exonuclease iii  (New England Biolabs)


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  • 99
    Name:
    Exonuclease III (E.coli) - 2
    Description:

    Catalog Number:
    M0206L
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    Score:
    85
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    Structured Review

    New England Biolabs exonuclease iii
    Fluorescence-emission spectra of zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex supramolecular fluorescent labels under different conditions: ( a ) Buffer; ( b ) Buffer + ZnPPIX; ( c ) RP (DNA duplex probe) + Circle DNA + hairpin probes (GHP) + Exonuclease <t>III</t> <t>(Exo</t> III) + ZnPPIX; ( d ) BPA + RP + Circle DNA + GHP + Exo III + ZnPPIX; C BPA = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, C ZnPPIX = 20 μM, RCA reaction time 1.5 h.

    https://www.bioz.com/result/exonuclease iii/product/New England Biolabs
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    Images

    1) Product Images from "A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification"

    Article Title: A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

    Journal: Nanomaterials

    doi: 10.3390/nano6100190

    Fluorescence-emission spectra of zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex supramolecular fluorescent labels under different conditions: ( a ) Buffer; ( b ) Buffer + ZnPPIX; ( c ) RP (DNA duplex probe) + Circle DNA + hairpin probes (GHP) + Exonuclease III (Exo III) + ZnPPIX; ( d ) BPA + RP + Circle DNA + GHP + Exo III + ZnPPIX; C BPA = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, C ZnPPIX = 20 μM, RCA reaction time 1.5 h.
    Figure Legend Snippet: Fluorescence-emission spectra of zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex supramolecular fluorescent labels under different conditions: ( a ) Buffer; ( b ) Buffer + ZnPPIX; ( c ) RP (DNA duplex probe) + Circle DNA + hairpin probes (GHP) + Exonuclease III (Exo III) + ZnPPIX; ( d ) BPA + RP + Circle DNA + GHP + Exo III + ZnPPIX; C BPA = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, C ZnPPIX = 20 μM, RCA reaction time 1.5 h.

    Techniques Used: Fluorescence

    Schematic illustration the principle of the fluorescent assay of Bisphenol A (BPA) based on the rolling circle amplification (RCA)/Exo III-combined cascade signal amplification strategy.
    Figure Legend Snippet: Schematic illustration the principle of the fluorescent assay of Bisphenol A (BPA) based on the rolling circle amplification (RCA)/Exo III-combined cascade signal amplification strategy.

    Techniques Used: Fluorescence, Amplification

    ( a ) Agarose gel (0.7%) electrophoresis: (1) DNA 1 alone; (2) P 1 alone; (3) Circle DNA alone; (4) GHP alone; (5) RP + Circle DNA; (6) BPA + RP + Circle DNA; (7) RP + Circle DNA + GHP; (8) BPA + RP + Circle DNA + GHP; ( b ) Atomic force microscope (AFM) images of amplification products of RCA/Exo III-combined cascade signal amplification reaction. C DNA1 = 1.0 μM, C P1 = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, RCA reaction time 1.5 h.
    Figure Legend Snippet: ( a ) Agarose gel (0.7%) electrophoresis: (1) DNA 1 alone; (2) P 1 alone; (3) Circle DNA alone; (4) GHP alone; (5) RP + Circle DNA; (6) BPA + RP + Circle DNA; (7) RP + Circle DNA + GHP; (8) BPA + RP + Circle DNA + GHP; ( b ) Atomic force microscope (AFM) images of amplification products of RCA/Exo III-combined cascade signal amplification reaction. C DNA1 = 1.0 μM, C P1 = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, RCA reaction time 1.5 h.

    Techniques Used: Agarose Gel Electrophoresis, Electrophoresis, Microscopy, Amplification

    2) Product Images from "DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION"

    Article Title: DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION

    Journal:

    doi: 10.1016/j.virol.2009.12.026

    Principle of the luciferase SV40 DNA replication assay (A) Schematic representation of the three plasmids used in the assay. The name of each plasmid is written on the left. The location of the SV40 origin of replication is represented by a black box with the position of the core (grey) and 21 bp-repeat regions (black) enlarged above. The nucleotide (nt) sequence boundaries of the origin are indicated. The locations of the CMV promoter and intron are indicated by dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of LT are indicated by white boxes. Amino acid boundaries of each protein are indicated below each box. (B) Schematic representation of the assay. A plasmid expressing SV40 LT (pLT) is co-transfected in cells along with a second plasmid containing the SV40 origin of replication (pFLORI40) and a firefly luciferase reporter gene. A third plasmid expressing Renilla luciferase (pRL) is also transfected as an internal control to normalize for variations in transfection efficiency. Viral DNA replication is measured using a dual-luciferase assay, at different times post-transfection.
    Figure Legend Snippet: Principle of the luciferase SV40 DNA replication assay (A) Schematic representation of the three plasmids used in the assay. The name of each plasmid is written on the left. The location of the SV40 origin of replication is represented by a black box with the position of the core (grey) and 21 bp-repeat regions (black) enlarged above. The nucleotide (nt) sequence boundaries of the origin are indicated. The locations of the CMV promoter and intron are indicated by dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of LT are indicated by white boxes. Amino acid boundaries of each protein are indicated below each box. (B) Schematic representation of the assay. A plasmid expressing SV40 LT (pLT) is co-transfected in cells along with a second plasmid containing the SV40 origin of replication (pFLORI40) and a firefly luciferase reporter gene. A third plasmid expressing Renilla luciferase (pRL) is also transfected as an internal control to normalize for variations in transfection efficiency. Viral DNA replication is measured using a dual-luciferase assay, at different times post-transfection.

    Techniques Used: Luciferase, Plasmid Preparation, Sequencing, Expressing, Transfection

    3) Product Images from "MukB-mediated Catenation of DNA Is ATP and MukEF Independent"

    Article Title: MukB-mediated Catenation of DNA Is ATP and MukEF Independent

    Journal:

    doi: 10.1074/jbc.M116.749994

    MukB-mediated catenation of DNA. A , the gapped DNA substrate has gaps that are less than 325 nucleotides in length. Either supercoiled pCG09 form I DNA (F1 DNA) or gapped pCG09 DNA prepared as described under “Experimental Procedures,” were treated with the indicated restriction enzymes that cut the plasmid DNA only once at the distance indicated from the nicking site. Complete digestion to linear product indicates that the DNA must be double-stranded at the indicated site. B, in the presence of Topo III, MukB mediates knotting of single DNA rings at low Mg2+ concentrations, but mediates catenation of DNA rings at moderate Mg2+ concentrations. Reaction mixtures containing 0.7 n m gapped DNA, 25 n m Topo III (a plus above the lane followed by an arrow indicates that all reactions analyzed in lanes covered by the arrow contained the indicated component), 300 n m MukB (as dimer), as indicated, and the indicated concentrations of Mg(OAc)2 , were incubated for 30 min at 37 °C, processed, and analyzed as described under “Experimental Procedures.” C , a gap is required to observe DNA catenation. Standard reaction mixtures containing either gapped DNA or DNA that had only been nicked with Nb.BbVCI (nicked DNA), 25 n m Topo III, as indicated, and the indicated concentration of MukB were incubated for 30 min at 37 °C, processed, and analyzed as described under “Experimental Procedures.” D , MukB-mediated catenation of DNA in the presence of Topo III as a function of MukB concentration. Standard reaction mixtures containing 25 n m Topo III and the indicated concentrations of MukB were incubated for 5 min at 37 °C, processed, and analyzed as described under “Experimental Procedures.” E, time course of DNA catenation at 300 n m MukB. A standard reaction mixture was increased 8-fold in size in volume and incubated at 37 °C. Aliquots (20 μl) were removed at the indicated times and processed and analyzed as described under “Experimental Procedures.” Note that the zero time aliquot was removed after mixing all components of the reaction together, accounting for the slight catenation and knotting observed. Catenated, large networks of catenated DNA rings that do not enter the gel, some of this material will wash off the top of the gels during handling. Intermediates , less complicated catenated networks of DNA rings were probably made of chains of DNA rings linked once. Gapped , gDNA starting material. Knotted , single DNA rings that have become knotted. Linear , a trace amount of linear pCG09 DNA present in the gDNA preparation. All assays were repeated at least three times, representative gels are shown.
    Figure Legend Snippet: MukB-mediated catenation of DNA. A , the gapped DNA substrate has gaps that are less than 325 nucleotides in length. Either supercoiled pCG09 form I DNA (F1 DNA) or gapped pCG09 DNA prepared as described under “Experimental Procedures,” were treated with the indicated restriction enzymes that cut the plasmid DNA only once at the distance indicated from the nicking site. Complete digestion to linear product indicates that the DNA must be double-stranded at the indicated site. B, in the presence of Topo III, MukB mediates knotting of single DNA rings at low Mg2+ concentrations, but mediates catenation of DNA rings at moderate Mg2+ concentrations. Reaction mixtures containing 0.7 n m gapped DNA, 25 n m Topo III (a plus above the lane followed by an arrow indicates that all reactions analyzed in lanes covered by the arrow contained the indicated component), 300 n m MukB (as dimer), as indicated, and the indicated concentrations of Mg(OAc)2 , were incubated for 30 min at 37 °C, processed, and analyzed as described under “Experimental Procedures.” C , a gap is required to observe DNA catenation. Standard reaction mixtures containing either gapped DNA or DNA that had only been nicked with Nb.BbVCI (nicked DNA), 25 n m Topo III, as indicated, and the indicated concentration of MukB were incubated for 30 min at 37 °C, processed, and analyzed as described under “Experimental Procedures.” D , MukB-mediated catenation of DNA in the presence of Topo III as a function of MukB concentration. Standard reaction mixtures containing 25 n m Topo III and the indicated concentrations of MukB were incubated for 5 min at 37 °C, processed, and analyzed as described under “Experimental Procedures.” E, time course of DNA catenation at 300 n m MukB. A standard reaction mixture was increased 8-fold in size in volume and incubated at 37 °C. Aliquots (20 μl) were removed at the indicated times and processed and analyzed as described under “Experimental Procedures.” Note that the zero time aliquot was removed after mixing all components of the reaction together, accounting for the slight catenation and knotting observed. Catenated, large networks of catenated DNA rings that do not enter the gel, some of this material will wash off the top of the gels during handling. Intermediates , less complicated catenated networks of DNA rings were probably made of chains of DNA rings linked once. Gapped , gDNA starting material. Knotted , single DNA rings that have become knotted. Linear , a trace amount of linear pCG09 DNA present in the gDNA preparation. All assays were repeated at least three times, representative gels are shown.

    Techniques Used: Plasmid Preparation, Incubation, Concentration Assay

    4) Product Images from "Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen"

    Article Title: Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen

    Journal: Protein Engineering, Design and Selection

    doi: 10.1093/protein/gzq057

    Asymmetric cell division and the role of SpoIIE. ( A ) In the pre-divisional cell (upper) and the mother cell (MC) following cell division (lower), SpoIIAA (AA) is phosphorylated and σ F is in a complex with SpoIIAB (AB 2 ). In the forespore (FS), σ F is free and AA and AB are in complex. SpoIIE (E) accumulates at the asymmetric septum, a double membrane structure. ( B ) The putative three domain structure of SpoIIE. The limits of the putative FtsZ-binding domain are uncertain.
    Figure Legend Snippet: Asymmetric cell division and the role of SpoIIE. ( A ) In the pre-divisional cell (upper) and the mother cell (MC) following cell division (lower), SpoIIAA (AA) is phosphorylated and σ F is in a complex with SpoIIAB (AB 2 ). In the forespore (FS), σ F is free and AA and AB are in complex. SpoIIE (E) accumulates at the asymmetric septum, a double membrane structure. ( B ) The putative three domain structure of SpoIIE. The limits of the putative FtsZ-binding domain are uncertain.

    Techniques Used: Binding Assay

    Assays of SpoIIE fragments. ( A ) SpoIIAA-phosphate dephosphorylation by the H1 and B1′ fragments monitored by native gel electrophoresis. Lane 1, SpoIIAA∼P (5 μg); lane 2, SpoIIAA (5 μg); lanes 3 and 4, SpoIIAA∼P (5 μg) incubated in the presence of the H1 fragment at 100:1 and 400:1 molar ratios, respectively; lanes 5 and 6, SpoIIAA∼P (5 μg) incubated in the presence of the B1′ fragment at 100:1 and 400:1 molar ratios, respectively. The conversion of SpoIIAA∼P to the lower mobility SpoIIAA species upon incubation with the SpoIIE fragments is evident. ( B ) Gel mobility shift assay of FtsZ binding by the H1 and B1′ fragments. Lane 1, FtsZ (7 μg); lane 2, FtsZ (7 μg) + 1 mM GTP; lane 3, SpoIIE H1 fragment (7 μg); lane 4, FtsZ (7 μg) + SpoIIE H1 (7 μg) + 1 mM GTP; lane 5, SpoIIE B1′ fragment (7 μg); lane 6, FtsZ (7 μg) + SpoIIE B1′ (7 μg) + 1 mM GTP; lane 7, FtsZ (7 μg) + 1mM GTP. There is no mobility shift evident from these gels other than the additional staining of material at the top of lane 4. ( C ) SEC-MALLS traces of the molecular mass and differential refractive index (dRI) versus time, of the eluate from a Superdex S200 column. The bold lines give molecular mass of the eluting species calculated from measurements of the refractive index and the multi-angle laser light scattering. Three traces for the (i) H1 (red), (ii) B1′ (green) and (iii) B2–B1 (blue) fragments are overlaid.
    Figure Legend Snippet: Assays of SpoIIE fragments. ( A ) SpoIIAA-phosphate dephosphorylation by the H1 and B1′ fragments monitored by native gel electrophoresis. Lane 1, SpoIIAA∼P (5 μg); lane 2, SpoIIAA (5 μg); lanes 3 and 4, SpoIIAA∼P (5 μg) incubated in the presence of the H1 fragment at 100:1 and 400:1 molar ratios, respectively; lanes 5 and 6, SpoIIAA∼P (5 μg) incubated in the presence of the B1′ fragment at 100:1 and 400:1 molar ratios, respectively. The conversion of SpoIIAA∼P to the lower mobility SpoIIAA species upon incubation with the SpoIIE fragments is evident. ( B ) Gel mobility shift assay of FtsZ binding by the H1 and B1′ fragments. Lane 1, FtsZ (7 μg); lane 2, FtsZ (7 μg) + 1 mM GTP; lane 3, SpoIIE H1 fragment (7 μg); lane 4, FtsZ (7 μg) + SpoIIE H1 (7 μg) + 1 mM GTP; lane 5, SpoIIE B1′ fragment (7 μg); lane 6, FtsZ (7 μg) + SpoIIE B1′ (7 μg) + 1 mM GTP; lane 7, FtsZ (7 μg) + 1mM GTP. There is no mobility shift evident from these gels other than the additional staining of material at the top of lane 4. ( C ) SEC-MALLS traces of the molecular mass and differential refractive index (dRI) versus time, of the eluate from a Superdex S200 column. The bold lines give molecular mass of the eluting species calculated from measurements of the refractive index and the multi-angle laser light scattering. Three traces for the (i) H1 (red), (ii) B1′ (green) and (iii) B2–B1 (blue) fragments are overlaid.

