lambda exonuclease Search Results


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  • 99
    New England Biolabs exonuclease λ
    Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with <t>λ-exonuclease</t> (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.
    Exonuclease λ, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lambda λ exonuclease
    Copper(I)-oxygen efficiently reveals incorporated BrdU; the revelation can be further increased by means of exonucleases. A ) The results of the detection of the BrdU labeling of replicated DNA using acid (4 N HCl) or hydroxide (0.07 M NaOH) or DNase I treatment or the one-step or the two-step procedure are shown. All of the images were taken using 99-ms time to be able to compare the signal intensity. In the one-step procedure (the image labeled as Cu), the 30-minute treatment with copper(I)-oxygen was used exclusively. In the two-step protocol, a 10-minute treatment of the samples with copper(I)-oxygen was followed by incubation with exonuclease III or exonuclease λ. The model shows the situation for both one-step and two-step procedures. Note that <t>exonuclease</t> λ reveals BrdU-labeled parts in the proximity of close single gaps as it has no activity at nicks and limited activity at gaps. Only close single gaps can result into the formation of double-strand break. Although only one strand is usually labeled by BrdU, the situation is shown as if both strands were labeled in the schematic picture. The revealed parts of distinct strands are distinguished by colors. Bar: 20 µm. B ) Relative signal intensity is shown in the graph.
    Lambda λ Exonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    GE Healthcare lambda exonuclease
    Model of DNA degradation by λ exonuclease. Initiation is achieved by two methods: plug-in at the blunt-end of linear DNA and trimer ring assembly at the single-/double-stranded junction of a partial duplex. The order of processive degradation is cleavage, product release, and translocation by electrostatic attraction with concomitant melting.
    Lambda Exonuclease, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore lambda exonuclease
    Other exonucleases can perform strand exchange with ICP8. A, Strand exchange with full-length M13mp18 substrates was performed as described in Materials and Methods. Incubations were at 37 °C for 10–40 minutes, as indicated. All of the lanes included 100 ng of ssM13mp18 DNA and 100 ng of dsM13mp18 DNA linearized by EcoRI. Lane 1, no protein control; lane 2, 40 minutes incubation with ICP8 only; lanes 3–5, incubation with ICP8 and 13.9 nM UL12 for 10, 20, and 40 minutes, respectively; lanes 6–8, incubation with ICP8 and five units of <t>lambda</t> exonuclease for 10, 20, and 40 minutes, respectively; lanes 9–11, incubation with ICP8 and 100 units of ExoIII for 10, 20, and 40 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, M13mp18 dsDNA linearized by EcoRI; ss, M13mp18 ssDNA. B–E, Visualization of ICP8 catalyzed strand exchange reactions using dsDNA preresected with lambda exonuclease and ExoIII. Linear double-stranded ϕX174 DNA was subjected to digestion by lambda exonuclease (B and C) or ExoIII (D and E) as described in Materials and Methods. The nuclease-treated DNA was then used in strand exchange reactions. The classic strand exchange products are seen: sigma (B), alpha (D), and gapped circles (C and E). The scale bar represents the length of 1000 bp of dsDNA.
    Lambda Exonuclease, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega lambda exonuclease
    Other exonucleases can perform strand exchange with ICP8. A, Strand exchange with full-length M13mp18 substrates was performed as described in Materials and Methods. Incubations were at 37 °C for 10–40 minutes, as indicated. All of the lanes included 100 ng of ssM13mp18 DNA and 100 ng of dsM13mp18 DNA linearized by EcoRI. Lane 1, no protein control; lane 2, 40 minutes incubation with ICP8 only; lanes 3–5, incubation with ICP8 and 13.9 nM UL12 for 10, 20, and 40 minutes, respectively; lanes 6–8, incubation with ICP8 and five units of <t>lambda</t> exonuclease for 10, 20, and 40 minutes, respectively; lanes 9–11, incubation with ICP8 and 100 units of ExoIII for 10, 20, and 40 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, M13mp18 dsDNA linearized by EcoRI; ss, M13mp18 ssDNA. B–E, Visualization of ICP8 catalyzed strand exchange reactions using dsDNA preresected with lambda exonuclease and ExoIII. Linear double-stranded ϕX174 DNA was subjected to digestion by lambda exonuclease (B and C) or ExoIII (D and E) as described in Materials and Methods. The nuclease-treated DNA was then used in strand exchange reactions. The classic strand exchange products are seen: sigma (B), alpha (D), and gapped circles (C and E). The scale bar represents the length of 1000 bp of dsDNA.
    Lambda Exonuclease, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Boehringer Mannheim λ exonuclease
    Other exonucleases can perform strand exchange with ICP8. A, Strand exchange with full-length M13mp18 substrates was performed as described in Materials and Methods. Incubations were at 37 °C for 10–40 minutes, as indicated. All of the lanes included 100 ng of ssM13mp18 DNA and 100 ng of dsM13mp18 DNA linearized by EcoRI. Lane 1, no protein control; lane 2, 40 minutes incubation with ICP8 only; lanes 3–5, incubation with ICP8 and 13.9 nM UL12 for 10, 20, and 40 minutes, respectively; lanes 6–8, incubation with ICP8 and five units of <t>lambda</t> exonuclease for 10, 20, and 40 minutes, respectively; lanes 9–11, incubation with ICP8 and 100 units of ExoIII for 10, 20, and 40 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, M13mp18 dsDNA linearized by EcoRI; ss, M13mp18 ssDNA. B–E, Visualization of ICP8 catalyzed strand exchange reactions using dsDNA preresected with lambda exonuclease and ExoIII. Linear double-stranded ϕX174 DNA was subjected to digestion by lambda exonuclease (B and C) or ExoIII (D and E) as described in Materials and Methods. The nuclease-treated DNA was then used in strand exchange reactions. The classic strand exchange products are seen: sigma (B), alpha (D), and gapped circles (C and E). The scale bar represents the length of 1000 bp of dsDNA.
    λ Exonuclease, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Boehringer Mannheim λ exonuclease digestion
    Other exonucleases can perform strand exchange with ICP8. A, Strand exchange with full-length M13mp18 substrates was performed as described in Materials and Methods. Incubations were at 37 °C for 10–40 minutes, as indicated. All of the lanes included 100 ng of ssM13mp18 DNA and 100 ng of dsM13mp18 DNA linearized by EcoRI. Lane 1, no protein control; lane 2, 40 minutes incubation with ICP8 only; lanes 3–5, incubation with ICP8 and 13.9 nM UL12 for 10, 20, and 40 minutes, respectively; lanes 6–8, incubation with ICP8 and five units of <t>lambda</t> exonuclease for 10, 20, and 40 minutes, respectively; lanes 9–11, incubation with ICP8 and 100 units of ExoIII for 10, 20, and 40 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, M13mp18 dsDNA linearized by EcoRI; ss, M13mp18 ssDNA. B–E, Visualization of ICP8 catalyzed strand exchange reactions using dsDNA preresected with lambda exonuclease and ExoIII. Linear double-stranded ϕX174 DNA was subjected to digestion by lambda exonuclease (B and C) or ExoIII (D and E) as described in Materials and Methods. The nuclease-treated DNA was then used in strand exchange reactions. The classic strand exchange products are seen: sigma (B), alpha (D), and gapped circles (C and E). The scale bar represents the length of 1000 bp of dsDNA.
    λ Exonuclease Digestion, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Roche lambda exonuclease
    Gel analysis of the effects of <t>lambda</t> exonuclease treatment on adapter assembly. Lanes 1 and 6, phosphorylated core adapter without overhang oligonucleotides; lanes 2 and 7, Adapter 1 without phosphorylation; lanes 3 and 8, Adapter 1 with phosphorylation;
    Lambda Exonuclease, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Brookhaven Instruments λ exonuclease
    Gel analysis of the effects of <t>lambda</t> exonuclease treatment on adapter assembly. Lanes 1 and 6, phosphorylated core adapter without overhang oligonucleotides; lanes 2 and 7, Adapter 1 without phosphorylation; lanes 3 and 8, Adapter 1 with phosphorylation;
    λ Exonuclease, supplied by Brookhaven Instruments, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lambda exonuclease iii
    Gel analysis of the effects of <t>lambda</t> exonuclease treatment on adapter assembly. Lanes 1 and 6, phosphorylated core adapter without overhang oligonucleotides; lanes 2 and 7, Adapter 1 without phosphorylation; lanes 3 and 8, Adapter 1 with phosphorylation;
    Lambda Exonuclease Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs λ exonuclease reaction buffer
    ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the <t>λ</t> exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )
    λ Exonuclease Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    GE Healthcare lambda exonuclease buffer
    ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the <t>λ</t> exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )
    Lambda Exonuclease Buffer, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs lambda exonucelase
    ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the <t>λ</t> exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )
    Lambda Exonucelase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher bacteriophage λ exonuclease
    Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 DNA. DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease III (lanes 8 – 11 ) or 10 units <t>λ</t> exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.
    Bacteriophage λ Exonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs e coli exonuclease
    Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda <t>exonuclease</t> I and E. coli ).
    E Coli Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher exonuclease thermoscientific digestion
    Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda <t>exonuclease</t> I and E. coli ).
    Exonuclease Thermoscientific Digestion, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs bal 31 exonuclease
    Polymorphic fragments are <t>BAL</t> 31 sensitive. Agarose blocks of spleen DNA from individual VII were treated with Agarase, mixed with lambda DNA, and treated with BAL 31 for the indicated numbers of minutes. Samples were then digested with Mse I, run on a 0.4% gel, blotted, and hybridized to a (TTAGGG) n probe. A portion of the BAL 31 reaction mixture was digested with Hin dIII and hybridized to a lambda probe (data not shown). The sizes of the marker fragments are indicated on the left in kilobases.
    Bal 31 Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs recjf
    Polymorphic fragments are <t>BAL</t> 31 sensitive. Agarose blocks of spleen DNA from individual VII were treated with Agarase, mixed with lambda DNA, and treated with BAL 31 for the indicated numbers of minutes. Samples were then digested with Mse I, run on a 0.4% gel, blotted, and hybridized to a (TTAGGG) n probe. A portion of the BAL 31 reaction mixture was digested with Hin dIII and hybridized to a lambda probe (data not shown). The sizes of the marker fragments are indicated on the left in kilobases.
    Recjf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with λ-exonuclease (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.

