fused with a human BRC4 (NM-BRC4), BRC3-BRC4 (NM-BRC3-4), or three repeats of BRC4 (NM-BRC4 x3 ). (C) Same amount (2 μg) of purified yRad52 and its derivatives were separated by SDS-PAGE and stained with coomassie brilliant blue. Asterisk indicates a contaminating protein present in all preparations. (D and E) DNA strand exchange was performed in the absence (lane 2) or presence of 1, 2, 3, 4 μM (lane 3 to 6) of yRad52 (D) or yRad52NM (E). DNA products were separated through agarose gel and visualized with ethidium bromide staining. Lane 1 shows a control reaction without any protein. (F) The products (nicked circles (nc) and joint molecules (jm)) were quantified from D (yRad52) and E (yRad52NM) and repeated experiments and relative product formation was plotted against the mediator concentration. Product formation in the absence of the mediator was 31.2%, which was defined as 1.0. Error bars are standard deviations (n = 3). (G) hRAD51 and His-tagged yRad52 were mixed as indicated and precipitated with Ni-beads. Proteins on the beads were then eluted and analyzed by SDS-PAGE.
    Figure Legend Snippet: Mediator assay of yRad52 with hRAD51. (A) Illustration of the DNA strand exchange experiment to analyze the mediator activity. ΦX-174 ssDNA was incubated with yRPA, yRad52, hRAD51, and then with ΦX-174 dsDNA in the indicated order. The reaction produced joint molecules (jm) and a nicked circular (nc) dsDNA. (B) Derivatives of yRad52. N-terminal (N), middle (M), and C-terminal (C) region have been described in previous paper [ 18 ]. For bottom three constructs, yRad52NM was fused with a human BRC4 (NM-BRC4), BRC3-BRC4 (NM-BRC3-4), or three repeats of BRC4 (NM-BRC4 x3 ). (C) Same amount (2 μg) of purified yRad52 and its derivatives were separated by SDS-PAGE and stained with coomassie brilliant blue. Asterisk indicates a contaminating protein present in all preparations. (D and E) DNA strand exchange was performed in the absence (lane 2) or presence of 1, 2, 3, 4 μM (lane 3 to 6) of yRad52 (D) or yRad52NM (E). DNA products were separated through agarose gel and visualized with ethidium bromide staining. Lane 1 shows a control reaction without any protein. (F) The products (nicked circles (nc) and joint molecules (jm)) were quantified from D (yRad52) and E (yRad52NM) and repeated experiments and relative product formation was plotted against the mediator concentration. Product formation in the absence of the mediator was 31.2%, which was defined as 1.0. Error bars are standard deviations (n = 3). (G) hRAD51 and His-tagged yRad52 were mixed as indicated and precipitated with Ni-beads. Proteins on the beads were then eluted and analyzed by SDS-PAGE.

    Techniques Used: Activity Assay, Incubation, Produced, Construct, Purification, SDS Page, Staining, Agarose Gel Electrophoresis, Concentration Assay

    Related Articles

    Clone Assay:

    Article Title: RecA requires two molecules of Mg2+ ions for its optimal strand exchange activity in vitro
    Article Snippet: The recA gene was cloned into a His-smt3 fusion protein expression vector, a gift from Dr. T.-F. Wang. .. RecA ΔC-tail was then purified by using TOYOPEARL Butyl-650M column (TOSOH), P11 column (Whatman) and MonoQ 5/50 GL column (GE Healthcare). ϕX174 circular ssDNA and dsDNA were purchased from New England Biolabs.

    Cell Culture:

    Article Title: RecA requires two molecules of Mg2+ ions for its optimal strand exchange activity in vitro
    Article Snippet: The protein was expressed in E. coli BL21 ΔrecA (DE3) cells transformed with the pET3a-RecA ΔC-tail vector, induced with IPTG (0.5 mM) and cultured for 5 h at 37°C. .. RecA ΔC-tail was then purified by using TOYOPEARL Butyl-650M column (TOSOH), P11 column (Whatman) and MonoQ 5/50 GL column (GE Healthcare). ϕX174 circular ssDNA and dsDNA were purchased from New England Biolabs.