    Techniques Used: De-Phosphorylation Assay, Nucleic Acid Electrophoresis, Incubation, Mobility Shift, Binding Assay, Staining, Size-exclusion Chromatography

    5) Product Images from "Concentration-dependent organization of DNA by the dinoflagellate histone-like protein HCc3"

    Article Title: Concentration-dependent organization of DNA by the dinoflagellate histone-like protein HCc3

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm165

    HCc3 promotes ligase-mediated DNA concatenation. All images are presented as negative images. ( a ) Concatenation of 125-bp (left panel) and 2.8-kb (right panel) DNA fragments to a higher level in the presence of HCc3 at respective dimer/bp ratios. The linearity of the resulting products was confirmed by Exo III digestion (marked with ‘+/−’ signs). The 125-bp product was resolved on a 8% non-denaturing polyacrylamide gel, and the 2.8-kb product on a 1% TAE agarose gel. ( b ) Variations in intensity of DNA concatenation in the presence of HCc3 at different dimer/bp ratios. The marker bands indicate DNA sizes from 3 to 12 kb at 1-kb increments.
    Figure Legend Snippet: HCc3 promotes ligase-mediated DNA concatenation. All images are presented as negative images. ( a ) Concatenation of 125-bp (left panel) and 2.8-kb (right panel) DNA fragments to a higher level in the presence of HCc3 at respective dimer/bp ratios. The linearity of the resulting products was confirmed by Exo III digestion (marked with ‘+/−’ signs). The 125-bp product was resolved on a 8% non-denaturing polyacrylamide gel, and the 2.8-kb product on a 1% TAE agarose gel. ( b ) Variations in intensity of DNA concatenation in the presence of HCc3 at different dimer/bp ratios. The marker bands indicate DNA sizes from 3 to 12 kb at 1-kb increments.

    Techniques Used: Agarose Gel Electrophoresis, Marker

    6) Product Images from "Evolution of linear chromosomes and multipartite genomes in yeast mitochondria"

    Article Title: Evolution of linear chromosomes and multipartite genomes in yeast mitochondria

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1345

    PFGE analysis of the yeast mtDNAs. The whole-cell DNA samples were separated by PFGE using a CHEF Mapper XA Chiller System (Biorad), blotted onto a nylon membrane and hybridized with mtDNA-derived probes as described in ‘Material and Methods’ section. Lane 1— C. viswanathii CBS 4024; lane 2— C. sojae CBS 7871; lane 3— C. maltosa CBS 5611; lane 4— C. neerlandica NRRL Y-27057; lane 5— C. alai NRRL Y-27739; lane 6— C. labiduridarum NRRL Y-27940; lane 7— C. frijolesensis NRRL Y-48060; lane 8— C. subhashii CBS 10753; lane 9— C. jiufengensis CBS 10846; lane 10— C. albicans CBS 562. Note that three discrete bands migrating in the region
    Figure Legend Snippet: PFGE analysis of the yeast mtDNAs. The whole-cell DNA samples were separated by PFGE using a CHEF Mapper XA Chiller System (Biorad), blotted onto a nylon membrane and hybridized with mtDNA-derived probes as described in ‘Material and Methods’ section. Lane 1— C. viswanathii CBS 4024; lane 2— C. sojae CBS 7871; lane 3— C. maltosa CBS 5611; lane 4— C. neerlandica NRRL Y-27057; lane 5— C. alai NRRL Y-27739; lane 6— C. labiduridarum NRRL Y-27940; lane 7— C. frijolesensis NRRL Y-48060; lane 8— C. subhashii CBS 10753; lane 9— C. jiufengensis CBS 10846; lane 10— C. albicans CBS 562. Note that three discrete bands migrating in the region

    Techniques Used: Derivative Assay

    Multipartite linear-mapping genomes in C. labiduridarum and C. frijolesensis ( A ) PFGE separated samples of C. labiduridarum NRRL Y-27940 (lane 1) and C. frijolesensis NRRL Y-48060 (lane 2) were blotted onto a nylon membrane and hybridized with the radioactively labeled probes P-668 and H-1030 (regions hybridizing with both probes are shown as dashed lines). Presumed master (I) and two smaller chromosomes (II and III) are indicated. Note that the master chromosome occurs in four isomers (i.e. L III − R III − L II − R II (shown in the scheme), L III − R III − R II − L II , R III − L III − L II − R II and R III − L III − R II − L II . ‘L’ and ‘R’ indicate the left and the right telomere, respectively). The C. frijolesensis mtDNA (∼1 µg) was digested with BAL-31 nuclease ( B ) or exonuclease III (ExoIII) ( C ) as indicated. After nuclease inactivation, the DNA was digested with EcoRV, separated in 0.9% (w/v) agarose gel. The Southern blots were hybridized with the P-668 and EH-1350 probes specific for the left and the right arm of the master chromosome, respectively (see ‘Materials and Methods’ section). Arrows show the positions of the left (L) and right (R) terminal fragments and their fusions (R + R, R + L and L + L). Note that after ExoIII treatment the telomeric fragments form two subpopulations that differ in their sensitivity to the ExoIII treatment. This indicates that the linear mtDNA molecules possess an open structure with 5′ overhang or blunt end or covalently closed t-hairpin. ( D ) The C. frijolesensis mtDNA was treated with antarctic phosphatase and labeled with [γ 32 P]ATP and T4 polynucleotide kinase. The mtDNA was then digested with restriction endonuclease EcoRV (lane 1) or BglII (lane 2) and separated in 0.8% (w/v) agarose gel (left panel). The gel was fixed in 10% (v/v) methanol/10% (v/v) acetic acid for 30 min, dried overnight and autoradiographed (right panel). Arrows indicate the position of telomeric fragments containing the open structures accessible to terminal labeling.
    Figure Legend Snippet: Multipartite linear-mapping genomes in C. labiduridarum and C. frijolesensis ( A ) PFGE separated samples of C. labiduridarum NRRL Y-27940 (lane 1) and C. frijolesensis NRRL Y-48060 (lane 2) were blotted onto a nylon membrane and hybridized with the radioactively labeled probes P-668 and H-1030 (regions hybridizing with both probes are shown as dashed lines). Presumed master (I) and two smaller chromosomes (II and III) are indicated. Note that the master chromosome occurs in four isomers (i.e. L III − R III − L II − R II (shown in the scheme), L III − R III − R II − L II , R III − L III − L II − R II and R III − L III − R II − L II . ‘L’ and ‘R’ indicate the left and the right telomere, respectively). The C. frijolesensis mtDNA (∼1 µg) was digested with BAL-31 nuclease ( B ) or exonuclease III (ExoIII) ( C ) as indicated. After nuclease inactivation, the DNA was digested with EcoRV, separated in 0.9% (w/v) agarose gel. The Southern blots were hybridized with the P-668 and EH-1350 probes specific for the left and the right arm of the master chromosome, respectively (see ‘Materials and Methods’ section). Arrows show the positions of the left (L) and right (R) terminal fragments and their fusions (R + R, R + L and L + L). Note that after ExoIII treatment the telomeric fragments form two subpopulations that differ in their sensitivity to the ExoIII treatment. This indicates that the linear mtDNA molecules possess an open structure with 5′ overhang or blunt end or covalently closed t-hairpin. ( D ) The C. frijolesensis mtDNA was treated with antarctic phosphatase and labeled with [γ 32 P]ATP and T4 polynucleotide kinase. The mtDNA was then digested with restriction endonuclease EcoRV (lane 1) or BglII (lane 2) and separated in 0.8% (w/v) agarose gel (left panel). The gel was fixed in 10% (v/v) methanol/10% (v/v) acetic acid for 30 min, dried overnight and autoradiographed (right panel). Arrows indicate the position of telomeric fragments containing the open structures accessible to terminal labeling.

    Techniques Used: Labeling, Agarose Gel Electrophoresis

    7) Product Images from "A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta"

    Article Title: A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta

    Journal:

    doi:

    Identification and Mapping of the AVR-Pita Telomere by Using Virulent Mutants. Genomic DNAs were digested with EcoRI (A) or NcoI or SacI (B) , electrophoresed on 0.7% agarose gels, blotted to a Hybond-N membrane, and hybridized with the 32 P-labeled telomere repeat oligonucleotide 5′-(AACCCT)4 -3′. (A) Lanes 1 and 2 contain the DNAs of 6043 (virulent on Yashiro-mochi) and 4224-7-8 ( AVR-Pita ), respectively, which are the parental strains of cross number 4360 . DNAs loaded in the remaining lanes are from three pairs of avirulent strains and virulent mutants derived from them: 4360-17-1/CP917 (lanes 3 and 4); 4375-R-6/CP983 (lanes 5 and 6); and 4375-R-26/CP984 (lanes 7 and 8). The arrow marks the band corresponding to Tel 5. Bars at left indicate positions of λ HindIII DNA fragments used as length standards; from the top, they are 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb. (B) Distal restriction fragments of the AVR-Pita telomere were identified by restriction digestion and hybridization of genomic DNAs from two avirulent strain/mutant pairs (4375-R-26/CP984 in lanes 1 and 2, and 4360-17-1/CP917 in lanes 3 and 4). No changes were observed in telomere fragments > 4.4 kb, so these fragments are not shown. The telomeric 2.0-kb NcoI fragment and the telomeric 0.8-kb SacI fragment (arrows) were altered in each of the two independent mutants. Not shown are data identifying the distal 9-kb SalI fragment, 6.9-kb BamHI fragment, 6.5-kb BglII fragment, 4-kb EcoRV fragment, and 1.3-kb HindIII fragment. Bars at left indicate positions of λ HindIII DNA length standards in kilobases.
    Figure Legend Snippet: Identification and Mapping of the AVR-Pita Telomere by Using Virulent Mutants. Genomic DNAs were digested with EcoRI (A) or NcoI or SacI (B) , electrophoresed on 0.7% agarose gels, blotted to a Hybond-N membrane, and hybridized with the 32 P-labeled telomere repeat oligonucleotide 5′-(AACCCT)4 -3′. (A) Lanes 1 and 2 contain the DNAs of 6043 (virulent on Yashiro-mochi) and 4224-7-8 ( AVR-Pita ), respectively, which are the parental strains of cross number 4360 . DNAs loaded in the remaining lanes are from three pairs of avirulent strains and virulent mutants derived from them: 4360-17-1/CP917 (lanes 3 and 4); 4375-R-6/CP983 (lanes 5 and 6); and 4375-R-26/CP984 (lanes 7 and 8). The arrow marks the band corresponding to Tel 5. Bars at left indicate positions of λ HindIII DNA fragments used as length standards; from the top, they are 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb. (B) Distal restriction fragments of the AVR-Pita telomere were identified by restriction digestion and hybridization of genomic DNAs from two avirulent strain/mutant pairs (4375-R-26/CP984 in lanes 1 and 2, and 4360-17-1/CP917 in lanes 3 and 4). No changes were observed in telomere fragments > 4.4 kb, so these fragments are not shown. The telomeric 2.0-kb NcoI fragment and the telomeric 0.8-kb SacI fragment (arrows) were altered in each of the two independent mutants. Not shown are data identifying the distal 9-kb SalI fragment, 6.9-kb BamHI fragment, 6.5-kb BglII fragment, 4-kb EcoRV fragment, and 1.3-kb HindIII fragment. Bars at left indicate positions of λ HindIII DNA length standards in kilobases.

    Techniques Used: Labeling, Derivative Assay, Hybridization, Mutagenesis

    8) Product Images from "The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription"

    Article Title: The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20082678

    Sequences of pKMP2 plasmids treated with AID during transcription and amplified in E. coli in the presence of ampicillin. The WT sequence is shown on the top lines. The two MP2 nucleosome positioning sequences are in green (BamHI sites underlined). The in-frame ACG initiation codons are in italics and underlined. The in-frame stop codons are underlined and not italicized. The AID-created Ts resulting in a potential TATA box present in all 35 clones are in bold font. The Shine-Dalgarno sequence (AGGAAG) is in blue. The transcription start site of unmutated pKMP2 plasmids in E. coli is in purple and underlined. The 5′ end of the Amp r coding region is in red. The ACG start codon for Amp r is at 3041–3043. Dashed lines represent agreement with the unmutated pKMP2 with mutations indicated by lowercase font. The sequences were obtained from one of three experiments.
    Figure Legend Snippet: Sequences of pKMP2 plasmids treated with AID during transcription and amplified in E. coli in the presence of ampicillin. The WT sequence is shown on the top lines. The two MP2 nucleosome positioning sequences are in green (BamHI sites underlined). The in-frame ACG initiation codons are in italics and underlined. The in-frame stop codons are underlined and not italicized. The AID-created Ts resulting in a potential TATA box present in all 35 clones are in bold font. The Shine-Dalgarno sequence (AGGAAG) is in blue. The transcription start site of unmutated pKMP2 plasmids in E. coli is in purple and underlined. The 5′ end of the Amp r coding region is in red. The ACG start codon for Amp r is at 3041–3043. Dashed lines represent agreement with the unmutated pKMP2 with mutations indicated by lowercase font. The sequences were obtained from one of three experiments.