    Journal: Nucleic Acids Research

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries

    doi: 10.1093/nar/gkv544

    Figure Lengend Snippet: Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with λ-exonuclease (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.

    Article Snippet: Splinted ligation was performed by first annealing tailed DNA2 with DNA5/6 and DNA7 by heating to 90°C for 30 s and cooling to room temperature for 5 min, adding all other components after this step [final concentrations: 10 μM DNA2, 22.5 μM DNA5/6, 25 μM blocked and phosphorylated DNA7, 50 μM ATP, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 1.5 U/μl T4 DNA ligase] incubating at 37°C for 4 h and heating to 80°C for 10 min. DNA5/6 and DNA7 were optionally removed from reaction mixtures to obtain pure, ligated/extended ssDNA by adding λ-exonuclease (0.25 U/μl for primer extension or 0.5 U/μl for ligation; New England Biolabs) directly into the reaction mixture and incubating at 37°C for 1 h, followed by 80°C for 10 min. DNA was purified by ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.5).

    Techniques: Modification, Polyacrylamide Gel Electrophoresis, Staining

    ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Journal: Nature Communications

    Article Title: Simplified ChIP-exo assays

    doi: 10.1038/s41467-018-05265-7

    Figure Lengend Snippet: ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Article Snippet: The λ exonuclease digestion (100 µl) containing: 20 U λ exonuclease (NEB), 1 × λ exonuclease reaction buffer (NEB), 0.1% Triton-X 100, and 5% DMSO was incubated for 30 min at 37 °C; then washed with 10 mM Tris-HCl, pH 8.0 at 4 °C.

    Techniques: Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Produced

    ( A ) Fluorescence responses of Nanoprobe A (0.1 mg/ml) to APE1 at different concentrations. ( B ) Linear calibration curve for detection of the activity of APE1. The linear regression equation is F = 0.20 c (U/ml) – 2.1 × 10 −4 , and the detection limit is 0.01 U/ml. ( C ) Selectivity of Nanoprobe A toward APE1 (2.0 U/ml) over other nucleases. (DNase I: 5.0 U/ml; Exo III: 4.0 U/ml; lambda exo: 66.7 U/ml; Exo I: 12.5 U/ml; T5: 5.0 U/ml; T7: 50 U/ml). All experiments were repeated at least three times.