    Purification:

    Article Title: RecA requires two molecules of Mg2+ ions for its optimal strand exchange activity in vitro
    Article Snippet: .. RecA ΔC-tail was then purified by using TOYOPEARL Butyl-650M column (TOSOH), P11 column (Whatman) and MonoQ 5/50 GL column (GE Healthcare). ϕX174 circular ssDNA and dsDNA were purchased from New England Biolabs. ..

    Incubation:

    Article Title: Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing
    Article Snippet: .. Mediator assay ΦX174 phage circular ssDNA and dsDNA were purchased from New England Biolabs and the dsDNA was linearized using Xho I. RPA and ssDNA were incubated for 2 minutes at 37°C in buffer containing 40 mM Tris-HCl (pH 7.5), 1 mM DTT, 2 mM ATP, 1 mM MgCl2 , 8 mM creatine phosphate, and 28 μg/ml creatine phosphokinase. .. Then Rad52 or its derivatives was added to the reaction and incubation continued for 3 min. Then hRAD51 was added to start hRAD51-ssDNA filament formation.

    Expressing:

    Article Title: RecA requires two molecules of Mg2+ ions for its optimal strand exchange activity in vitro
    Article Snippet: The recA gene was cloned into a His-smt3 fusion protein expression vector, a gift from Dr. T.-F. Wang. .. RecA ΔC-tail was then purified by using TOYOPEARL Butyl-650M column (TOSOH), P11 column (Whatman) and MonoQ 5/50 GL column (GE Healthcare). ϕX174 circular ssDNA and dsDNA were purchased from New England Biolabs.

    Transformation Assay:

    Article Title: RecA requires two molecules of Mg2+ ions for its optimal strand exchange activity in vitro
    Article Snippet: The protein was expressed in E. coli BL21 ΔrecA (DE3) cells transformed with the pET3a-RecA ΔC-tail vector, induced with IPTG (0.5 mM) and cultured for 5 h at 37°C. .. RecA ΔC-tail was then purified by using TOYOPEARL Butyl-650M column (TOSOH), P11 column (Whatman) and MonoQ 5/50 GL column (GE Healthcare). ϕX174 circular ssDNA and dsDNA were purchased from New England Biolabs.

    Recombinase Polymerase Amplification:

    Article Title: Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing
    Article Snippet: .. Mediator assay ΦX174 phage circular ssDNA and dsDNA were purchased from New England Biolabs and the dsDNA was linearized using Xho I. RPA and ssDNA were incubated for 2 minutes at 37°C in buffer containing 40 mM Tris-HCl (pH 7.5), 1 mM DTT, 2 mM ATP, 1 mM MgCl2 , 8 mM creatine phosphate, and 28 μg/ml creatine phosphokinase. .. Then Rad52 or its derivatives was added to the reaction and incubation continued for 3 min. Then hRAD51 was added to start hRAD51-ssDNA filament formation.

    Plasmid Preparation:

    Article Title: RecA requires two molecules of Mg2+ ions for its optimal strand exchange activity in vitro
    Article Snippet: The protein was expressed in E. coli BL21 ΔrecA (DE3) cells transformed with the pET3a-RecA ΔC-tail vector, induced with IPTG (0.5 mM) and cultured for 5 h at 37°C. .. RecA ΔC-tail was then purified by using TOYOPEARL Butyl-650M column (TOSOH), P11 column (Whatman) and MonoQ 5/50 GL column (GE Healthcare). ϕX174 circular ssDNA and dsDNA were purchased from New England Biolabs.