    Techniques Used: Amplification, Sequencing, Clone Assay

    9) Product Images from "Quantum Dot Doping-Induced Photoluminescence for Facile, Label-Free, and Sensitive Pyrophosphatase Activity Assay and Inhibitor Screening"

    Article Title: Quantum Dot Doping-Induced Photoluminescence for Facile, Label-Free, and Sensitive Pyrophosphatase Activity Assay and Inhibitor Screening

    Journal: Nanomaterials

    doi: 10.3390/nano9010111

    ( A ) Fluorescence spectra of QD upon incubation of different concentrations of PPase from 0 to 20 mU/mL; ( B ) Relationship of the fluorescence intensity of QD at 510 nm, with the PPase concentration. Inset shows the corresponding linear range. ( C ) The specificity of the proposed sensing strategy toward PPase, against Exo I, Exo III, GOx, and lysozyme. The concentration of PPase was 10 mU/mL, and the concentrations for all other proteins were 0.1 U/mL. ( D ) The fluorescence intensities of QD, into the mixture of Cu 2+ (10 µM), Cu 2+ (10 µM) + PPi (20 µM), and Cu 2+ (10 µM) + PPi (20 µM) + PPase (1, 10 mU/mL), respectively, in the buffer solution and 5% diluted fetal bovine serum (FBS). Error bars in ( B – D ), for each data point, indicate the standard deviations, which were calculated on the basis of three repetitive experiments.
    Figure Legend Snippet: ( A ) Fluorescence spectra of QD upon incubation of different concentrations of PPase from 0 to 20 mU/mL; ( B ) Relationship of the fluorescence intensity of QD at 510 nm, with the PPase concentration. Inset shows the corresponding linear range. ( C ) The specificity of the proposed sensing strategy toward PPase, against Exo I, Exo III, GOx, and lysozyme. The concentration of PPase was 10 mU/mL, and the concentrations for all other proteins were 0.1 U/mL. ( D ) The fluorescence intensities of QD, into the mixture of Cu 2+ (10 µM), Cu 2+ (10 µM) + PPi (20 µM), and Cu 2+ (10 µM) + PPi (20 µM) + PPase (1, 10 mU/mL), respectively, in the buffer solution and 5% diluted fetal bovine serum (FBS). Error bars in ( B – D ), for each data point, indicate the standard deviations, which were calculated on the basis of three repetitive experiments.

    Techniques Used: Fluorescence, Incubation, Concentration Assay

    10) Product Images from "Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification"

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn1014

    Real-time quantification of hly gene in L. monocytogenes genomic DNA by PG–RCA. ( A ) Circular probe LM for detection of pathogenic L. monocytogenes genomic DNA. The probe targets the complementary strand of virulence gene, hly (GeneBank GeneID 2797098), encoding a cholesterol-dependent cytolysin, listeriolysin O (LLO). Circular probe LM contains three repeats of a 26-base sequence complementary to the gene including a nicking site for Nb.BsmI. Since these repeat sequences have 5-base overlaps each other, the circular probe comprises three repeats of a 21-base sequence (red, blue and green). ( B ) Genomic DNA from L. monocytogenes (0.1–100 pg) was analyzed by real-time PG–RCA with circular probe LM. Threshold time ( T T ) was plotted against the L. monocytogenes genomic DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 0.1 and 100 pg L. monocytogenes genomic DNA and its formulation is T T = −19.1 log 10 (S) + 233 ( R 2 =0.964). Perforated line indicates average T T value of the negative controls ( n =2). Limit of detection is 0.163 pg (∼60 molecules) of L. monocytogenes genomic DNA by calculation from the intersection of both lines. ( C ) Genomic DNA (100 pg) from L. monocytogenes , L. innocua , E. coli and S. enterica were analyzed by real-time PG–RCA with circular probe LM and their threshold times were compared with the values for L. monocytogenes (100 pg). ‘No DNA’ indicates the negative controls.
    Figure Legend Snippet: Real-time quantification of hly gene in L. monocytogenes genomic DNA by PG–RCA. ( A ) Circular probe LM for detection of pathogenic L. monocytogenes genomic DNA. The probe targets the complementary strand of virulence gene, hly (GeneBank GeneID 2797098), encoding a cholesterol-dependent cytolysin, listeriolysin O (LLO). Circular probe LM contains three repeats of a 26-base sequence complementary to the gene including a nicking site for Nb.BsmI. Since these repeat sequences have 5-base overlaps each other, the circular probe comprises three repeats of a 21-base sequence (red, blue and green). ( B ) Genomic DNA from L. monocytogenes (0.1–100 pg) was analyzed by real-time PG–RCA with circular probe LM. Threshold time ( T T ) was plotted against the L. monocytogenes genomic DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 0.1 and 100 pg L. monocytogenes genomic DNA and its formulation is T T = −19.1 log 10 (S) + 233 ( R 2 =0.964). Perforated line indicates average T T value of the negative controls ( n =2). Limit of detection is 0.163 pg (∼60 molecules) of L. monocytogenes genomic DNA by calculation from the intersection of both lines. ( C ) Genomic DNA (100 pg) from L. monocytogenes , L. innocua , E. coli and S. enterica were analyzed by real-time PG–RCA with circular probe LM and their threshold times were compared with the values for L. monocytogenes (100 pg). ‘No DNA’ indicates the negative controls.

    Techniques Used: Sequencing, Concentration Assay

    11) Product Images from "Molecular interactions of Escherichia coli ExoIX and identification of its associated 3?-5? exonuclease activity"

    Article Title: Molecular interactions of Escherichia coli ExoIX and identification of its associated 3?-5? exonuclease activity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm396

    Chromatographic separation of 3′-5′ exodeoxyribonuclease activity associated with preparations of Exonuclease IX. ( A ) SDS–PAGE analysis of the purification of ExoIX from cell lysate of induced BL21 (pJONEX/ xni , pcI857). SPL, cleared cell lysate applied to SP/first Heparin column (28 µg); QL, Q load (6 µg); H2L, second Heparin column load (5 µg); IX, concentrated ExoIX eluate from second Heparin (7.5 µg). (B–D). Eluted fractions from first Heparin column were separated by SDS–PAGE. ( B ) Ethidium bromide stained substrate gel. High molecular weight DNA cast in the gel fluoresces with UV, while regions of DNA degradation appear as darker bands. Early fractions (lanes 1–4), contain detectable exonuclease activity. ( C ) The same gel counter-stained with Coomassie G250. Over-expressed ExoIX is eluted in later fractions (lanes 5 and 6). ( D ) Superimposition of images in panels B and C, demonstrating that exonuclease activity can be resolved from ExoIX. A fraction represented in lane 4 was used for subsequent enrichment and identification of the co-purifying nuclease. Lanes, 1–6, heparin fractions (2.5 µl); 7, loading sample (5 µl); 8, flow through (5 µl). ( E ) Highly purified ExoIX lacks activity on a single-stranded DNA substrate (34-mer). Protein samples taken during the purification of ExoIX were incubated with 15 fmol 32 P-labelled 34-mer at 37°C for 10 min in the presence of 10 mM MgCl 2 and the reaction products separated by denaturing PAGE. Reactions (10 µl) contained varying amounts of protein. SPL, 0.7 and 0.07 µg of protein from cell-free extract of induced cells expressing ExoIX; QL, 0.1 and 0.01 µg of protein loaded on to first anion exchange column; H2L, 3 and 0.3 µg of protein from sample loaded onto second heparin column; IX, contains samples from final purified fraction of ExoIX eluted from second heparin column, 5 and 0.5 µg; two positive controls are also shown, bacteriophage T5 D15 exonuclease (T5), 0.1 and 0.01 µg and exonuclease III (III), 0.03 and 0.003 µg.
    Figure Legend Snippet: Chromatographic separation of 3′-5′ exodeoxyribonuclease activity associated with preparations of Exonuclease IX. ( A ) SDS–PAGE analysis of the purification of ExoIX from cell lysate of induced BL21 (pJONEX/ xni , pcI857). SPL, cleared cell lysate applied to SP/first Heparin column (28 µg); QL, Q load (6 µg); H2L, second Heparin column load (5 µg); IX, concentrated ExoIX eluate from second Heparin (7.5 µg). (B–D). Eluted fractions from first Heparin column were separated by SDS–PAGE. ( B ) Ethidium bromide stained substrate gel. High molecular weight DNA cast in the gel fluoresces with UV, while regions of DNA degradation appear as darker bands. Early fractions (lanes 1–4), contain detectable exonuclease activity. ( C ) The same gel counter-stained with Coomassie G250. Over-expressed ExoIX is eluted in later fractions (lanes 5 and 6). ( D ) Superimposition of images in panels B and C, demonstrating that exonuclease activity can be resolved from ExoIX. A fraction represented in lane 4 was used for subsequent enrichment and identification of the co-purifying nuclease. Lanes, 1–6, heparin fractions (2.5 µl); 7, loading sample (5 µl); 8, flow through (5 µl). ( E ) Highly purified ExoIX lacks activity on a single-stranded DNA substrate (34-mer). Protein samples taken during the purification of ExoIX were incubated with 15 fmol 32 P-labelled 34-mer at 37°C for 10 min in the presence of 10 mM MgCl 2 and the reaction products separated by denaturing PAGE. Reactions (10 µl) contained varying amounts of protein. SPL, 0.7 and 0.07 µg of protein from cell-free extract of induced cells expressing ExoIX; QL, 0.1 and 0.01 µg of protein loaded on to first anion exchange column; H2L, 3 and 0.3 µg of protein from sample loaded onto second heparin column; IX, contains samples from final purified fraction of ExoIX eluted from second heparin column, 5 and 0.5 µg; two positive controls are also shown, bacteriophage T5 D15 exonuclease (T5), 0.1 and 0.01 µg and exonuclease III (III), 0.03 and 0.003 µg.

    Techniques Used: Activity Assay, SDS Page, Purification, Staining, Molecular Weight, Flow Cytometry, Incubation, Polyacrylamide Gel Electrophoresis, Expressing

    12) Product Images from "Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification"

    Article Title: Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr909

    RNA detection mechanism by three-way junction probe and primer generation-rolling circle amplification. ( A ) Three-way junction (3WJ) probes (primer and template) are designed to form a 3WJ structure on target RNA, however they do not interact each other without target RNA because their complementary sequence is only 6–8 bases. ( B ) Addition of DNA polymerase and nicking enzyme initiates a reaction cycle of primer extension, nicking reaction and signal primer generation under an isothermal condition to generate signal primers. ( C ) The generated signal primers can be detected by primer generation-rolling circle amplification.
    Figure Legend Snippet: RNA detection mechanism by three-way junction probe and primer generation-rolling circle amplification. ( A ) Three-way junction (3WJ) probes (primer and template) are designed to form a 3WJ structure on target RNA, however they do not interact each other without target RNA because their complementary sequence is only 6–8 bases. ( B ) Addition of DNA polymerase and nicking enzyme initiates a reaction cycle of primer extension, nicking reaction and signal primer generation under an isothermal condition to generate signal primers. ( C ) The generated signal primers can be detected by primer generation-rolling circle amplification.

    Techniques Used: RNA Detection, Amplification, Sequencing, Generated

    13) Product Images from "The mitochondrial genome of the pathogenic yeast Candida subhashii: GC-rich linear DNA with a protein covalently attached to the 5? termini"

    Article Title: The mitochondrial genome of the pathogenic yeast Candida subhashii: GC-rich linear DNA with a protein covalently attached to the 5? termini

    Journal: Microbiology

    doi: 10.1099/mic.0.038646-0

    The termini of the linear mtDNA are bound by a protein. (a) DNA samples were prepared from C. subhashii (see Methods) and separated by PFGE in a 1.5 % agarose gel. The gel was stained with 0.5 μg ml −1 ethidium bromide (EtBr) and transferred onto a nylon membrane. The blot was hybridized with radioactively labelled mtDNA from C. subhashii . Lane 1, isolated mtDNA; lanes 2 and 3, total cellular DNA prepared in agarose blocks treated or untreated with proteinase K, respectively. (b) Approximately 1 μg of isolated mtDNA was treated with exonuclease III (ExoIII) (left panel) or BAL-31 nuclease (right panel), as indicated. The mtDNA was then extracted from reactions, digested with Xba I endonuclease and electrophoretically separated. Note that the terminal fragments are sensitive to ExoIII but apparently not to BAL-31, indicating the possibility that the linear molecules have their 5′ termini blocked. L and R, positions of the 1527 and 2803 bp terminal restriction enzyme fragments, respectively; C, position of the internal control (a 1040 bp long linear blunt-ended DNA fragment) mixed with mtDNA prior to digestion with BAL-31 nuclease. (c) The mtDNA–protein complexes were isolated as described in Methods, digested with restriction endonucleases Cla I or Pvu I, and treated or not treated with proteinase K. The positions of terminal restriction enzyme fragments generated by Pvu I (833 and 2339 bp) and Cla I (547 bp) are indicated as L, R and L+R, respectively. Note that both terminal Cla I fragments have identical sizes, as this enzyme digests the C. subhashii mtDNA within TIRs.
    Figure Legend Snippet: The termini of the linear mtDNA are bound by a protein. (a) DNA samples were prepared from C. subhashii (see Methods) and separated by PFGE in a 1.5 % agarose gel. The gel was stained with 0.5 μg ml −1 ethidium bromide (EtBr) and transferred onto a nylon membrane. The blot was hybridized with radioactively labelled mtDNA from C. subhashii . Lane 1, isolated mtDNA; lanes 2 and 3, total cellular DNA prepared in agarose blocks treated or untreated with proteinase K, respectively. (b) Approximately 1 μg of isolated mtDNA was treated with exonuclease III (ExoIII) (left panel) or BAL-31 nuclease (right panel), as indicated. The mtDNA was then extracted from reactions, digested with Xba I endonuclease and electrophoretically separated. Note that the terminal fragments are sensitive to ExoIII but apparently not to BAL-31, indicating the possibility that the linear molecules have their 5′ termini blocked. L and R, positions of the 1527 and 2803 bp terminal restriction enzyme fragments, respectively; C, position of the internal control (a 1040 bp long linear blunt-ended DNA fragment) mixed with mtDNA prior to digestion with BAL-31 nuclease. (c) The mtDNA–protein complexes were isolated as described in Methods, digested with restriction endonucleases Cla I or Pvu I, and treated or not treated with proteinase K. The positions of terminal restriction enzyme fragments generated by Pvu I (833 and 2339 bp) and Cla I (547 bp) are indicated as L, R and L+R, respectively. Note that both terminal Cla I fragments have identical sizes, as this enzyme digests the C. subhashii mtDNA within TIRs.