    Journal: Nucleic Acids Research

    Article Title: A specific DNA-nanoprobe for tracking the activities of human apurinic/apyrimidinic endonuclease 1 in living cells

    doi: 10.1093/nar/gkw1205

    Figure Lengend Snippet: ( A ) Fluorescence responses of Nanoprobe A (0.1 mg/ml) to APE1 at different concentrations. ( B ) Linear calibration curve for detection of the activity of APE1. The linear regression equation is F = 0.20 c (U/ml) – 2.1 × 10 −4 , and the detection limit is 0.01 U/ml. ( C ) Selectivity of Nanoprobe A toward APE1 (2.0 U/ml) over other nucleases. (DNase I: 5.0 U/ml; Exo III: 4.0 U/ml; lambda exo: 66.7 U/ml; Exo I: 12.5 U/ml; T5: 5.0 U/ml; T7: 50 U/ml). All experiments were repeated at least three times.

    Article Snippet: Apurinic/apyrimidinic endonuclease I (APE1), uracil–DNA glycocasylase (UDG), deoxyribonuclease I (DNase I), exonuclease III (Exo III), exonuclease I (Exo I), lambda exonuclease (λ exo), T5 exonuclease (T5 Exo), T7 exonuclease (T7 Exo) and their corresponding buffers ( ) were all purchased from New England Biolabs (NEB, USA).

    Techniques: Fluorescence, Activity Assay

    PFGE and Southern blot hybridization with IS 1245 -derived probe of M. avium and M. kansasii colonies. (A) PFGE with undigested DNA; (B) Southern blot hybridization with IS 1245 -derived probe. Open arrow indicates pMA100; closed arrow indicates the uncharacterized smaller hybridization band. 88.1 to 88.4 = M . avium ; 88.5 to 88.75 = PCR−IS 1245- negative M . kansasii ; 88.8 to 88.15 = PCR−IS 1245- positive M. kansasii. On the left, Lambda Ladder PFG Marker (NewEngland BioLabs) molecular size markers.

    Journal: PLoS ONE

    Article Title: First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid

    doi: 10.1371/journal.pone.0029884

    Figure Lengend Snippet: PFGE and Southern blot hybridization with IS 1245 -derived probe of M. avium and M. kansasii colonies. (A) PFGE with undigested DNA; (B) Southern blot hybridization with IS 1245 -derived probe. Open arrow indicates pMA100; closed arrow indicates the uncharacterized smaller hybridization band. 88.1 to 88.4 = M . avium ; 88.5 to 88.75 = PCR−IS 1245- negative M . kansasii ; 88.8 to 88.15 = PCR−IS 1245- positive M. kansasii. On the left, Lambda Ladder PFG Marker (NewEngland BioLabs) molecular size markers.

    Article Snippet: Besides that, the pMA100 band excised from PFGE agarose gels was treated in separate experiments with 30 U exonuclease III, 30 U exonuclease lambda or 30 U topoiosomerase I (all enzymes from New England BioLabs) for 3 h at 37°C, according to the manufacturer's protocols.

    Techniques: Southern Blot, Hybridization, Derivative Assay, Polymerase Chain Reaction, Marker

    PFGE of DNA genomic preparations. (A) PFGE with undigested DNAs from M. avium 88.3 (1) and M. kansasii 88.8 (2) under different switch times, indicated below each figure; (B) pMA100 extracted from PFGE gels and treated with exonuclease III (3) or exonuclease lambda (4); (C) pMA100 extracted from PFGE gels and treated (+) or not (-) with topoisomerase I; (D) DNA prepared with (+) or without (-) adding proteinase K to the lysis buffer; (E) same as in (D) in PFGE gels and running buffer prepared with 0.2% SDS. λ: DNA concatemers of the bacteriophage λ genome.

    Journal: PLoS ONE

    Article Title: First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid

    doi: 10.1371/journal.pone.0029884

    Figure Lengend Snippet: PFGE of DNA genomic preparations. (A) PFGE with undigested DNAs from M. avium 88.3 (1) and M. kansasii 88.8 (2) under different switch times, indicated below each figure; (B) pMA100 extracted from PFGE gels and treated with exonuclease III (3) or exonuclease lambda (4); (C) pMA100 extracted from PFGE gels and treated (+) or not (-) with topoisomerase I; (D) DNA prepared with (+) or without (-) adding proteinase K to the lysis buffer; (E) same as in (D) in PFGE gels and running buffer prepared with 0.2% SDS. λ: DNA concatemers of the bacteriophage λ genome.

    Article Snippet: Besides that, the pMA100 band excised from PFGE agarose gels was treated in separate experiments with 30 U exonuclease III, 30 U exonuclease lambda or 30 U topoiosomerase I (all enzymes from New England BioLabs) for 3 h at 37°C, according to the manufacturer's protocols.

    Techniques: Lysis

    In situ detection of DNA using padlock probes and target primed rolling circle DNA synthesis . (A) The samples are cleaved with a restriction enzyme having a restriction site positioned 3' to the probe binding sequence. It is important that the enzyme does not have any other cleavage sites in close proximity to the 5'-end of the probe binding sequence to avoid degradation of the recognition sequence during exonuclease treatment. (B) The target sequence is made single stranded using the lambda exonuclease which digests duplex DNA in the 5'→3' direction in a highly processive manner, thereby making the target sequence single stranded. (C) The padlock probe is hybridized and ligated on the target sequence. Only padlock probes which are correctly hybridized at the point of ligation will be circularized. (D-E) The rolling circle reaction is initiated by using the target sequence as a primer, thereby locking the rolling circle product to the target sequence. (F) The rolling circle product is visualized by hybridizing a labeled oligonucleotide to the part of the padlock probe not recognizing the genomic hybridization target.

    Journal: BMC Molecular Biology

    Article Title: Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

    doi: 10.1186/1471-2199-8-103

    Figure Lengend Snippet: In situ detection of DNA using padlock probes and target primed rolling circle DNA synthesis . (A) The samples are cleaved with a restriction enzyme having a restriction site positioned 3' to the probe binding sequence. It is important that the enzyme does not have any other cleavage sites in close proximity to the 5'-end of the probe binding sequence to avoid degradation of the recognition sequence during exonuclease treatment. (B) The target sequence is made single stranded using the lambda exonuclease which digests duplex DNA in the 5'→3' direction in a highly processive manner, thereby making the target sequence single stranded. (C) The padlock probe is hybridized and ligated on the target sequence. Only padlock probes which are correctly hybridized at the point of ligation will be circularized. (D-E) The rolling circle reaction is initiated by using the target sequence as a primer, thereby locking the rolling circle product to the target sequence. (F) The rolling circle product is visualized by hybridizing a labeled oligonucleotide to the part of the padlock probe not recognizing the genomic hybridization target.