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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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    New England Biolabs 2 7 kb dsdna substrate
    Sgs1 does not stimulate resection of <t>dsDNA</t> by Exo1. ( A ) Nuclease assays with Exo1 (0.35, 0.53, 0.8, 1.2, and 1.8 nM), RPA (0.4 μM), and either without (lanes 2–6) or with Sgs1 (0.1 nM, lanes 8–13) in low-salt buffer. Blunt-ended pUC19 dsDNA (1 nM), 32 P labeled at the 3′ end, was used. ( B ) Quantification of experiments as shown in A . Error bars show SE. ( C ) Nuclease assays with Exo1 (0.53, 0.8, 1.2, 1.8, and <t>2.7</t> nM), RPA (0.4 μM), and either without (lanes 2–6) or with Sgs1 (0.5 nM) and Top3-Rmi1 (5 nM, lanes 9–14, respectively), in standard buffer. Substrate is as in A . ( D ) Quantification of experiments as shown in C . Error bars show SE. ( E ) Nuclease assay carried out with Exo1 (0.5, 1, 2, 3, and 4 nM), RPA (0.4 μM), and either without (lanes 2–6) or with helicase-dead Sgs1 K706A (20 nM, lanes 8–12). Substrate is as in A . ( F ) Increasing amounts of nuclease-dead Exo1 D173A (0.53, 0.8, 1.2, 1.8, 2.7, 4, and 8 nM) were added to reactions containing Sgs1 (0.5 nM) and/or Top3-Rmi1 (5 nM), as indicated, in the presence of RPA (0.4 μM). Substrate is as in A .
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    New England Biolabs dsdna donor cassettes
    Modification of 5’ ends of long <t>dsDNA</t> fragments promotes HDR-mediated single-copy integration. ( A ) GFP expression in the respective expression domain after HDR-mediated integration of modified dsDNA gfp donor cassettes into rx2 , rx1 , actb and dnmt1 ORFs in the injected generation. ( B ) Individual embryo <t>PCR</t> genotyping highlights efficient HDR-mediated single-copy integration of 5’Biotin modified long dsDNA donors, but not unmodified donor cassettes. Locus PCR reveals band size indicative of single-copy gfp integration (asterisk) besides alleles without gfp integration (open arrowhead). Amplification of gfp donor (white arrow) for control.
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    New England Biolabs m13mp18 dna sequencing standard m13mp18 dsdna
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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

    Sgs1 does not stimulate resection of dsDNA by Exo1. ( A ) Nuclease assays with Exo1 (0.35, 0.53, 0.8, 1.2, and 1.8 nM), RPA (0.4 μM), and either without (lanes 2–6) or with Sgs1 (0.1 nM, lanes 8–13) in low-salt buffer. Blunt-ended pUC19 dsDNA (1 nM), 32 P labeled at the 3′ end, was used. ( B ) Quantification of experiments as shown in A . Error bars show SE. ( C ) Nuclease assays with Exo1 (0.53, 0.8, 1.2, 1.8, and 2.7 nM), RPA (0.4 μM), and either without (lanes 2–6) or with Sgs1 (0.5 nM) and Top3-Rmi1 (5 nM, lanes 9–14, respectively), in standard buffer. Substrate is as in A . ( D ) Quantification of experiments as shown in C . Error bars show SE. ( E ) Nuclease assay carried out with Exo1 (0.5, 1, 2, 3, and 4 nM), RPA (0.4 μM), and either without (lanes 2–6) or with helicase-dead Sgs1 K706A (20 nM, lanes 8–12). Substrate is as in A . ( F ) Increasing amounts of nuclease-dead Exo1 D173A (0.53, 0.8, 1.2, 1.8, 2.7, 4, and 8 nM) were added to reactions containing Sgs1 (0.5 nM) and/or Top3-Rmi1 (5 nM), as indicated, in the presence of RPA (0.4 μM). Substrate is as in A .