    Techniques Used: Agarose Gel Electrophoresis, Staining, Isolation, Generated

    14) Product Images from "In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium"

    Article Title: In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177751

    DdrC protects DNA against degradation by nucleases. Protection of supercoiled pBR322 plasmid (3.5 nM) from DNase I activity (0.1 U) (panel a), linear pBR322 (3.5 nM) from Exonuclease III activity (200 U) (panel b) and phiX174 ssDNA (5.9 nM) from Mung Bean Nuclease activity (1 U) (panel c) by 7 μM, 7 μM, and 2 μM DdrC, respectively. Lanes C: DNA controls without protein. Lanes 1: DNA incubation with nuclease alone. Lanes 2: DNA incubation with DdrC alone. Lanes 3: DNA pre-incubated with DdrC 15 min at 4°C before addition of nuclease. Lanes 4: Reaction products corresponding to lane 3 were further treated with Proteinase K/SDS. Panel a, lane 5: DdrC and DNase I were simultaneously incubated with supercoiled DNA before treatment with Proteinase K/SDS.
    Figure Legend Snippet: DdrC protects DNA against degradation by nucleases. Protection of supercoiled pBR322 plasmid (3.5 nM) from DNase I activity (0.1 U) (panel a), linear pBR322 (3.5 nM) from Exonuclease III activity (200 U) (panel b) and phiX174 ssDNA (5.9 nM) from Mung Bean Nuclease activity (1 U) (panel c) by 7 μM, 7 μM, and 2 μM DdrC, respectively. Lanes C: DNA controls without protein. Lanes 1: DNA incubation with nuclease alone. Lanes 2: DNA incubation with DdrC alone. Lanes 3: DNA pre-incubated with DdrC 15 min at 4°C before addition of nuclease. Lanes 4: Reaction products corresponding to lane 3 were further treated with Proteinase K/SDS. Panel a, lane 5: DdrC and DNase I were simultaneously incubated with supercoiled DNA before treatment with Proteinase K/SDS.

    Techniques Used: Plasmid Preparation, Activity Assay, Incubation

    15) Product Images from "Sequence analysis of an Archaeal virus isolated from a hypersaline lake in Inner Mongolia, China"

    Article Title: Sequence analysis of an Archaeal virus isolated from a hypersaline lake in Inner Mongolia, China

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-8-410

    Panel a. 0.8% TAE agarose gel showing virus BJ1 genome sensitivity to nucleases. Lanes 1 and 4 , undigested controls; Lane 2, DNAse treated; Lane 3 RNase treated; Lane 5, exonuclease III treated. Panel b, 1% agarose 0.5× TBE pulse field gel; lanes 1 and 4 size markers (kbps), lanes 2 and 3 BJ1 virus genome. Panel c, Bam H1 enzyme digest of virus BJ1 genomic DNA, DNA size markers are shown on the left (kbps). The image has been overexposed to show the smaller bands.
    Figure Legend Snippet: Panel a. 0.8% TAE agarose gel showing virus BJ1 genome sensitivity to nucleases. Lanes 1 and 4 , undigested controls; Lane 2, DNAse treated; Lane 3 RNase treated; Lane 5, exonuclease III treated. Panel b, 1% agarose 0.5× TBE pulse field gel; lanes 1 and 4 size markers (kbps), lanes 2 and 3 BJ1 virus genome. Panel c, Bam H1 enzyme digest of virus BJ1 genomic DNA, DNA size markers are shown on the left (kbps). The image has been overexposed to show the smaller bands.

    Techniques Used: Agarose Gel Electrophoresis

    16) Product Images from "Elongation complexes of Thermus thermophilus RNA polymerase that possess distinct translocation conformations"

    Article Title: Elongation complexes of Thermus thermophilus RNA polymerase that possess distinct translocation conformations

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl559

    Probing of the EC translocation conformations. ( A ) Exo III footprints of EC14 and EC15. Tth EC14 (lanes 1 and 2) was assembled as described in Materials and Methods. EC15 (lanes 3 and 4) was obtained by incubation of EC14 with substrate ATP for 2 min at 60°C. Exo III (0.02 U/μl) was added for 5 (lanes 1 and 3) or 10 (lanes 2 and 4) min at 37°C. ( B ) Schematics of RNAP front edge oscillations in EC14 and EC15. ( C ) Photo cross-linking patterns of Tth EC14 and EC15, EC14 and EC15 containing 5′ 32 P-labeled RNA primers were prepared as illustrated (left). The photo cross-linking analog 4-thio UTP (50 μM) was incorporated into the transcript for 2 min at 60°C followed UV light irradiation for 5 min at RT. The cross-linked species were separated using gel electrophoresis (right).
    Figure Legend Snippet: Probing of the EC translocation conformations. ( A ) Exo III footprints of EC14 and EC15. Tth EC14 (lanes 1 and 2) was assembled as described in Materials and Methods. EC15 (lanes 3 and 4) was obtained by incubation of EC14 with substrate ATP for 2 min at 60°C. Exo III (0.02 U/μl) was added for 5 (lanes 1 and 3) or 10 (lanes 2 and 4) min at 37°C. ( B ) Schematics of RNAP front edge oscillations in EC14 and EC15. ( C ) Photo cross-linking patterns of Tth EC14 and EC15, EC14 and EC15 containing 5′ 32 P-labeled RNA primers were prepared as illustrated (left). The photo cross-linking analog 4-thio UTP (50 μM) was incorporated into the transcript for 2 min at 60°C followed UV light irradiation for 5 min at RT. The cross-linked species were separated using gel electrophoresis (right).

    Techniques Used: Translocation Assay, Incubation, Labeling, Irradiation, Nucleic Acid Electrophoresis

    17) Product Images from "The Binding Site of Transcription Factor YY1 Is Required for Intramolecular Recombination between Terminally Repeated Sequences of Linear Replicative Hepatitis B Virus DNA"

    Article Title: The Binding Site of Transcription Factor YY1 Is Required for Intramolecular Recombination between Terminally Repeated Sequences of Linear Replicative Hepatitis B Virus DNA

    Journal:

    doi:

    Detection of the recombination junction in Mbo I-resistant cccDNA. (A) Schematic representation of PCR products (911 ± α bp: nt 1135 to 2045), containing the recombination junction (arrowhead), amplified from Mbo I-resistant cccDNA with the F and R primers. The DNA region (647 bp; nt 1399 to 2045) amplified from pBS-HBV3 DNA, using the F* and R primers, is also shown on the top. The locations of the three primers are shown in Fig. . Numbers above the open rectangles show the nucleotide numbers of Tsp 509I recognition sites in the HBV DNA sequence , and the numbers in or under the open rectangles indicate the sizes (in base pairs) of Tsp 509I-digested DNA fragments. (B) Detection of PCR products. Shown are PCR products generated by 2% agarose gel electrophoresis (lanes 3 to 6) from Mbo I-resistant cccDNA in cells transfected with WT HBV, HBV ΔDR, HBV Δr, or HBV ΔYY (Fig. B). Products generated with pBS-HBV3 (lane 1) and WT HBV DNA (lane 2) by the same treatment are also shown as controls. Lane M and numbers at the left indicate the size markers in base pairs. (C) Detection of the DNA fragment containing the recombination junction. The PCR products obtained in panel B were digested with Tsp 509I and subjected to 15 to 25% gradient polyacrylamide gel electrophoresis. Lanes 2 to 5 contain the samples from Mbo I-resistant cccDNA in WT-HBV-, HBV ΔDR-, HBV Δr-, and HBV ΔYY-transfected cells, respectively. The PCR product generated from pBS-HBV3 DNA by the same treatment was also loaded as the size marker control (lane 1).
    Figure Legend Snippet: Detection of the recombination junction in Mbo I-resistant cccDNA. (A) Schematic representation of PCR products (911 ± α bp: nt 1135 to 2045), containing the recombination junction (arrowhead), amplified from Mbo I-resistant cccDNA with the F and R primers. The DNA region (647 bp; nt 1399 to 2045) amplified from pBS-HBV3 DNA, using the F* and R primers, is also shown on the top. The locations of the three primers are shown in Fig. . Numbers above the open rectangles show the nucleotide numbers of Tsp 509I recognition sites in the HBV DNA sequence , and the numbers in or under the open rectangles indicate the sizes (in base pairs) of Tsp 509I-digested DNA fragments. (B) Detection of PCR products. Shown are PCR products generated by 2% agarose gel electrophoresis (lanes 3 to 6) from Mbo I-resistant cccDNA in cells transfected with WT HBV, HBV ΔDR, HBV Δr, or HBV ΔYY (Fig. B). Products generated with pBS-HBV3 (lane 1) and WT HBV DNA (lane 2) by the same treatment are also shown as controls. Lane M and numbers at the left indicate the size markers in base pairs. (C) Detection of the DNA fragment containing the recombination junction. The PCR products obtained in panel B were digested with Tsp 509I and subjected to 15 to 25% gradient polyacrylamide gel electrophoresis. Lanes 2 to 5 contain the samples from Mbo I-resistant cccDNA in WT-HBV-, HBV ΔDR-, HBV Δr-, and HBV ΔYY-transfected cells, respectively. The PCR product generated from pBS-HBV3 DNA by the same treatment was also loaded as the size marker control (lane 1).

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Generated, Agarose Gel Electrophoresis, Transfection, Polyacrylamide Gel Electrophoresis, Marker

    18) Product Images from "Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase"

    Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0157270

    TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.
    Figure Legend Snippet: TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.

    Techniques Used: Activity Assay, Labeling, Incubation, Negative Control, Positive Control, Purification, Transformation Assay, Transfection

    19) Product Images from "Reverse transcription of the pFOXC mitochondrial retroplasmids of Fusarium oxysporum is protein primed"

    Article Title: Reverse transcription of the pFOXC mitochondrial retroplasmids of Fusarium oxysporum is protein primed

    Journal: Mobile DNA

    doi: 10.1186/1759-8753-2-1

    Model for protein-primed reverse transcription by the pFOXC-reverse transcriptase (RT) . Transcription of the pFOXC plasmid DNA molecules produces full-length RNAs that appear to function as both mRNAs for the synthesis of the RT and as templates for (-) strand cDNA synthesis [ 6 ]. Transcripts of pFOXC3 terminate in approximately three pentameric repeats, whereas transcripts of pFOXC1 terminate in approximately four copies of a 3 bp sequence (the 3' terminus of in vitro RNA used in this study is shown). Following production of the plasmid-encoded RT, deoxynucleotidylation occurs with the covalent addition of dAMP to a tyrosine residue of the 60 kDa pFOXC3-RT, followed by incorporation of deoxyguanosine monophosphate (dGMP) and a third nucleotide. Deoxynucleotidylation of the pFOXC1-RT results in the addition of thymidine monophosphate (TMP) to the RT, followed by one or more deoxynucleotide monophosphates (dNMPs) (a second TMP is shown). The resulting RT-(dNMP) n complex would have complementarity to the corresponding terminal repeat. Based on studies of protein-primed DNA elements, the model predicts that the complex anneals to the penultimate 3' repeat of the template (shown for pFOXC1 only). Following the synthesis of a unit-length repeat, the RT-(dNMP) n complex undergoes a slideback and is repositioned opposite the terminal repeat. The nascent cDNA is elongated via reverse transcription of the template by the 5'-linked RT or by a separate RT recruited to the complex. The model could also accommodate an increase in the number of repeats, depending on the number of slideback events that occur.
    Figure Legend Snippet: Model for protein-primed reverse transcription by the pFOXC-reverse transcriptase (RT) . Transcription of the pFOXC plasmid DNA molecules produces full-length RNAs that appear to function as both mRNAs for the synthesis of the RT and as templates for (-) strand cDNA synthesis [ 6 ]. Transcripts of pFOXC3 terminate in approximately three pentameric repeats, whereas transcripts of pFOXC1 terminate in approximately four copies of a 3 bp sequence (the 3' terminus of in vitro RNA used in this study is shown). Following production of the plasmid-encoded RT, deoxynucleotidylation occurs with the covalent addition of dAMP to a tyrosine residue of the 60 kDa pFOXC3-RT, followed by incorporation of deoxyguanosine monophosphate (dGMP) and a third nucleotide. Deoxynucleotidylation of the pFOXC1-RT results in the addition of thymidine monophosphate (TMP) to the RT, followed by one or more deoxynucleotide monophosphates (dNMPs) (a second TMP is shown). The resulting RT-(dNMP) n complex would have complementarity to the corresponding terminal repeat. Based on studies of protein-primed DNA elements, the model predicts that the complex anneals to the penultimate 3' repeat of the template (shown for pFOXC1 only). Following the synthesis of a unit-length repeat, the RT-(dNMP) n complex undergoes a slideback and is repositioned opposite the terminal repeat. The nascent cDNA is elongated via reverse transcription of the template by the 5'-linked RT or by a separate RT recruited to the complex. The model could also accommodate an increase in the number of repeats, depending on the number of slideback events that occur.

    Techniques Used: Plasmid Preparation, Sequencing, In Vitro

    20) Product Images from "High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells"

    Article Title: High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells

    Journal: Theranostics

    doi: 10.7150/thno.23852

    Enzymatic resistance of phosphorothioate-modified Au-TDNNs. (A, B) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by DNase I. (C, D) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by Exo III.
    Figure Legend Snippet: Enzymatic resistance of phosphorothioate-modified Au-TDNNs. (A, B) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by DNase I. (C, D) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by Exo III.

    Techniques Used: Modification, Fluorescence

    Related Articles

    Clone Assay:

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    Article Snippet: These subclones were tested for phenotypic rescue of lf3-2 , lf3-5 , and lf3-6 by cotransformation. .. Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to . .. The sequence of the LF3 gene was obtained by sequencing plasmid clones with universal sequencing primers or gene-specific primers (DNA Sequencing and Synthesis Facility, Iowa State University, or Advanced Genetics Analysis Center, University of Minnesota).