    Article Snippet: Lambda exonuclease Exonuclease digestion was performed in a buffer containing 1× lambda exonuclease buffer (NEB), 0.2 μg/μl BSA (NEB) and 1u/μl lambda exonuclease (NEB) for 1 min at 37°C.

    Techniques: In Situ, DNA Synthesis, Binding Assay, Sequencing, Ligation, Labeling, Hybridization

    Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for lambda exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.

    Journal: Nature biotechnology

    Article Title: ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints

    doi: 10.1038/nbt.3121

    Figure Lengend Snippet: Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for lambda exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.

    Article Snippet: For lambda exonuclease digestion, each sample was incubated in 0.2 u/μl lambda exonuclease (New England Biolabs, M0262), 5% DMSO and 0.1% triton X-100 in 100 μl 1x NEB Lambda exonuclease reaction buffer at 37 °C for 60 min with constant agitation, followed by washing steps as above.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Amplification, Functional Assay, Ligation, Purification, Polymerase Chain Reaction, Sequencing, Genomic Sequencing, Generated, In Vitro, Footprinting, In Vivo

    Analysis of the Dorsal, Twist and Max in vivo footprint (a–c) For each factor, the top 200 motifs with the highest ChIP-nexus read counts were selected and are shown in descending order as heat map. The footprints show a consistent boundary on the positive strand (red) and negative strand (blue) around each motif. The zoomed-in average profile below reveals that the footprints are wider than the motif. A schematic representation of the digestion pattern is shown below using Pacman symbols for lambda exonuclease. (a) The ChIP-nexus footprint for Dorsal (NFkB) on its canonical motif (GGRWWTTCC with up to one mismatch) extends on average 5 bp away from the motif edge. Thus, the average dorsal footprint is 18 bp long (horizontal black bar). (b) The Twist ChIP-nexus footprint on the E-box motif CABATG (no mismatch) has two outside boundaries, one at 11 bp, and one at 2 bp away from the motif edge, suggesting interactions with flanking DNA sequences. Each portion of the footprint is around 8–9bp long (horizontal black bar). (c) The Max ChIP-nexus footprint on its canonical E-box motif (CACGTG, no mismatch) has an outside boundary at 8 bp away from the motif edge, as well as a boundary inside the motif (at the A/T base), suggesting two partial footprints (horizontal black bars). (d, e) Average Max and Twist ChIP-nexus footprints at the top 200 sites for all possible E-box variants (CANNTG). Each variant profile includes its reverse complement. (d) Max binds specifically to the canonical CACGTG motif and to a lesser extent to the CACATG motif. Note that the Max footprint shape looks identical between the two motifs. (e) In contrast, the Twist binding specificity and the footprint shape is more complex. Notably, the outer boundary at -11bp is stronger at the CATATG and CACATG motif, whereas the inner boundary at -2 bp is stronger at the CAGATG motif.

    Journal: Nature biotechnology

    Article Title: ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints

    doi: 10.1038/nbt.3121

    Figure Lengend Snippet: Analysis of the Dorsal, Twist and Max in vivo footprint (a–c) For each factor, the top 200 motifs with the highest ChIP-nexus read counts were selected and are shown in descending order as heat map. The footprints show a consistent boundary on the positive strand (red) and negative strand (blue) around each motif. The zoomed-in average profile below reveals that the footprints are wider than the motif. A schematic representation of the digestion pattern is shown below using Pacman symbols for lambda exonuclease. (a) The ChIP-nexus footprint for Dorsal (NFkB) on its canonical motif (GGRWWTTCC with up to one mismatch) extends on average 5 bp away from the motif edge. Thus, the average dorsal footprint is 18 bp long (horizontal black bar). (b) The Twist ChIP-nexus footprint on the E-box motif CABATG (no mismatch) has two outside boundaries, one at 11 bp, and one at 2 bp away from the motif edge, suggesting interactions with flanking DNA sequences. Each portion of the footprint is around 8–9bp long (horizontal black bar). (c) The Max ChIP-nexus footprint on its canonical E-box motif (CACGTG, no mismatch) has an outside boundary at 8 bp away from the motif edge, as well as a boundary inside the motif (at the A/T base), suggesting two partial footprints (horizontal black bars). (d, e) Average Max and Twist ChIP-nexus footprints at the top 200 sites for all possible E-box variants (CANNTG). Each variant profile includes its reverse complement. (d) Max binds specifically to the canonical CACGTG motif and to a lesser extent to the CACATG motif. Note that the Max footprint shape looks identical between the two motifs. (e) In contrast, the Twist binding specificity and the footprint shape is more complex. Notably, the outer boundary at -11bp is stronger at the CATATG and CACATG motif, whereas the inner boundary at -2 bp is stronger at the CAGATG motif.

    Article Snippet: For lambda exonuclease digestion, each sample was incubated in 0.2 u/μl lambda exonuclease (New England Biolabs, M0262), 5% DMSO and 0.1% triton X-100 in 100 μl 1x NEB Lambda exonuclease reaction buffer at 37 °C for 60 min with constant agitation, followed by washing steps as above.

    Techniques: In Vivo, Chromatin Immunoprecipitation, Variant Assay, Binding Assay

    Lambda exonuclease mapping of the upstream borders of HIV-1 RT bound to P/T

    Journal:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

    doi: 10.1016/j.jmb.2007.03.006

    Figure Lengend Snippet: Lambda exonuclease mapping of the upstream borders of HIV-1 RT bound to P/T

    Article Snippet: For lambda exonuclease mapping experiments the 5'-end of the L32 primer was phosphorylated by T4 polynucleotide kinase (New England Biolabs) and unlabeled ATP prior to annealing with template and adding the 3′ label.

    Techniques:

    Copper(I)-oxygen efficiently reveals incorporated BrdU; the revelation can be further increased by means of exonucleases. A ) The results of the detection of the BrdU labeling of replicated DNA using acid (4 N HCl) or hydroxide (0.07 M NaOH) or DNase I treatment or the one-step or the two-step procedure are shown. All of the images were taken using 99-ms time to be able to compare the signal intensity. In the one-step procedure (the image labeled as Cu), the 30-minute treatment with copper(I)-oxygen was used exclusively. In the two-step protocol, a 10-minute treatment of the samples with copper(I)-oxygen was followed by incubation with exonuclease III or exonuclease λ. The model shows the situation for both one-step and two-step procedures. Note that exonuclease λ reveals BrdU-labeled parts in the proximity of close single gaps as it has no activity at nicks and limited activity at gaps. Only close single gaps can result into the formation of double-strand break. Although only one strand is usually labeled by BrdU, the situation is shown as if both strands were labeled in the schematic picture. The revealed parts of distinct strands are distinguished by colors. Bar: 20 µm. B ) Relative signal intensity is shown in the graph.