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

    Article Title: Relationship of DNA degradation by Saccharomyces cerevisiae Exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection

    doi: 10.1073/pnas.1305166110

    Figure Lengend Snippet: Sgs1 does not stimulate resection of dsDNA by Exo1. ( A ) Nuclease assays with Exo1 (0.35, 0.53, 0.8, 1.2, and 1.8 nM), RPA (0.4 μM), and either without (lanes 2–6) or with Sgs1 (0.1 nM, lanes 8–13) in low-salt buffer. Blunt-ended pUC19 dsDNA (1 nM), 32 P labeled at the 3′ end, was used. ( B ) Quantification of experiments as shown in A . Error bars show SE. ( C ) Nuclease assays with Exo1 (0.53, 0.8, 1.2, 1.8, and 2.7 nM), RPA (0.4 μM), and either without (lanes 2–6) or with Sgs1 (0.5 nM) and Top3-Rmi1 (5 nM, lanes 9–14, respectively), in standard buffer. Substrate is as in A . ( D ) Quantification of experiments as shown in C . Error bars show SE. ( E ) Nuclease assay carried out with Exo1 (0.5, 1, 2, 3, and 4 nM), RPA (0.4 μM), and either without (lanes 2–6) or with helicase-dead Sgs1 K706A (20 nM, lanes 8–12). Substrate is as in A . ( F ) Increasing amounts of nuclease-dead Exo1 D173A (0.53, 0.8, 1.2, 1.8, 2.7, 4, and 8 nM) were added to reactions containing Sgs1 (0.5 nM) and/or Top3-Rmi1 (5 nM), as indicated, in the presence of RPA (0.4 μM). Substrate is as in A .

    Article Snippet: The 2.7-kb dsDNA substrate with 4-nt overhangs at the 5′ end was pUC19 dsDNA linearized with HindIII (New England Biolabs).

    Techniques: Recombinase Polymerase Amplification, Labeling, Nuclease Assay

    Modification of 5’ ends of long dsDNA fragments promotes HDR-mediated single-copy integration. ( A ) GFP expression in the respective expression domain after HDR-mediated integration of modified dsDNA gfp donor cassettes into rx2 , rx1 , actb and dnmt1 ORFs in the injected generation. ( B ) Individual embryo PCR genotyping highlights efficient HDR-mediated single-copy integration of 5’Biotin modified long dsDNA donors, but not unmodified donor cassettes. Locus PCR reveals band size indicative of single-copy gfp integration (asterisk) besides alleles without gfp integration (open arrowhead). Amplification of gfp donor (white arrow) for control.

    Journal: eLife

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors

    doi: 10.7554/eLife.39468

    Figure Lengend Snippet: Modification of 5’ ends of long dsDNA fragments promotes HDR-mediated single-copy integration. ( A ) GFP expression in the respective expression domain after HDR-mediated integration of modified dsDNA gfp donor cassettes into rx2 , rx1 , actb and dnmt1 ORFs in the injected generation. ( B ) Individual embryo PCR genotyping highlights efficient HDR-mediated single-copy integration of 5’Biotin modified long dsDNA donors, but not unmodified donor cassettes. Locus PCR reveals band size indicative of single-copy gfp integration (asterisk) besides alleles without gfp integration (open arrowhead). Amplification of gfp donor (white arrow) for control.

    Article Snippet: The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs).

    Techniques: Modification, Expressing, Injection, Polymerase Chain Reaction, Amplification

    Single-copy integration of long dsDNA donor establishes stably transmitted gfp-rx2 fusion gene. ( A ) F2 homozygous embryos exhibit GFP-Rx2 fusion protein expression in the pattern of the endogenous gene in the retina. ( B ) Southern Blot analysis of F2 gfp-rx2 embryos reveals a single band for a digestion scheme cutting outside the donor cassette (BglII/HindIII) or within the 5’ donor cassette and in intron 2 (ScaI/HindIII) indicating precise single-copy donor integration. ( B’ ) Schematic representation of the modified locus indicating the restriction sites and the domain complementary to the probe used in ( B ). ( C ) RT-PCR analysis on mRNA isolated from individual homozygous F3 embryos indicates the transcription of a single gfp-rx2 fusion mRNA in comparison to the shorter wild-type rx2 mRNA as schematically represented in ( C’ ).