    Centrifugation:

    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: For denaturing polyacrylamide gel electrophoresis (PAGE) analysis, samples were extracted with 150 μl of CHCl3 containing 1/25 volume of isoamyl alcohol, the aqueous phase was precipitated with 2.5 volumes of 95% ethanol, and the DNA was recovered by centrifugation. .. Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above.

    Amplification:

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    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: Subsequently, 0.75 μl Exonuclease I (NEB, M0293S) and 0.75 μl Exonuclease III (NEB, M0206S) were added into the reaction and jointly incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min. .. The successfully circularized DNA was purified using the Oligo Clean & concentrator kit (Zymo, D4060).

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: HIS4 (−96/+112) was amplified by PCR in a 1–2-ml reaction using the 32 P-labeled upstream primer and downstream primer (5′-TATTCCATGAGGCCAGATC-3′) and purified by electrophoresis on a 2% agarose gel. .. Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
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    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each). .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

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    Article Title: Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation, Replication, and Persistence
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    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to . .. The sequence of the LF3 gene was obtained by sequencing plasmid clones with universal sequencing primers or gene-specific primers (DNA Sequencing and Synthesis Facility, Iowa State University, or Advanced Genetics Analysis Center, University of Minnesota).

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Second-strand cDNA was then bulk amplified with LongAmp HotStart Taq using Pac_for_dU and Pac_rev_dU primers, which contain an internal 5′-dUTP. .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates.

    Autoradiography:

    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above. .. To recover intact nucleosomes, 32 μg of salmon testes DNA (Sigma) was added to each 12-μl sample after the DNase I digestion, and then native electrophoresis was performed with gels containing 3.8% polyacrylamide (0.1% bisacrylamide) and 25% Tris-borate-EDTA ( ).

    Electrophoresis:

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
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    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
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    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: Samples were incubated for several minutes at 65°C and then subjected to electrophoresis through 0.4-mm gels consisting of 8% polyacrylamide (0.4% bisacrylamide), 7 M urea, and 70% Tris-borate-EDTA ( ). .. Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above.

    Microarray:

    Article Title: Structural and Regulatory Characterization of the Placental Epigenome at Its Maternal Interface
    Article Snippet: Paragraph title: Target DNA Preparation for Agilent Microarray Analysis ... Reactions were then incubated further with 75U (0.75 µl) Exonuclease III (NEB) and incubated at 30°C for 1 hour.

    Random Hexamer Labeling:

    Article Title: A paired-end sequencing strategy to map the complex landscape of transcription initiation
    Article Snippet: Ligation was performed with 5 cycles of 95°C for 30 sec, 68°C for 2 min, 55°C for 1 min and 60°C for 5 min, followed by 5 cycles of 95°C for 30 sec, 65°C for 2 min, 55°C for 1 min and 60°C for 5 min. To remove excess oligonucleotides and unligated DNA fragments, we added 3 μl Exonuclease I (NEB) and 0.6 μl Exonuclease III (NEB) to the ligation reaction and the mixture was incubated at 37°C for 45 min followed by 80°C for 20 min. .. This removes all linear DNA fragments (ssDNAs and dsDNAs) and the remaining circular DNAs were further amplified by rolling circle amplification (RCA).

    Activity Assay:

    Article Title: Dissection of the sequence-specific DNA binding and exonuclease activities reveals a superactive yet apoptotically impaired mutant p53 protein
    Article Snippet: Typically, 2–4 times molar excess of tetrameric p53 was incubated with DNA substrates in an exonuclease activity assay buffer containing 50 mM TrisHCl (pH 8.0), and 10 mM MgCl2 at 30°C for 30 min. .. For exonuclease III (New England BioLabs, Beverly, MA), 1–1 ×10−7 pmol of protein was incubated with 0.25 pmol of substrate as described above.

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Subsequently, 50 U (5 μl) of XmaI endonuclease (NEB) was added, and digestion continued for an additional 16 h. The digested DNA was purified using a QIAquick PCR purification kit (Qiagen, CA). .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each). .. Illumina paired-end sequencing adaptors were ligated using Rapid T4 DNA ligase (Enzymatics, MA).

    Expressing:

    Article Title: A survey of Type III restriction-modification systems reveals numerous, novel epigenetic regulators controlling phase-variable regulons; phasevarions
    Article Snippet: Plasmid midi-preps from E. coli cells expressing STEC mod methyltransferase and the negative control expressing a non-methyltransferase (SiaB), were prepared using the Qiagen plasmid midi kit according to the manufacturer's instructions. .. Incompletely formed SMRTbell templates were degraded with a combination of Exonuclease III (New England Biolabs; Ipswich, MA, USA) and Exonuclease VII (USB; Cleveland, OH, USA).

    Genome Wide:

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Genome-wide DNA methylation analysis using next-generation sequencing ( , ) was performed for 20 samples for which a sufficient amount of DNA was available. .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

    Modification:

    Article Title: A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta
    Article Snippet: An Exonuclease III deletion procedure, modified from , was used to produce progressive deletions from the telomere repeat end of the putative AVR-Pita clone. .. Plasmid pCB783, containing the 791-bp telomeric SacI fragment, was digested first with KpnI and XhoI and then with Exonuclease III (New England Biolabs).

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
    Article Snippet: In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer. .. Circular DNA was then purified on Macherey-Nagel columns following a 25-min heating step at 85°C to inactivate the enzymes.

    Article Title: A highly selective, label-free, homogenous luminescent switch-on probe for the detection of nanomolar transcription factor NF-kappaB
    Article Snippet: The p50 protein was expressed and purified based on a modified procedure from Leung et al. ( ). .. Exonuclease III (ExoIII) was purchased from New England Biolabs.

    Hybridization:

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: A 4-kb PstI–SstI fragment from the cloned region was used as a hybridization probe to screen a λ phage library of Chlamydomonas WT genomic DNA and positive clones were tested for phenotypic rescue of lf3-5 or lf3-6 mutants by cotransformation using the pARG7.8 plasmid ( ) as described previously ( ). .. Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to .

    High Performance Liquid Chromatography:

    Article Title: Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes
    Article Snippet: The synthetic oligodeoxyribonucleotides and biotinylated oligodeoxyribonucleotides purchased from VBC Biotech (Vienna, Austria) were purified by HPLC as described previously . .. T4 DNA ligase and Exonuclease III were from New England Biolabs (Beverly, MA, USA).

    Sequencing:

    Article Title: A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta
    Article Snippet: Plasmid pCB783, containing the 791-bp telomeric SacI fragment, was digested first with KpnI and XhoI and then with Exonuclease III (New England Biolabs). .. Plasmid pCB783, containing the 791-bp telomeric SacI fragment, was digested first with KpnI and XhoI and then with Exonuclease III (New England Biolabs).

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: These subclones were tested for phenotypic rescue of lf3-2 , lf3-5 , and lf3-6 by cotransformation. .. Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to . .. The sequence of the LF3 gene was obtained by sequencing plasmid clones with universal sequencing primers or gene-specific primers (DNA Sequencing and Synthesis Facility, Iowa State University, or Advanced Genetics Analysis Center, University of Minnesota).

    Article Title: A survey of Type III restriction-modification systems reveals numerous, novel epigenetic regulators controlling phase-variable regulons; phasevarions
    Article Snippet: Paragraph title: Single-molecule, real-time (SMRT) sequencing and methylome analysis ... Incompletely formed SMRTbell templates were degraded with a combination of Exonuclease III (New England Biolabs; Ipswich, MA, USA) and Exonuclease VII (USB; Cleveland, OH, USA).

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Paragraph title: SMRTbell library preparation and SMRT Sequencing ... After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates.

    Ligation:

    Article Title: A paired-end sequencing strategy to map the complex landscape of transcription initiation
    Article Snippet: 9 pmol “bridge” oligonucleotide (5′-GCC GTC TTC AGC CGC CTCA AGC TTC TAA CGA TGT ACG-3′) and 7.5 units of Ampligase (Epicentre) were then added. .. Ligation was performed with 5 cycles of 95°C for 30 sec, 68°C for 2 min, 55°C for 1 min and 60°C for 5 min, followed by 5 cycles of 95°C for 30 sec, 65°C for 2 min, 55°C for 1 min and 60°C for 5 min. To remove excess oligonucleotides and unligated DNA fragments, we added 3 μl Exonuclease I (NEB) and 0.6 μl Exonuclease III (NEB) to the ligation reaction and the mixture was incubated at 37°C for 45 min followed by 80°C for 20 min. .. This removes all linear DNA fragments (ssDNAs and dsDNAs) and the remaining circular DNAs were further amplified by rolling circle amplification (RCA).

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
    Article Snippet: The cross-linking of either 3C or 4C DNA products was then reversed overnight at 65°C following the addition of EDTA (final concentration, 1 mM), NaCl (final concentration, 0.2 M), and 10 or 20 μl of 10 μg/μl proteinase K. The samples were then subjected to three successive phenol-chloroform-isoamyl alcohol (25:24:1) extractions, followed by a chloroform washing step, diluted 4 times in water, and precipitated at −20°C for 2 h by 2 volumes of isopropanol. .. In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer. .. Circular DNA was then purified on Macherey-Nagel columns following a 25-min heating step at 85°C to inactivate the enzymes.

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each). .. Illumina paired-end sequencing adaptors were ligated using Rapid T4 DNA ligase (Enzymatics, MA).

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: Following ligation, the beads were washed three times with Dynabead wash buffer: twice at 58°C and once at room temperature. .. 1 μl Exo III (NEB catalog no. M0206S)) was added to the MIP reaction and samples were incubated at 37°C for 45 minutes.

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: The the sticky ended cDNA for each size-bin was then ligated to 1 μM hairpin adapters that contain complementary sticky ends to create SMRTbell templates using 2000 Units T4 DNA ligase (New England Biolabs). .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates. .. SMRTbell libraries for the 2–3 kb and 3–6 kb size-bins were subjected to an additional round of size selection on the Sage BluePippin to remove trace amounts of small inserts.

    SYBR Green Assay:

    Article Title: Introducing structure-switching functionality into small-molecule-binding aptamers via nuclease-directed truncation
    Article Snippet: Exonuclease III and exonuclease I were purchased from New England Biolabs. .. Exonuclease III and exonuclease I were purchased from New England Biolabs.

    Footprinting:

    Article Title: Multiple sequence-directed possibilities provide a pool of nucleosome position choices in different states of activity of a gene
    Article Snippet: Paragraph title: Exonuclease III footprinting ... The reconstituted mononucleosomes were digested with 20 U/ml Exonuclease III (NEB), for 0, 3, 6, and 9 minutes as compared with 0, .5, 1.5, and 2 minutes for the naked DNA samples.

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: Paragraph title: Exonuclease Footprinting ... Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    Cell Culture:

    Article Title: DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication
    Article Snippet: For chromosome orientation FISH (CO-FISH), cells were cultured in 7.5 mM of 5-bromo-2′-deoxyuridine (BrdU) and 2.5 mM of 5-bromo-2′-deoxycytosine (BrdC) for 20 h prior to colcemid treatment and chromosome spread. .. Slides were stained with Hoechst 33258 (10 μg/ml), exposed to ultraviolet light (365-nm UV-A, 30 min), incubated with exonuclease III (1.6% v/v, New England Biolabs) for 10 min at RT, hybridized with Cy3-labeled C-rich and fluorescein isothiocyanate-labeled G-rich telomeric PNA probes (Panagene), followed by PBS washes and dehydrated as described.

    Generated:

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
    Article Snippet: The cross-linking of either 3C or 4C DNA products was then reversed overnight at 65°C following the addition of EDTA (final concentration, 1 mM), NaCl (final concentration, 0.2 M), and 10 or 20 μl of 10 μg/μl proteinase K. The samples were then subjected to three successive phenol-chloroform-isoamyl alcohol (25:24:1) extractions, followed by a chloroform washing step, diluted 4 times in water, and precipitated at −20°C for 2 h by 2 volumes of isopropanol. .. In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer. .. Circular DNA was then purified on Macherey-Nagel columns following a 25-min heating step at 85°C to inactivate the enzymes.

    Imaging:

    Article Title: Multiple sequence-directed possibilities provide a pool of nucleosome position choices in different states of activity of a gene
    Article Snippet: The reconstituted mononucleosomes were digested with 20 U/ml Exonuclease III (NEB), for 0, 3, 6, and 9 minutes as compared with 0, .5, 1.5, and 2 minutes for the naked DNA samples. .. The reconstituted mononucleosomes were digested with 20 U/ml Exonuclease III (NEB), for 0, 3, 6, and 9 minutes as compared with 0, .5, 1.5, and 2 minutes for the naked DNA samples.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to . .. The sequence of the LF3 gene was obtained by sequencing plasmid clones with universal sequencing primers or gene-specific primers (DNA Sequencing and Synthesis Facility, Iowa State University, or Advanced Genetics Analysis Center, University of Minnesota).

    Binding Assay:

    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: Paragraph title: Binding and DNase I assays. ... Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above.

    Fluorescence:

    Article Title: DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication
    Article Snippet: Paragraph title: Fluorescence in situ hybridization and immuno-fluorescence in situ hybridization ... Slides were stained with Hoechst 33258 (10 μg/ml), exposed to ultraviolet light (365-nm UV-A, 30 min), incubated with exonuclease III (1.6% v/v, New England Biolabs) for 10 min at RT, hybridized with Cy3-labeled C-rich and fluorescein isothiocyanate-labeled G-rich telomeric PNA probes (Panagene), followed by PBS washes and dehydrated as described.

    Methylation:

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Paragraph title: Digital restriction enzyme analysis of methylation (DREAM) ... The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

    Mutagenesis:

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: Paragraph title: Cloning and sequencing of the LF3 gene and the mutant lf3-2 allele ... Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to .

    Size-exclusion Chromatography:

    Article Title: A paired-end sequencing strategy to map the complex landscape of transcription initiation
    Article Snippet: 9 pmol “bridge” oligonucleotide (5′-GCC GTC TTC AGC CGC CTCA AGC TTC TAA CGA TGT ACG-3′) and 7.5 units of Ampligase (Epicentre) were then added. .. Ligation was performed with 5 cycles of 95°C for 30 sec, 68°C for 2 min, 55°C for 1 min and 60°C for 5 min, followed by 5 cycles of 95°C for 30 sec, 65°C for 2 min, 55°C for 1 min and 60°C for 5 min. To remove excess oligonucleotides and unligated DNA fragments, we added 3 μl Exonuclease I (NEB) and 0.6 μl Exonuclease III (NEB) to the ligation reaction and the mixture was incubated at 37°C for 45 min followed by 80°C for 20 min. .. This removes all linear DNA fragments (ssDNAs and dsDNAs) and the remaining circular DNAs were further amplified by rolling circle amplification (RCA).