    Journal: PLoS ONE

    Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ

    doi: 10.1371/journal.pone.0052584

    Figure Lengend Snippet: Copper(I)-oxygen efficiently reveals incorporated BrdU; the revelation can be further increased by means of exonucleases. A ) The results of the detection of the BrdU labeling of replicated DNA using acid (4 N HCl) or hydroxide (0.07 M NaOH) or DNase I treatment or the one-step or the two-step procedure are shown. All of the images were taken using 99-ms time to be able to compare the signal intensity. In the one-step procedure (the image labeled as Cu), the 30-minute treatment with copper(I)-oxygen was used exclusively. In the two-step protocol, a 10-minute treatment of the samples with copper(I)-oxygen was followed by incubation with exonuclease III or exonuclease λ. The model shows the situation for both one-step and two-step procedures. Note that exonuclease λ reveals BrdU-labeled parts in the proximity of close single gaps as it has no activity at nicks and limited activity at gaps. Only close single gaps can result into the formation of double-strand break. Although only one strand is usually labeled by BrdU, the situation is shown as if both strands were labeled in the schematic picture. The revealed parts of distinct strands are distinguished by colors. Bar: 20 µm. B ) Relative signal intensity is shown in the graph.

    Article Snippet: Enzymes used These enzymes and condition were used: Terminal deoxynucleotidyl transferase (TdT; 2 U/µl, 10 minutes, 37°C, Fermentas), buffer for TdT, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa Fluor® 555-aha-2′-deoxyuridine-5′-triphosphate (Alexa-dUTP); DNA polymerase I (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for DNA polymerase I, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment Exo- (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment Exo-, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Exonuclease III (1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease III; Exonuclease λ (0.1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease λ; Shrimp alkaline phosphomonoesterase (phosphatase; SAP; 1 U/µl, 20 minutes, 37°C, Fermentas), buffer for SAP.

    Techniques: Labeling, Mass Spectrometry, Incubation, Activity Assay

    GR binding recruits Oct-2 to octamer motifs adjacent to GR binding sites in the nucleus. (A) Nuclei prepared from CHO cells transfected with the MMTV promoter construct pHCWT, GR, and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Hin ) and GR and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Sma I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T3 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif sequence and GRE sequences in the MMTV LTR are summarized schematically. The Dex-, GR-, and Oct-2-specific λ pause site is indicated by the arrow. (C) Nuclei prepared from CHO cells transfected with pBluescript containing either a Gal4 binding site separated by 8 nucleotides from the octamer motif sequence from the MMTV LTR (left) or a nonspecific oligonucleotide encoding an IAP enhancer core (right) along with Gal-GR WT , Gal-GR L501P , and/or Oct-2 expression plasmids were restricted with Xho I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T7 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif-IAP sequence and of the Gal4 sequence are summarized schematically. The Gal-GR WT -, Oct-2-, and octamer motif-dependent specific λ pause site is indicated by the arrow. Western blots of cellular extracts verified that Gal-GR WT and Gal-GR L501P ).

    Journal: Molecular and Cellular Biology

    Article Title: Recruitment of Octamer Transcription Factors to DNA by Glucocorticoid Receptor

    doi:

    Figure Lengend Snippet: GR binding recruits Oct-2 to octamer motifs adjacent to GR binding sites in the nucleus. (A) Nuclei prepared from CHO cells transfected with the MMTV promoter construct pHCWT, GR, and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Hin ) and GR and/or Oct-2 expression plasmids and treated with 10 −6 M Dex or vehicle for 15 min were restricted with Sma I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T3 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif sequence and GRE sequences in the MMTV LTR are summarized schematically. The Dex-, GR-, and Oct-2-specific λ pause site is indicated by the arrow. (C) Nuclei prepared from CHO cells transfected with pBluescript containing either a Gal4 binding site separated by 8 nucleotides from the octamer motif sequence from the MMTV LTR (left) or a nonspecific oligonucleotide encoding an IAP enhancer core (right) along with Gal-GR WT , Gal-GR L501P , and/or Oct-2 expression plasmids were restricted with Xho I and digested with λ exonuclease as indicated. Digestion was revealed by linear PCR extension of a T7 polymerase primer, and pause sites were positioned relative to an A sequencing track amplified with the same primer. The positions of the octamer motif-IAP sequence and of the Gal4 sequence are summarized schematically. The Gal-GR WT -, Oct-2-, and octamer motif-dependent specific λ pause site is indicated by the arrow. Western blots of cellular extracts verified that Gal-GR WT and Gal-GR L501P ).

    Article Snippet: Each sample was simultaneously digested with 100 U of restriction enzyme and 15 U of λ exonuclease (Life Technologies) for 15 min at 30°C.

    Techniques: Binding Assay, Transfection, Construct, Expressing, Polymerase Chain Reaction, Sequencing, Amplification, Western Blot

    λ-exonuclease treatment of nascent DNA does not alter the array profile. Array hybridizations results with MCF-7 short nascent DNA untreated or treated with λ-exonuclease, prior to hybridization, were compared along a 90 kb region of Chr17. The top panel shows the profile of a λ-exonuclease untreated preparation (NS fraction 10–12). The lower panels are two independently isolated samples (NS71 and NS73) after λ-exonuclease treatment (NS Lambda exo, and NS lambda exo, rep 2 , respectively).

    Journal: PLoS ONE

    Article Title: Preferential Localization of Human Origins of DNA Replication at the 5?-Ends of Expressed Genes and at Evolutionarily Conserved DNA Sequences

    doi: 10.1371/journal.pone.0017308

    Figure Lengend Snippet: λ-exonuclease treatment of nascent DNA does not alter the array profile. Array hybridizations results with MCF-7 short nascent DNA untreated or treated with λ-exonuclease, prior to hybridization, were compared along a 90 kb region of Chr17. The top panel shows the profile of a λ-exonuclease untreated preparation (NS fraction 10–12). The lower panels are two independently isolated samples (NS71 and NS73) after λ-exonuclease treatment (NS Lambda exo, and NS lambda exo, rep 2 , respectively).