    Journal: eLife

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors

    doi: 10.7554/eLife.39468

    Figure Lengend Snippet: Single-copy integration of long dsDNA donor establishes stably transmitted gfp-rx2 fusion gene. ( A ) F2 homozygous embryos exhibit GFP-Rx2 fusion protein expression in the pattern of the endogenous gene in the retina. ( B ) Southern Blot analysis of F2 gfp-rx2 embryos reveals a single band for a digestion scheme cutting outside the donor cassette (BglII/HindIII) or within the 5’ donor cassette and in intron 2 (ScaI/HindIII) indicating precise single-copy donor integration. ( B’ ) Schematic representation of the modified locus indicating the restriction sites and the domain complementary to the probe used in ( B ). ( C ) RT-PCR analysis on mRNA isolated from individual homozygous F3 embryos indicates the transcription of a single gfp-rx2 fusion mRNA in comparison to the shorter wild-type rx2 mRNA as schematically represented in ( C’ ).

    Article Snippet: The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs).

    Techniques: Stable Transfection, Expressing, Southern Blot, Modification, Reverse Transcription Polymerase Chain Reaction, Isolation

    Modification of 5’ ends of long dsDNA fragments prevents in vivo multimerization. ( A ) Schematic representation of long dsDNA donor cassette PCR amplification with universal primers (black arrows) complementary to the cloning vector backbone outside of the assembled donor cassette (e. g. gfp with homology flanks). Bulky moieties like Biotin at the 5’ ends of both modified primers (red octagon) prevent multimerization/NHEJ of dsDNA, providing optimal conditions for HDR-mediated single-copy integration following CRISPR/Cas9-introduced DSB at the target locus (grey scissors). Representation of locus (Lf/Lr) and internal gfp (Gf/Gr) primers for PCR genotyping of putative HDR-mediated gfp integration events. ( B ) Southern blot analysis reveals the monomeric state of injected dsDNA fragments in vivo for 5’ modification with Biotin or Spacer C3. Long dsDNAs generated with control unmodified primers or Amino-dT attached primers multimerize as indicated by a high molecular weight ladder apparent already within two hours post-injection (hpi). Note: 5’ moieties did not enhance the stability of injected DNA.

    Journal: eLife

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors

    doi: 10.7554/eLife.39468

    Figure Lengend Snippet: Modification of 5’ ends of long dsDNA fragments prevents in vivo multimerization. ( A ) Schematic representation of long dsDNA donor cassette PCR amplification with universal primers (black arrows) complementary to the cloning vector backbone outside of the assembled donor cassette (e. g. gfp with homology flanks). Bulky moieties like Biotin at the 5’ ends of both modified primers (red octagon) prevent multimerization/NHEJ of dsDNA, providing optimal conditions for HDR-mediated single-copy integration following CRISPR/Cas9-introduced DSB at the target locus (grey scissors). Representation of locus (Lf/Lr) and internal gfp (Gf/Gr) primers for PCR genotyping of putative HDR-mediated gfp integration events. ( B ) Southern blot analysis reveals the monomeric state of injected dsDNA fragments in vivo for 5’ modification with Biotin or Spacer C3. Long dsDNAs generated with control unmodified primers or Amino-dT attached primers multimerize as indicated by a high molecular weight ladder apparent already within two hours post-injection (hpi). Note: 5’ moieties did not enhance the stability of injected DNA.

    Article Snippet: The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs).

    Techniques: Modification, In Vivo, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Non-Homologous End Joining, CRISPR, Southern Blot, Injection, Generated, Molecular Weight

    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