    Labeling:

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: The labeled DNA (1 pmol) was incubated for 1 h at room temperature with 2 pmol of TFIIB, 1.6 pmol of TFIIA, 1.1 pmol of TBP, 2.4 pmol of TFIIE, 1.5 pmol of TFIIH*, and 1.2 pmol of pol II-TFIIF complex in 8 μl of buffer A; combined with 12 μl of buffer B; and incubated for 1 h at 4 °C. .. Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    Article Title: Dissection of the sequence-specific DNA binding and exonuclease activities reveals a superactive yet apoptotically impaired mutant p53 protein
    Article Snippet: Duplex substrates were prepared by purifying annealed DNA from 10% (w/v) polyacrylamide gels and quantitated as described in Ahn et al. DNA substrates were labeled at the 5'-end with 32 P by incubating with T4 polynucleotide kinase and [γ-32 P] ATP (4,500 Ci/mmol). .. For exonuclease III (New England BioLabs, Beverly, MA), 1–1 ×10−7 pmol of protein was incubated with 0.25 pmol of substrate as described above.

    Purification:

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: The size-selected and purified DNA was concentrated to 12 μl, then denatured at 95 °C for 3 min followed by incubation on ice for 3 min. Then the sample was supplemented with a mixture of 10X Cirligase buffer (1.5 μl), 50 mM MnCl2 (0.75 μl), Cirligase (0.75 μl) (Epicentre CL9025K) and further incubated at 60°Cfor 14 h before the reaction was stopped by heating at 80 °C for 10 min. .. Subsequently, 0.75 μl Exonuclease I (NEB, M0293S) and 0.75 μl Exonuclease III (NEB, M0206S) were added into the reaction and jointly incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Article Title: Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes
    Article Snippet: The synthetic oligodeoxyribonucleotides and biotinylated oligodeoxyribonucleotides purchased from VBC Biotech (Vienna, Austria) were purified by HPLC as described previously . .. T4 DNA ligase and Exonuclease III were from New England Biolabs (Beverly, MA, USA).

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: HIS4 (−96/+112) was amplified by PCR in a 1–2-ml reaction using the 32 P-labeled upstream primer and downstream primer (5′-TATTCCATGAGGCCAGATC-3′) and purified by electrophoresis on a 2% agarose gel. .. Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
    Article Snippet: The cross-linking of either 3C or 4C DNA products was then reversed overnight at 65°C following the addition of EDTA (final concentration, 1 mM), NaCl (final concentration, 0.2 M), and 10 or 20 μl of 10 μg/μl proteinase K. The samples were then subjected to three successive phenol-chloroform-isoamyl alcohol (25:24:1) extractions, followed by a chloroform washing step, diluted 4 times in water, and precipitated at −20°C for 2 h by 2 volumes of isopropanol. .. In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer. .. Circular DNA was then purified on Macherey-Nagel columns following a 25-min heating step at 85°C to inactivate the enzymes.

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Subsequently, 50 U (5 μl) of XmaI endonuclease (NEB) was added, and digestion continued for an additional 16 h. The digested DNA was purified using a QIAquick PCR purification kit (Qiagen, CA). .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: When comparing nucleosome positions in different strains or conditions it is critical to match the digestion levels for each sample analyzed ( ). .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification .. 1 Dilute overnight culture to OD600 = 0.1 in 250 ml fresh YPD media.

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: The ligated MIPs were further purified and fully eluted from the immobilized cDNA by digesting all linear DNA with exonucleases; 4 μl of exonuclease mix (3.5 μl 10× Exonuclease Buffer, 0.5 μl Exo I (NEB catalog no. M0293S. .. 1 μl Exo III (NEB catalog no. M0206S)) was added to the MIP reaction and samples were incubated at 37°C for 45 minutes.

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to . .. The gene prediction programs GeneMark ( http://opal.biology.gatech.edu/GeneMark/eukhmm.cgi ) or Genscan ( http://genes.mit.edu/GENSCAN.html ) were used to predict the exon/intron structure of LF3 . cDNA sequences were obtained by RT-PCR using primers designed for putative exons under conditions described previously , except that RNA was treated with amplification-grade DNaseI (GIBCO BRL) for 15 min at RT to remove DNA contamination before reverse transcription.

    Article Title: A highly selective, label-free, homogenous luminescent switch-on probe for the detection of nanomolar transcription factor NF-kappaB
    Article Snippet: The p50 protein was expressed and purified based on a modified procedure from Leung et al. ( ). .. Exonuclease III (ExoIII) was purchased from New England Biolabs.

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Purified, size-selected cDNA was eluted in 20 μl Low TE and then digested using 1 unit USER (New England Biolabs) to create sticky ends. .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates.

    Polymerase Chain Reaction:

    Article Title: A paired-end sequencing strategy to map the complex landscape of transcription initiation
    Article Snippet: First-strand cDNAs were amplified by 2-5 cycles of PCR with high-fidelity DNA polymerase (Finnzymes) to generate blunt-end dsDNAs. .. Ligation was performed with 5 cycles of 95°C for 30 sec, 68°C for 2 min, 55°C for 1 min and 60°C for 5 min, followed by 5 cycles of 95°C for 30 sec, 65°C for 2 min, 55°C for 1 min and 60°C for 5 min. To remove excess oligonucleotides and unligated DNA fragments, we added 3 μl Exonuclease I (NEB) and 0.6 μl Exonuclease III (NEB) to the ligation reaction and the mixture was incubated at 37°C for 45 min followed by 80°C for 20 min.

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: HIS4 (−96/+112) was amplified by PCR in a 1–2-ml reaction using the 32 P-labeled upstream primer and downstream primer (5′-TATTCCATGAGGCCAGATC-3′) and purified by electrophoresis on a 2% agarose gel. .. Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Subsequently, 50 U (5 μl) of XmaI endonuclease (NEB) was added, and digestion continued for an additional 16 h. The digested DNA was purified using a QIAquick PCR purification kit (Qiagen, CA). .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: When comparing nucleosome positions in different strains or conditions it is critical to match the digestion levels for each sample analyzed ( ). .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification .. 1 Dilute overnight culture to OD600 = 0.1 in 250 ml fresh YPD media.

    Article Title: Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation, Replication, and Persistence
    Article Snippet: To detect total p8TR-gB, 2 μg of Hirt DNA was digested with 30 U of EcoRV (New England Biolabs [NEB]) in a 50-μl reaction mixture overnight at 37°C, followed by heat inactivation of EcoRV at 80°C for 30 min. PCR amplification was performed in triplicate using 3.75 μl of the EcoRV-digested product as the template. .. To detect replicated p8TR-gB, 6 μg of Hirt DNA was digested with 60 U DpnI in NEB buffer 2 in a 50-μl reaction mixture overnight at 37°C, followed by exonuclease III (ExoIII) (NEB) treatment (120 U) for 30 min at 37°C to reduce the background due to incompletely DpnI digested DNA ( , ).

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to . .. Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to .

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Large-scale PCR products were purified with AMPure PB beads (Pacific Biosciences) and quality control was performed on a BioAnalyzer (Agilent). cDNA was then subjected to size fractionation using the Sage BluePippin system (Sage Science), collecting three size-bins: 1–2 kb, 2–3 kb, and 3–6 kb. .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)). .. The DNA pellet was recovered by centrifugation at maximum speed for 1 h, dried at 37 °C, and resuspended in 10 μl of gel loading buffer (95% formamide, 0.02% bromphenol blue, 5 m m EDTA, and 0.025% xylene cyanol).

    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: Gels were fixed in 12% methanol and 10% acetic acid, dried, and then autoradiographed. .. Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above. .. DraI, HhaI, and StyI (New England Biolabs) were used in the same way except the NaCl was omitted from the binding reaction and replaced with 1× reaction buffer supplied with the enzyme, and 20 U of each enzyme was added per 100 fmol of DNA.

    Gel Extraction:

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: Subsequently, the gel regions with DNAs in length of 60 ~ 100 bp marked by the 20 bp DNA ladder (Takara) were specifically cut out, and further extracted using QIAGEN MinElute Gel Extraction Kit (6X buffer QG). .. Subsequently, 0.75 μl Exonuclease I (NEB, M0293S) and 0.75 μl Exonuclease III (NEB, M0206S) were added into the reaction and jointly incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: When comparing nucleosome positions in different strains or conditions it is critical to match the digestion levels for each sample analyzed ( ). .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification .. 1 Dilute overnight culture to OD600 = 0.1 in 250 ml fresh YPD media.

    Agarose Gel Electrophoresis:

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: HIS4 (−96/+112) was amplified by PCR in a 1–2-ml reaction using the 32 P-labeled upstream primer and downstream primer (5′-TATTCCATGAGGCCAGATC-3′) and purified by electrophoresis on a 2% agarose gel. .. Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    In Situ Hybridization:

    Article Title: DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication
    Article Snippet: Paragraph title: Fluorescence in situ hybridization and immuno-fluorescence in situ hybridization ... Slides were stained with Hoechst 33258 (10 μg/ml), exposed to ultraviolet light (365-nm UV-A, 30 min), incubated with exonuclease III (1.6% v/v, New England Biolabs) for 10 min at RT, hybridized with Cy3-labeled C-rich and fluorescein isothiocyanate-labeled G-rich telomeric PNA probes (Panagene), followed by PBS washes and dehydrated as described.

    Plasmid Preparation:

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: Alternatively, if the plasmid DNA less than 100 ng, the fragmentation step should be performed in the following shearing conditions: duty cycle: 10%, intensity: 5, cycles per burst: 100, time: 900 s, temperature < 4 °C, and purified with MinElute Reaction Cleanup Kit (3X buffer ERC) to remove small fragments (less than 70 bp). .. Subsequently, 0.75 μl Exonuclease I (NEB, M0293S) and 0.75 μl Exonuclease III (NEB, M0206S) were added into the reaction and jointly incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Article Title: A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta
    Article Snippet: An Exonuclease III deletion procedure, modified from , was used to produce progressive deletions from the telomere repeat end of the putative AVR-Pita clone. .. Plasmid pCB783, containing the 791-bp telomeric SacI fragment, was digested first with KpnI and XhoI and then with Exonuclease III (New England Biolabs). .. The ExoIII-digested DNAs were treated with S1 nuclease (Sigma) followed by a fill-in reaction with the Klenow fragment of DNA polymerase I, ligation, and transformation into DH5αMCR cells (Gibco BRL).

    Article Title: Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation, Replication, and Persistence
    Article Snippet: Amplified DNA was quantified using a standard curve from a p8TR-gB plasmid dilution series. .. To detect replicated p8TR-gB, 6 μg of Hirt DNA was digested with 60 U DpnI in NEB buffer 2 in a 50-μl reaction mixture overnight at 37°C, followed by exonuclease III (ExoIII) (NEB) treatment (120 U) for 30 min at 37°C to reduce the background due to incompletely DpnI digested DNA ( , ).

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: Fragments of a lambda clone λ12D9, which was able to rescue the mutant phenotype, were subcloned into the vector pBluescript KS+ (Stratagene) using restriction sites indicated in A. .. Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to .

    Article Title: A survey of Type III restriction-modification systems reveals numerous, novel epigenetic regulators controlling phase-variable regulons; phasevarions
    Article Snippet: Plasmid midi-preps from E. coli cells expressing STEC mod methyltransferase and the negative control expressing a non-methyltransferase (SiaB), were prepared using the Qiagen plasmid midi kit according to the manufacturer's instructions. .. Incompletely formed SMRTbell templates were degraded with a combination of Exonuclease III (New England Biolabs; Ipswich, MA, USA) and Exonuclease VII (USB; Cleveland, OH, USA).

    Software:

    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above. .. DraI, HhaI, and StyI (New England Biolabs) were used in the same way except the NaCl was omitted from the binding reaction and replaced with 1× reaction buffer supplied with the enzyme, and 20 U of each enzyme was added per 100 fmol of DNA.

    Real-time Polymerase Chain Reaction:

    Article Title: Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation, Replication, and Persistence
    Article Snippet: PCRs were performed using an ABI 7300 real-time PCR system (Applied Biosystems) (1 cycle at 50°C for 2 min; 1 cycle at 95°C for 10 min; 40 cycles of 95°C for 30 s and 60°C for 1 min). .. To detect replicated p8TR-gB, 6 μg of Hirt DNA was digested with 60 U DpnI in NEB buffer 2 in a 50-μl reaction mixture overnight at 37°C, followed by exonuclease III (ExoIII) (NEB) treatment (120 U) for 30 min at 37°C to reduce the background due to incompletely DpnI digested DNA ( , ).

    Negative Control:

    Article Title: A survey of Type III restriction-modification systems reveals numerous, novel epigenetic regulators controlling phase-variable regulons; phasevarions
    Article Snippet: Plasmid midi-preps from E. coli cells expressing STEC mod methyltransferase and the negative control expressing a non-methyltransferase (SiaB), were prepared using the Qiagen plasmid midi kit according to the manufacturer's instructions. .. Incompletely formed SMRTbell templates were degraded with a combination of Exonuclease III (New England Biolabs; Ipswich, MA, USA) and Exonuclease VII (USB; Cleveland, OH, USA).

    Selection:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Following size selection, size-selected cDNA was re-amplified using LongAmp HotStart Taq. .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates.

    Sample Prep:

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: When comparing nucleosome positions in different strains or conditions it is critical to match the digestion levels for each sample analyzed ( ). .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification .. 1 Dilute overnight culture to OD600 = 0.1 in 250 ml fresh YPD media.