    Article Snippet: After λ-exonuclease treatment of the DNA pool in the size range of 400–800 bp, we synthesized a double stranded DNA population required for massively parallel sequencing using the Klenow fragment of DNA polymerase I (Invitrogen, Carlsbed, CA) and random primers (Invitrogen, Carlsbad, CA).

    Techniques: Hybridization, Isolation

    Correlation of nascent DNA enrichment profiles by DNA microarray assay, and DNA sequencing. Average array enrichment results (NS-chip) from two independent MCF-7 preparations were compared to those obtained from high throughput DNA sequencing of MCF-7 short nascent DNA after λ-exonuclease digestion (NS-seq). The region of comparison comprises about 90 kb on Chr17. A comparison over a larger region is presented in Figure S2 .

    Journal: PLoS ONE

    Article Title: Preferential Localization of Human Origins of DNA Replication at the 5?-Ends of Expressed Genes and at Evolutionarily Conserved DNA Sequences

    doi: 10.1371/journal.pone.0017308

    Figure Lengend Snippet: Correlation of nascent DNA enrichment profiles by DNA microarray assay, and DNA sequencing. Average array enrichment results (NS-chip) from two independent MCF-7 preparations were compared to those obtained from high throughput DNA sequencing of MCF-7 short nascent DNA after λ-exonuclease digestion (NS-seq). The region of comparison comprises about 90 kb on Chr17. A comparison over a larger region is presented in Figure S2 .

    Article Snippet: After λ-exonuclease treatment of the DNA pool in the size range of 400–800 bp, we synthesized a double stranded DNA population required for massively parallel sequencing using the Klenow fragment of DNA polymerase I (Invitrogen, Carlsbed, CA) and random primers (Invitrogen, Carlsbad, CA).

    Techniques: Microarray, DNA Sequencing, Chromatin Immunoprecipitation, High Throughput Screening Assay

    Schematic overview of the enzymatic steps of the probe generation protocol Templates were amplified from the template library pool by PCR using primers specific for each FISH genotyping round. Lambda exonuclease selectively digested the 5′‐phosphorylated strand, leaving only the 5′‐phosphorothioate strand. Fluorescently labeled and phosphorothioate‐modified elongation probes were hybridized to the ssDNA template and elongated with DreamTaq polymerase. The dsDNA product was digested with restriction enzyme SchI, removing the phosphorothioate bonds from the unlabeled strand. Lambda exonuclease digestion produced the final FISH probe.

    Journal: Molecular Systems Biology

    Article Title: In situ genotyping of a pooled strain library after characterizing complex phenotypes

    doi: 10.15252/msb.20177951

    Figure Lengend Snippet: Schematic overview of the enzymatic steps of the probe generation protocol Templates were amplified from the template library pool by PCR using primers specific for each FISH genotyping round. Lambda exonuclease selectively digested the 5′‐phosphorylated strand, leaving only the 5′‐phosphorothioate strand. Fluorescently labeled and phosphorothioate‐modified elongation probes were hybridized to the ssDNA template and elongated with DreamTaq polymerase. The dsDNA product was digested with restriction enzyme SchI, removing the phosphorothioate bonds from the unlabeled strand. Lambda exonuclease digestion produced the final FISH probe.

    Article Snippet: The phosphorylated strand was selectively digested by lambda exonuclease (Thermo Scientific) treatment for 30 min at 37°C followed by heat inactivation at 80°C for 10 min.

    Techniques: Amplification, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Labeling, Modification, Produced

    Products from the different steps of the FISH probe production protocol Products were run on a 10% polyacrylamide gel and imaged in (A) Cy3 and (B) Cy5 channels. (M): Cy3‐ and Cy5‐labeled 39‐nt and 19‐nt ssDNA probes were used as size references. (1) and (2): the initial fluorescent elongation product for the two rounds of FISH probe generation, respectively. (3) and (4): SchI digestion of the elongation products for rounds one and two, respectively. (5) and (6): Lambda exonuclease treatment and gel‐purified product for rounds one and two, respectively.

    Journal: Molecular Systems Biology

    Article Title: In situ genotyping of a pooled strain library after characterizing complex phenotypes

    doi: 10.15252/msb.20177951

    Figure Lengend Snippet: Products from the different steps of the FISH probe production protocol Products were run on a 10% polyacrylamide gel and imaged in (A) Cy3 and (B) Cy5 channels. (M): Cy3‐ and Cy5‐labeled 39‐nt and 19‐nt ssDNA probes were used as size references. (1) and (2): the initial fluorescent elongation product for the two rounds of FISH probe generation, respectively. (3) and (4): SchI digestion of the elongation products for rounds one and two, respectively. (5) and (6): Lambda exonuclease treatment and gel‐purified product for rounds one and two, respectively.

    Article Snippet: The phosphorylated strand was selectively digested by lambda exonuclease (Thermo Scientific) treatment for 30 min at 37°C followed by heat inactivation at 80°C for 10 min.

    Techniques: Fluorescence In Situ Hybridization, Labeling, Purification

    Model of DNA degradation by λ exonuclease. Initiation is achieved by two methods: plug-in at the blunt-end of linear DNA and trimer ring assembly at the single-/double-stranded junction of a partial duplex. The order of processive degradation is cleavage, product release, and translocation by electrostatic attraction with concomitant melting.

    Journal: Nucleic Acids Research

    Article Title: Allosteric ring assembly and chemo-mechanical melting by the interaction between 5′-phosphate and λ exonuclease

    doi: 10.1093/nar/gkv1150

    Figure Lengend Snippet: Model of DNA degradation by λ exonuclease. Initiation is achieved by two methods: plug-in at the blunt-end of linear DNA and trimer ring assembly at the single-/double-stranded junction of a partial duplex. The order of processive degradation is cleavage, product release, and translocation by electrostatic attraction with concomitant melting.

    Article Snippet: The cell lysate was centrifuged for 30 min at 35 000 ×g , and re-suspended in 50 mM Tris–HCl (pH 8.0), 300 mM NaCl and 10 mM imidazole. λ exonuclease was purified by HisTrap FF (GE Healthcare) using a buffer (50 mM Tris–HCl (pH8.0), 300 mM NaCl and 300 mM Imidazol in a gradient method. (His)6 -tag-MBP was removed by TEV protease, which remained five glycines at the N-terminus of the protein, and purified again by HisTrap FF (GE Healthcare).

    Techniques: Translocation Assay

    Single molecule FRET assay for DNA degradation. ( A ) Top and side views of the crystal structure of λ exonuclease with DNA (PDB entry 3SM4) demonstrating that two nucleotides (violet) of the 5′-end strand are pre-melted at the ss-ds junction and inserted into the active site of one subunit. ( B ) Experimental scheme, depicting the DNA and protein tilted with an angle relative to the DNA. ( C ) FRET-time trace, demonstrating how degradation time is assigned. ( D ) FRET-histogram that was obtained as shown in (B) (top: dsDNA; middle: before the degradation without Mg 2+ ; bottom: after the degradation with Mg 2+ ). ( E ) FRET-histograms obtained before (black curve) and after (red curve) a 2-min reaction with DNA substrates with (top) and without (bottom) a 5′ terminal phosphate. ( F ) The 5′ terminal phosphate is essential for the formation of a catalytically active DNA and enzyme complex. The fitted exponential growth of 5′P-DNA (black line) and 5′OH-DNA (red line) are 20 s and 403 s, respectively. Error bars denote SEM.

    Journal: Nucleic Acids Research

    Article Title: Allosteric ring assembly and chemo-mechanical melting by the interaction between 5′-phosphate and λ exonuclease

    doi: 10.1093/nar/gkv1150

    Figure Lengend Snippet: Single molecule FRET assay for DNA degradation. ( A ) Top and side views of the crystal structure of λ exonuclease with DNA (PDB entry 3SM4) demonstrating that two nucleotides (violet) of the 5′-end strand are pre-melted at the ss-ds junction and inserted into the active site of one subunit. ( B ) Experimental scheme, depicting the DNA and protein tilted with an angle relative to the DNA. ( C ) FRET-time trace, demonstrating how degradation time is assigned. ( D ) FRET-histogram that was obtained as shown in (B) (top: dsDNA; middle: before the degradation without Mg 2+ ; bottom: after the degradation with Mg 2+ ). ( E ) FRET-histograms obtained before (black curve) and after (red curve) a 2-min reaction with DNA substrates with (top) and without (bottom) a 5′ terminal phosphate. ( F ) The 5′ terminal phosphate is essential for the formation of a catalytically active DNA and enzyme complex. The fitted exponential growth of 5′P-DNA (black line) and 5′OH-DNA (red line) are 20 s and 403 s, respectively. Error bars denote SEM.

    Article Snippet: The cell lysate was centrifuged for 30 min at 35 000 ×g , and re-suspended in 50 mM Tris–HCl (pH 8.0), 300 mM NaCl and 10 mM imidazole. λ exonuclease was purified by HisTrap FF (GE Healthcare) using a buffer (50 mM Tris–HCl (pH8.0), 300 mM NaCl and 300 mM Imidazol in a gradient method. (His)6 -tag-MBP was removed by TEV protease, which remained five glycines at the N-terminus of the protein, and purified again by HisTrap FF (GE Healthcare).

    Techniques:

    The lack of a phosphate at a nick in the DNA substrate prevents the formation of a stable association with λ exonuclease during degradation. ( A ) Schematic of a DNA substrate without a 5′ phosphate group at a nick in the hydrolyzed strand (nick-5′OH-DNA). ( B ) Exemplary FRET time trajectory demonstrating diffusive back and forth movement of the enzyme in the absence of a 5′ phosphate in the substrate. Approximately 90% of all trajectories exhibited diffusive movements on the nick-5′OH-DNA. The blue trace is an idealized fit that is used to extract association and dissociation rates based on the HaMMy ( 25 ) algorithm. ( C ) Transition density plots (TDP) generated from the FRET value before transition (association of the enzyme) on the x-axis and the FRET value after transition (dissociation of the enzyme) on the y-axis. To extract association and dissociation constants, HaMMy ( 25 ) analysis was performed based on 161 individual FRET time trajectories.

    Journal: Nucleic Acids Research

    Article Title: Allosteric ring assembly and chemo-mechanical melting by the interaction between 5′-phosphate and λ exonuclease

    doi: 10.1093/nar/gkv1150

    Figure Lengend Snippet: The lack of a phosphate at a nick in the DNA substrate prevents the formation of a stable association with λ exonuclease during degradation. ( A ) Schematic of a DNA substrate without a 5′ phosphate group at a nick in the hydrolyzed strand (nick-5′OH-DNA). ( B ) Exemplary FRET time trajectory demonstrating diffusive back and forth movement of the enzyme in the absence of a 5′ phosphate in the substrate. Approximately 90% of all trajectories exhibited diffusive movements on the nick-5′OH-DNA. The blue trace is an idealized fit that is used to extract association and dissociation rates based on the HaMMy ( 25 ) algorithm. ( C ) Transition density plots (TDP) generated from the FRET value before transition (association of the enzyme) on the x-axis and the FRET value after transition (dissociation of the enzyme) on the y-axis. To extract association and dissociation constants, HaMMy ( 25 ) analysis was performed based on 161 individual FRET time trajectories.

    Article Snippet: The cell lysate was centrifuged for 30 min at 35 000 ×g , and re-suspended in 50 mM Tris–HCl (pH 8.0), 300 mM NaCl and 10 mM imidazole. λ exonuclease was purified by HisTrap FF (GE Healthcare) using a buffer (50 mM Tris–HCl (pH8.0), 300 mM NaCl and 300 mM Imidazol in a gradient method. (His)6 -tag-MBP was removed by TEV protease, which remained five glycines at the N-terminus of the protein, and purified again by HisTrap FF (GE Healthcare).

    Techniques: Generated

    Other exonucleases can perform strand exchange with ICP8. A, Strand exchange with full-length M13mp18 substrates was performed as described in Materials and Methods. Incubations were at 37 °C for 10–40 minutes, as indicated. All of the lanes included 100 ng of ssM13mp18 DNA and 100 ng of dsM13mp18 DNA linearized by EcoRI. Lane 1, no protein control; lane 2, 40 minutes incubation with ICP8 only; lanes 3–5, incubation with ICP8 and 13.9 nM UL12 for 10, 20, and 40 minutes, respectively; lanes 6–8, incubation with ICP8 and five units of lambda exonuclease for 10, 20, and 40 minutes, respectively; lanes 9–11, incubation with ICP8 and 100 units of ExoIII for 10, 20, and 40 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, M13mp18 dsDNA linearized by EcoRI; ss, M13mp18 ssDNA. B–E, Visualization of ICP8 catalyzed strand exchange reactions using dsDNA preresected with lambda exonuclease and ExoIII. Linear double-stranded ϕX174 DNA was subjected to digestion by lambda exonuclease (B and C) or ExoIII (D and E) as described in Materials and Methods. The nuclease-treated DNA was then used in strand exchange reactions. The classic strand exchange products are seen: sigma (B), alpha (D), and gapped circles (C and E). The scale bar represents the length of 1000 bp of dsDNA.