    Next-Generation Sequencing:

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Genome-wide DNA methylation analysis using next-generation sequencing ( , ) was performed for 20 samples for which a sufficient amount of DNA was available. .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

    Incubation:

    Article Title: A paired-end sequencing strategy to map the complex landscape of transcription initiation
    Article Snippet: 9 pmol “bridge” oligonucleotide (5′-GCC GTC TTC AGC CGC CTCA AGC TTC TAA CGA TGT ACG-3′) and 7.5 units of Ampligase (Epicentre) were then added. .. Ligation was performed with 5 cycles of 95°C for 30 sec, 68°C for 2 min, 55°C for 1 min and 60°C for 5 min, followed by 5 cycles of 95°C for 30 sec, 65°C for 2 min, 55°C for 1 min and 60°C for 5 min. To remove excess oligonucleotides and unligated DNA fragments, we added 3 μl Exonuclease I (NEB) and 0.6 μl Exonuclease III (NEB) to the ligation reaction and the mixture was incubated at 37°C for 45 min followed by 80°C for 20 min. .. This removes all linear DNA fragments (ssDNAs and dsDNAs) and the remaining circular DNAs were further amplified by rolling circle amplification (RCA).

    Article Title: DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication
    Article Snippet: For chromosome orientation FISH (CO-FISH), cells were cultured in 7.5 mM of 5-bromo-2′-deoxyuridine (BrdU) and 2.5 mM of 5-bromo-2′-deoxycytosine (BrdC) for 20 h prior to colcemid treatment and chromosome spread. .. Slides were stained with Hoechst 33258 (10 μg/ml), exposed to ultraviolet light (365-nm UV-A, 30 min), incubated with exonuclease III (1.6% v/v, New England Biolabs) for 10 min at RT, hybridized with Cy3-labeled C-rich and fluorescein isothiocyanate-labeled G-rich telomeric PNA probes (Panagene), followed by PBS washes and dehydrated as described. .. For immuno-FISH, the slides were immunostained with anti-γH2AX antibody and fluorescein-labeled second antibody.

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: The size-selected and purified DNA was concentrated to 12 μl, then denatured at 95 °C for 3 min followed by incubation on ice for 3 min. Then the sample was supplemented with a mixture of 10X Cirligase buffer (1.5 μl), 50 mM MnCl2 (0.75 μl), Cirligase (0.75 μl) (Epicentre CL9025K) and further incubated at 60°Cfor 14 h before the reaction was stopped by heating at 80 °C for 10 min. .. Subsequently, 0.75 μl Exonuclease I (NEB, M0293S) and 0.75 μl Exonuclease III (NEB, M0206S) were added into the reaction and jointly incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min. .. The successfully circularized DNA was purified using the Oligo Clean & concentrator kit (Zymo, D4060).

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: The reconstituted PIC was combined with an equal volume of 2× NTP buffer (1.6 m m NTP(s) or 0.5 m m non-hydrolyzable analog ATPγS, 10 m m magnesium acetate, and 5 units of RNaseOUT) and incubated for 30 min at 30 °C. .. Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
    Article Snippet: Following these 5 days of ligation, 4C samples were further incubated for 1.5 h with 1 μl of T4 DNA ligase in order to ensure that the ligase filled any nicks in the circularized 3C products. .. In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer.

    Article Title: Dissection of the sequence-specific DNA binding and exonuclease activities reveals a superactive yet apoptotically impaired mutant p53 protein
    Article Snippet: Product formation was analyzed by PhosphoImager (Molecular Dynamics). .. For exonuclease III (New England BioLabs, Beverly, MA), 1–1 ×10−7 pmol of protein was incubated with 0.25 pmol of substrate as described above. .. For immunoprecipitation-exonuclease assays, each p53 protein (200 ng) purified from Ni-NTA column was immunoprecipitated with 20 µL of mAb1801 conjugated to Protein A-Agarose beads (Pharmacia LKB, Piscataway, NJ) in an exonuclease buffer.

    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: Gels were fixed in 12% methanol and 10% acetic acid, dried, and then autoradiographed. .. Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above. .. DraI, HhaI, and StyI (New England Biolabs) were used in the same way except the NaCl was omitted from the binding reaction and replaced with 1× reaction buffer supplied with the enzyme, and 20 U of each enzyme was added per 100 fmol of DNA.

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: The ligated MIPs were further purified and fully eluted from the immobilized cDNA by digesting all linear DNA with exonucleases; 4 μl of exonuclease mix (3.5 μl 10× Exonuclease Buffer, 0.5 μl Exo I (NEB catalog no. M0293S. .. 1 μl Exo III (NEB catalog no. M0206S)) was added to the MIP reaction and samples were incubated at 37°C for 45 minutes. .. The specific process used for amplifying circularized asMIPs was tailored to the method of quantitation; for arrays, MIPs were amplified with biotinylated PCR primers before being hybridized to microarrays.

    Article Title: Structural and Regulatory Characterization of the Placental Epigenome at Its Maternal Interface
    Article Snippet: Following overnight digestion, reactions were digested further with 5 uL (50U) of TspRI (NEB) at 65°C for three hours. .. Reactions were then incubated further with 75U (0.75 µl) Exonuclease III (NEB) and incubated at 30°C for 1 hour. .. Enzymatic activity was then nullified by heating at 70°C for 20 min after which 50U of RecJF (NEB) were added to remove single stranded DNA.

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: The the sticky ended cDNA for each size-bin was then ligated to 1 μM hairpin adapters that contain complementary sticky ends to create SMRTbell templates using 2000 Units T4 DNA ligase (New England Biolabs). .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates. .. SMRTbell libraries for the 2–3 kb and 3–6 kb size-bins were subjected to an additional round of size selection on the Sage BluePippin to remove trace amounts of small inserts.

    Spectrophotometry:

    Article Title: Structural and Regulatory Characterization of the Placental Epigenome at Its Maternal Interface
    Article Snippet: Reactions were then incubated further with 75U (0.75 µl) Exonuclease III (NEB) and incubated at 30°C for 1 hour. .. Reactions were then incubated further with 75U (0.75 µl) Exonuclease III (NEB) and incubated at 30°C for 1 hour.

    DNA Methylation Assay:

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Genome-wide DNA methylation analysis using next-generation sequencing ( , ) was performed for 20 samples for which a sufficient amount of DNA was available. .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

    Concentration Assay:

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: The ultimate DNA concentration was calibrated using Qubit 2.0 dsDNA HS Assay kit. .. Subsequently, 0.75 μl Exonuclease I (NEB, M0293S) and 0.75 μl Exonuclease III (NEB, M0206S) were added into the reaction and jointly incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
    Article Snippet: The cross-linking of either 3C or 4C DNA products was then reversed overnight at 65°C following the addition of EDTA (final concentration, 1 mM), NaCl (final concentration, 0.2 M), and 10 or 20 μl of 10 μg/μl proteinase K. The samples were then subjected to three successive phenol-chloroform-isoamyl alcohol (25:24:1) extractions, followed by a chloroform washing step, diluted 4 times in water, and precipitated at −20°C for 2 h by 2 volumes of isopropanol. .. In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer.

    Fractionation:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Large-scale PCR products were purified with AMPure PB beads (Pacific Biosciences) and quality control was performed on a BioAnalyzer (Agilent). cDNA was then subjected to size fractionation using the Sage BluePippin system (Sage Science), collecting three size-bins: 1–2 kb, 2–3 kb, and 3–6 kb. .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates.

    Staining:

    Article Title: DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication
    Article Snippet: For chromosome orientation FISH (CO-FISH), cells were cultured in 7.5 mM of 5-bromo-2′-deoxyuridine (BrdU) and 2.5 mM of 5-bromo-2′-deoxycytosine (BrdC) for 20 h prior to colcemid treatment and chromosome spread. .. Slides were stained with Hoechst 33258 (10 μg/ml), exposed to ultraviolet light (365-nm UV-A, 30 min), incubated with exonuclease III (1.6% v/v, New England Biolabs) for 10 min at RT, hybridized with Cy3-labeled C-rich and fluorescein isothiocyanate-labeled G-rich telomeric PNA probes (Panagene), followed by PBS washes and dehydrated as described. .. For immuno-FISH, the slides were immunostained with anti-γH2AX antibody and fluorescein-labeled second antibody.

    Fluorescence In Situ Hybridization:

    Article Title: DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication
    Article Snippet: For chromosome orientation FISH (CO-FISH), cells were cultured in 7.5 mM of 5-bromo-2′-deoxyuridine (BrdU) and 2.5 mM of 5-bromo-2′-deoxycytosine (BrdC) for 20 h prior to colcemid treatment and chromosome spread. .. Slides were stained with Hoechst 33258 (10 μg/ml), exposed to ultraviolet light (365-nm UV-A, 30 min), incubated with exonuclease III (1.6% v/v, New England Biolabs) for 10 min at RT, hybridized with Cy3-labeled C-rich and fluorescein isothiocyanate-labeled G-rich telomeric PNA probes (Panagene), followed by PBS washes and dehydrated as described.

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    New England Biolabs exonuclease iii
    C-circles and 5' C-overhangs are linked to <t>DNA</t> damage-induced replication fork collapse. (A) Replication fork stalling induced by HU or aphidicolin decreases abundance of C-circles in U2OS cells. U2OS cells were treated with HU (hydroxyurea, 2mM) or aphidicolin (Aphi, 1μg/mL) for 24h and genomic DNA was purified for C-circle assay. Error bars represent the mean ± SEM of <t>three</t> independent experiments. Two-tailed unpaired student’s t- test was used to calculate P-values. *P
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    C-circles and 5' C-overhangs are linked to DNA damage-induced replication fork collapse. (A) Replication fork stalling induced by HU or aphidicolin decreases abundance of C-circles in U2OS cells. U2OS cells were treated with HU (hydroxyurea, 2mM) or aphidicolin (Aphi, 1μg/mL) for 24h and genomic DNA was purified for C-circle assay. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t- test was used to calculate P-values. *P

    Journal: PLoS Genetics

    Article Title: Strand break-induced replication fork collapse leads to C-circles, C-overhangs and telomeric recombination

    doi: 10.1371/journal.pgen.1007925

    Figure Lengend Snippet: C-circles and 5' C-overhangs are linked to DNA damage-induced replication fork collapse. (A) Replication fork stalling induced by HU or aphidicolin decreases abundance of C-circles in U2OS cells. U2OS cells were treated with HU (hydroxyurea, 2mM) or aphidicolin (Aphi, 1μg/mL) for 24h and genomic DNA was purified for C-circle assay. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t- test was used to calculate P-values. *P

    Article Snippet: Purified DNA were digested with or without 200U Exonuclease III (New England Biolabs) overnight at 37°C, and then subjected to 0.7% agarose gel electrophoresis and in gel hybridization.

    Techniques: Purification, Two Tailed Test

    Endogenous ssDNA break/gap or induced ssDNA break in C-rich strand stimulates formation of C-circles and 5' C-overhangs. (A) Experimental protocol to study strand specific (G-rich or C-rich) breaks/gaps on telomere is shown schematically. HinfI and RsaI digested genomic DNA was purified and further digested with Exo III to examine potential breaks/gaps on G-strand or C-strand of telomeres. If breaks/gaps occur on C-strand, Exo III would degrade all C-strand, leaving single-stranded G-strand that can be detected by hybridization with C-rich probe under native or denatured condition. Contrariwise, only C-strand can be detected if breaks/gaps occur on G-strand. (B) Breaks/gaps occur more frequently on C-rich strand of telomere. Exo III digestion produces single-stranded DNA that is less in molecular weight than corresponding double-stranded DNA, thereby migrating faster during electrophoresis. (C) Methyl-methane sulfonate (MMS) stimulates formation of C-circle DNA in U2OS cells. U2OS cells were treated with MMS (0.25mM) for 24h and genomic DNA was purified for C-circle assay. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t -test was used to calculate P-values. ***P

    Journal: PLoS Genetics

    Article Title: Strand break-induced replication fork collapse leads to C-circles, C-overhangs and telomeric recombination

    doi: 10.1371/journal.pgen.1007925

    Figure Lengend Snippet: Endogenous ssDNA break/gap or induced ssDNA break in C-rich strand stimulates formation of C-circles and 5' C-overhangs. (A) Experimental protocol to study strand specific (G-rich or C-rich) breaks/gaps on telomere is shown schematically. HinfI and RsaI digested genomic DNA was purified and further digested with Exo III to examine potential breaks/gaps on G-strand or C-strand of telomeres. If breaks/gaps occur on C-strand, Exo III would degrade all C-strand, leaving single-stranded G-strand that can be detected by hybridization with C-rich probe under native or denatured condition. Contrariwise, only C-strand can be detected if breaks/gaps occur on G-strand. (B) Breaks/gaps occur more frequently on C-rich strand of telomere. Exo III digestion produces single-stranded DNA that is less in molecular weight than corresponding double-stranded DNA, thereby migrating faster during electrophoresis. (C) Methyl-methane sulfonate (MMS) stimulates formation of C-circle DNA in U2OS cells. U2OS cells were treated with MMS (0.25mM) for 24h and genomic DNA was purified for C-circle assay. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t -test was used to calculate P-values. ***P

    Article Snippet: Purified DNA were digested with or without 200U Exonuclease III (New England Biolabs) overnight at 37°C, and then subjected to 0.7% agarose gel electrophoresis and in gel hybridization.