    Journal: Journal of molecular biology

    Article Title: Catalysis of Strand Exchange by the HSV-1 UL12 and ICP8 Proteins: Potent ICP8 Recombinase Activity is Revealed upon Resection of dsDNA Substrate by Nuclease

    doi: 10.1016/j.jmb.2004.07.012

    Figure Lengend Snippet: Other exonucleases can perform strand exchange with ICP8. A, Strand exchange with full-length M13mp18 substrates was performed as described in Materials and Methods. Incubations were at 37 °C for 10–40 minutes, as indicated. All of the lanes included 100 ng of ssM13mp18 DNA and 100 ng of dsM13mp18 DNA linearized by EcoRI. Lane 1, no protein control; lane 2, 40 minutes incubation with ICP8 only; lanes 3–5, incubation with ICP8 and 13.9 nM UL12 for 10, 20, and 40 minutes, respectively; lanes 6–8, incubation with ICP8 and five units of lambda exonuclease for 10, 20, and 40 minutes, respectively; lanes 9–11, incubation with ICP8 and 100 units of ExoIII for 10, 20, and 40 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, M13mp18 dsDNA linearized by EcoRI; ss, M13mp18 ssDNA. B–E, Visualization of ICP8 catalyzed strand exchange reactions using dsDNA preresected with lambda exonuclease and ExoIII. Linear double-stranded ϕX174 DNA was subjected to digestion by lambda exonuclease (B and C) or ExoIII (D and E) as described in Materials and Methods. The nuclease-treated DNA was then used in strand exchange reactions. The classic strand exchange products are seen: sigma (B), alpha (D), and gapped circles (C and E). The scale bar represents the length of 1000 bp of dsDNA.

    Article Snippet: Some experiments were also performed with lambda exonuclease purchased from Novagen.

    Techniques: Incubation, Staining

    Gel analysis of the effects of lambda exonuclease treatment on adapter assembly. Lanes 1 and 6, phosphorylated core adapter without overhang oligonucleotides; lanes 2 and 7, Adapter 1 without phosphorylation; lanes 3 and 8, Adapter 1 with phosphorylation;

    Journal: Nucleic Acids Research

    Article Title: Selective DNA amplification from complex genomes using universal double-sided adapters

    doi: 10.1093/nar/gnh019

    Figure Lengend Snippet: Gel analysis of the effects of lambda exonuclease treatment on adapter assembly. Lanes 1 and 6, phosphorylated core adapter without overhang oligonucleotides; lanes 2 and 7, Adapter 1 without phosphorylation; lanes 3 and 8, Adapter 1 with phosphorylation;

    Article Snippet: Core adapter, adapter after ligation of overhang oligonucleotides and adapter after final phosphorylation was treated with 2.5 U of Lambda Exonuclease (Roche, Indianapolis, IN) in a reaction buffer of 67 mM glycine–KOH, pH 9.4, 2.5 mM MgCl2 and 50 µg/ml BSA in 10 µl.

    Techniques:

    ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Journal: Nature Communications

    Article Title: Simplified ChIP-exo assays

    doi: 10.1038/s41467-018-05265-7

    Figure Lengend Snippet: ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Article Snippet: The λ exonuclease digestion (100 µl) containing: 20 U λ exonuclease (NEB), 1 × λ exonuclease reaction buffer (NEB), 0.1% Triton-X 100, and 5% DMSO was incubated for 30 min at 37 °C; then washed with 10 mM Tris-HCl, pH 8.0 at 4 °C.

    Techniques: Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Produced

    Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 DNA. DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease III (lanes 8 – 11 ) or 10 units λ exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.

    Journal: Genes & Development

    Article Title: Long palindromes formed in Streptomyces by nonrecombinational intra-strand annealing

    doi:

    Figure Lengend Snippet: Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 DNA. DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease III (lanes 8 – 11 ) or 10 units λ exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.

    Article Snippet: Aliquots of DNA were incubated with 100 units of E. coli exonuclease III or 10 units of bacteriophage λ exonuclease (either purchased from Life Technologies, Inc. or a gift of Drs. Deb Chatterjee and Per Harbury) at 37°C for 1 hr and the completeness of their digestion was confirmed by gel electrophoresis.

    Techniques: Southern Blot, Derivative Assay, Isolation, Agarose Gel Electrophoresis, Electrophoresis, Migration, Neutralization

    Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda exonuclease I and E. coli ).

    Journal: Nature protocols

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq

    doi: 10.1038/s41596-018-0055-0

    Figure Lengend Snippet: Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda exonuclease I and E. coli ).

    Article Snippet: M0293L) Lambda Exonuclease (New England BioLabs, cat.no.

    Techniques:

    Polymorphic fragments are BAL 31 sensitive. Agarose blocks of spleen DNA from individual VII were treated with Agarase, mixed with lambda DNA, and treated with BAL 31 for the indicated numbers of minutes. Samples were then digested with Mse I, run on a 0.4% gel, blotted, and hybridized to a (TTAGGG) n probe. A portion of the BAL 31 reaction mixture was digested with Hin dIII and hybridized to a lambda probe (data not shown). The sizes of the marker fragments are indicated on the left in kilobases.

    Journal: Molecular and Cellular Biology

    Article Title: Telomere Variation in Xenopus laevis

    doi:

    Figure Lengend Snippet: Polymorphic fragments are BAL 31 sensitive. Agarose blocks of spleen DNA from individual VII were treated with Agarase, mixed with lambda DNA, and treated with BAL 31 for the indicated numbers of minutes. Samples were then digested with Mse I, run on a 0.4% gel, blotted, and hybridized to a (TTAGGG) n probe. A portion of the BAL 31 reaction mixture was digested with Hin dIII and hybridized to a lambda probe (data not shown). The sizes of the marker fragments are indicated on the left in kilobases.

    Article Snippet: After the solution was preheated to 30°C, a portion was removed and the remaining 75 to 80 μl was digested with 1.5 to 3 U of BAL 31 exonuclease (New England Biolabs or Boehringer Mannheim).

    Techniques: Lambda DNA Preparation, Marker