    Techniques: Purification, Hybridization, Molecular Weight, Electrophoresis, Two Tailed Test

    Nascent C-circles and 5' C-overhangs are generated during telomere replication. (A) FACS analysis of G1/S synchronized U2OS cells. Cells were synchronized by double thymidine block, then released and harvested at the indicated time. (B) C-circle assay was performed at the indicated time after release from G1/S. (C) Representative image and statistical analysis showing that RPA2 foci colocalize with telomere at each time point. Cells with more than 5 colocalized foci/cell were scored positively, > 100 cells were counted per time point. Error bars represent the mean ± SEM of three independent experiments.(D) BrdU pulse-labeling strategy. U2OS cells were synchronized at G1/S, released in presence of BrdU for 12h. (E) Leading, lagging and unreplicated telomeric fractions were resolved by CsCl gradient ultracentrifugation and hybridized with telomeric probe. Non-BrdU labeled U2OS was used as a negative control (upper figure). “Area under peak” for leading, lagging and unreplicated telomeres was analyzed by Graphpad Prism and the relative amount of telomeres was indicated above individual peak. (F) Nascent C-circle is predominantly associated with lagging strand DNA synthesis. C-circle assay analysis of CsCl gradient fractions in (E). The amount of C-circle in leading, lagging and unreplicated telomeres was calculated by determining

    Journal: PLoS Genetics

    Article Title: Strand break-induced replication fork collapse leads to C-circles, C-overhangs and telomeric recombination

    doi: 10.1371/journal.pgen.1007925

    Figure Lengend Snippet: Nascent C-circles and 5' C-overhangs are generated during telomere replication. (A) FACS analysis of G1/S synchronized U2OS cells. Cells were synchronized by double thymidine block, then released and harvested at the indicated time. (B) C-circle assay was performed at the indicated time after release from G1/S. (C) Representative image and statistical analysis showing that RPA2 foci colocalize with telomere at each time point. Cells with more than 5 colocalized foci/cell were scored positively, > 100 cells were counted per time point. Error bars represent the mean ± SEM of three independent experiments.(D) BrdU pulse-labeling strategy. U2OS cells were synchronized at G1/S, released in presence of BrdU for 12h. (E) Leading, lagging and unreplicated telomeric fractions were resolved by CsCl gradient ultracentrifugation and hybridized with telomeric probe. Non-BrdU labeled U2OS was used as a negative control (upper figure). “Area under peak” for leading, lagging and unreplicated telomeres was analyzed by Graphpad Prism and the relative amount of telomeres was indicated above individual peak. (F) Nascent C-circle is predominantly associated with lagging strand DNA synthesis. C-circle assay analysis of CsCl gradient fractions in (E). The amount of C-circle in leading, lagging and unreplicated telomeres was calculated by determining "area under peak" using Graphpad Prism. The relative amount of C-circles was indicated above individual peak. (G) Schematic of the migration of linear dsDNA, ssDNA (C-overhangs) and telomeric open circles (T-circle) during 2D agarose gel electrophoresis and hybridization to a telomere-specific G-rich probe under native or denatured condition. (H) 5' C-overhang DNA is predominantly associated with leading strand DNA synthesis. The fractions corresponding to leading, lagging or non-replication telomeres from 12h BrdU labeled sample in (E) were pooled. DNA was incubated with or without RecJf, analyzed by 2D agarose gel electrophoresis and hybridized with G-rich telomeric probe under native and denaturing conditions. C-overhangs were indicated by red arrows. C-overhangs abundance was expressed as a ratio between the native and denatured signals. Values were then normalized with leading C-overhangs to obtain relative abundance.

    Article Snippet: Purified DNA were digested with or without 200U Exonuclease III (New England Biolabs) overnight at 37°C, and then subjected to 0.7% agarose gel electrophoresis and in gel hybridization.

    Techniques: Generated, FACS, Blocking Assay, Labeling, Negative Control, DNA Synthesis, Migration, Agarose Gel Electrophoresis, Hybridization, Incubation

    Fluorescence-emission spectra of zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex supramolecular fluorescent labels under different conditions: ( a ) Buffer; ( b ) Buffer + ZnPPIX; ( c ) RP (DNA duplex probe) + Circle DNA + hairpin probes (GHP) + Exonuclease III (Exo III) + ZnPPIX; ( d ) BPA + RP + Circle DNA + GHP + Exo III + ZnPPIX; C BPA = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, C ZnPPIX = 20 μM, RCA reaction time 1.5 h.

    Journal: Nanomaterials

    Article Title: A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

    doi: 10.3390/nano6100190

    Figure Lengend Snippet: Fluorescence-emission spectra of zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex supramolecular fluorescent labels under different conditions: ( a ) Buffer; ( b ) Buffer + ZnPPIX; ( c ) RP (DNA duplex probe) + Circle DNA + hairpin probes (GHP) + Exonuclease III (Exo III) + ZnPPIX; ( d ) BPA + RP + Circle DNA + GHP + Exo III + ZnPPIX; C BPA = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, C ZnPPIX = 20 μM, RCA reaction time 1.5 h.

    Article Snippet: Exonuclease III (Exo III) was purchased from New England BioLabs (Ipswich, MA, USA).

    Techniques: Fluorescence

    Schematic illustration the principle of the fluorescent assay of Bisphenol A (BPA) based on the rolling circle amplification (RCA)/Exo III-combined cascade signal amplification strategy.

    Journal: Nanomaterials

    Article Title: A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

    doi: 10.3390/nano6100190

    Figure Lengend Snippet: Schematic illustration the principle of the fluorescent assay of Bisphenol A (BPA) based on the rolling circle amplification (RCA)/Exo III-combined cascade signal amplification strategy.

    Article Snippet: Exonuclease III (Exo III) was purchased from New England BioLabs (Ipswich, MA, USA).

    Techniques: Fluorescence, Amplification

    ( a ) Agarose gel (0.7%) electrophoresis: (1) DNA 1 alone; (2) P 1 alone; (3) Circle DNA alone; (4) GHP alone; (5) RP + Circle DNA; (6) BPA + RP + Circle DNA; (7) RP + Circle DNA + GHP; (8) BPA + RP + Circle DNA + GHP; ( b ) Atomic force microscope (AFM) images of amplification products of RCA/Exo III-combined cascade signal amplification reaction. C DNA1 = 1.0 μM, C P1 = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, RCA reaction time 1.5 h.

    Journal: Nanomaterials

    Article Title: A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

    doi: 10.3390/nano6100190

    Figure Lengend Snippet: ( a ) Agarose gel (0.7%) electrophoresis: (1) DNA 1 alone; (2) P 1 alone; (3) Circle DNA alone; (4) GHP alone; (5) RP + Circle DNA; (6) BPA + RP + Circle DNA; (7) RP + Circle DNA + GHP; (8) BPA + RP + Circle DNA + GHP; ( b ) Atomic force microscope (AFM) images of amplification products of RCA/Exo III-combined cascade signal amplification reaction. C DNA1 = 1.0 μM, C P1 = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, RCA reaction time 1.5 h.

    Article Snippet: Exonuclease III (Exo III) was purchased from New England BioLabs (Ipswich, MA, USA).

    Techniques: Agarose Gel Electrophoresis, Electrophoresis, Microscopy, Amplification

    Differential inhibition of dumbbell decoy activity by DNA adducts of BBR 3464 as compared with cisplatin and transplatin in HEK293-NF-кB-luciferase reporter cell line. The percentage of inhibition of the specific decoy activity was calculated by measuring, for each experiment, the difference between luciferase activities obtained with cells transfected with platinated DUMBBELL-кB and nonplatinated DUMBBELL-kB divided by the difference between luciferase activities obtained with cells transfected with nonplatinated scrambled and specific (κB-site containing) DUMBBELL decoy oligonucleotides. Data represent the mean ± SD obtained from triplicate wells and are representative of at least three independent experiments. Data for ciplatin and transplatin were taken from ref. 10 .

    Journal: Scientific Reports

    Article Title: Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes

    doi: 10.1038/srep28474

    Figure Lengend Snippet: Differential inhibition of dumbbell decoy activity by DNA adducts of BBR 3464 as compared with cisplatin and transplatin in HEK293-NF-кB-luciferase reporter cell line. The percentage of inhibition of the specific decoy activity was calculated by measuring, for each experiment, the difference between luciferase activities obtained with cells transfected with platinated DUMBBELL-кB and nonplatinated DUMBBELL-kB divided by the difference between luciferase activities obtained with cells transfected with nonplatinated scrambled and specific (κB-site containing) DUMBBELL decoy oligonucleotides. Data represent the mean ± SD obtained from triplicate wells and are representative of at least three independent experiments. Data for ciplatin and transplatin were taken from ref. 10 .

    Article Snippet: T4 DNA ligase and Exonuclease III were from New England Biolabs (Beverly, MA, USA).

    Techniques: Inhibition, Activity Assay, Luciferase, Transfection

    Binding of NF-кB proteins to the DNA duplex containing the кB site. ( A ) The nucleotide sequence of the 22-bp oligodeoxyribonucleotide duplex containing κB site (DUPLEX-κB). The bold letters in the sequence indicate the κB recognition sequence. Left panels in Fig. 2B–D. Binding of p50/p65 heterodimer and p50/p50 and p65/p65 homodimers to the DUPLEX-κB containing the кB site (( B–D ), respectively). The panels show autoradiograms of the EMSA experiments showing the binding of p50/p65 heterodimer (Fig. 2B), p50/p50 (Fig. 2C) and p65/p65 (Fig. 2D) homodimers to the DUPLEX-κB. Lanes 1 and 2, non-modified duplex; lanes 3–5, duplex globally modified by BBR3464 at r b = 0.023, 0.045, or 0.091, respectively. The gel mobility shift assay was performed as described in the section Materials and Methods; concentration of the oligonucleotide duplex was 1 nM and the concentrations of p50/p50, p65/p65 and p50/p65 were 10, 15 and 15 nM, respectively. Right panels in Fig. 2B–D. Plots of the amount of the DUPLEX-κB modified by BBR3464 (full line), cisplatin (dashed line) or transplatin (dotted line) in complex with p50/p65 heterodimer (Fig. 2B), p50/p50 (Fig. 2C) and p65/p65 homodimers (Fig. 2D) on r b ; the data for cisplatin and transplatin were taken from ref. 10 . Data are the mean ± SD obtained from three different experiments.

    Journal: Scientific Reports

    Article Title: Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes

    doi: 10.1038/srep28474

    Figure Lengend Snippet: Binding of NF-кB proteins to the DNA duplex containing the кB site. ( A ) The nucleotide sequence of the 22-bp oligodeoxyribonucleotide duplex containing κB site (DUPLEX-κB). The bold letters in the sequence indicate the κB recognition sequence. Left panels in Fig. 2B–D. Binding of p50/p65 heterodimer and p50/p50 and p65/p65 homodimers to the DUPLEX-κB containing the кB site (( B–D ), respectively). The panels show autoradiograms of the EMSA experiments showing the binding of p50/p65 heterodimer (Fig. 2B), p50/p50 (Fig. 2C) and p65/p65 (Fig. 2D) homodimers to the DUPLEX-κB. Lanes 1 and 2, non-modified duplex; lanes 3–5, duplex globally modified by BBR3464 at r b = 0.023, 0.045, or 0.091, respectively. The gel mobility shift assay was performed as described in the section Materials and Methods; concentration of the oligonucleotide duplex was 1 nM and the concentrations of p50/p50, p65/p65 and p50/p65 were 10, 15 and 15 nM, respectively. Right panels in Fig. 2B–D. Plots of the amount of the DUPLEX-κB modified by BBR3464 (full line), cisplatin (dashed line) or transplatin (dotted line) in complex with p50/p65 heterodimer (Fig. 2B), p50/p50 (Fig. 2C) and p65/p65 homodimers (Fig. 2D) on r b ; the data for cisplatin and transplatin were taken from ref. 10 . Data are the mean ± SD obtained from three different experiments.

    Article Snippet: T4 DNA ligase and Exonuclease III were from New England Biolabs (Beverly, MA, USA).

    Techniques: Binding Assay, Sequencing, Modification, Mobility Shift, Concentration Assay

    PFGE analysis of the yeast mtDNAs. The whole-cell DNA samples were separated by PFGE using a CHEF Mapper XA Chiller System (Biorad), blotted onto a nylon membrane and hybridized with mtDNA-derived probes as described in ‘Material and Methods’ section. Lane 1— C. viswanathii CBS 4024; lane 2— C. sojae CBS 7871; lane 3— C. maltosa CBS 5611; lane 4— C. neerlandica NRRL Y-27057; lane 5— C. alai NRRL Y-27739; lane 6— C. labiduridarum NRRL Y-27940; lane 7— C. frijolesensis NRRL Y-48060; lane 8— C. subhashii CBS 10753; lane 9— C. jiufengensis CBS 10846; lane 10— C. albicans CBS 562. Note that three discrete bands migrating in the region

    Journal: Nucleic Acids Research

    Article Title: Evolution of linear chromosomes and multipartite genomes in yeast mitochondria

    doi: 10.1093/nar/gkq1345

    Figure Lengend Snippet: PFGE analysis of the yeast mtDNAs. The whole-cell DNA samples were separated by PFGE using a CHEF Mapper XA Chiller System (Biorad), blotted onto a nylon membrane and hybridized with mtDNA-derived probes as described in ‘Material and Methods’ section. Lane 1— C. viswanathii CBS 4024; lane 2— C. sojae CBS 7871; lane 3— C. maltosa CBS 5611; lane 4— C. neerlandica NRRL Y-27057; lane 5— C. alai NRRL Y-27739; lane 6— C. labiduridarum NRRL Y-27940; lane 7— C. frijolesensis NRRL Y-48060; lane 8— C. subhashii CBS 10753; lane 9— C. jiufengensis CBS 10846; lane 10— C. albicans CBS 562. Note that three discrete bands migrating in the region

    Article Snippet: Approximately 1 µg mtDNA aliquots were treated with exonuclease III (ExoIII; New England Biolabs ) or BAL-31 nuclease (New England Biolabs ) according to the manufacturer’s instructions, for increasing time periods.

    Techniques: Derivative Assay

    Multipartite linear-mapping genomes in C. labiduridarum and C. frijolesensis ( A ) PFGE separated samples of C. labiduridarum NRRL Y-27940 (lane 1) and C. frijolesensis NRRL Y-48060 (lane 2) were blotted onto a nylon membrane and hybridized with the radioactively labeled probes P-668 and H-1030 (regions hybridizing with both probes are shown as dashed lines). Presumed master (I) and two smaller chromosomes (II and III) are indicated. Note that the master chromosome occurs in four isomers (i.e. L III − R III − L II − R II (shown in the scheme), L III − R III − R II − L II , R III − L III − L II − R II and R III − L III − R II − L II . ‘L’ and ‘R’ indicate the left and the right telomere, respectively). The C. frijolesensis mtDNA (∼1 µg) was digested with BAL-31 nuclease ( B ) or exonuclease III (ExoIII) ( C ) as indicated. After nuclease inactivation, the DNA was digested with EcoRV, separated in 0.9% (w/v) agarose gel. The Southern blots were hybridized with the P-668 and EH-1350 probes specific for the left and the right arm of the master chromosome, respectively (see ‘Materials and Methods’ section). Arrows show the positions of the left (L) and right (R) terminal fragments and their fusions (R + R, R + L and L + L). Note that after ExoIII treatment the telomeric fragments form two subpopulations that differ in their sensitivity to the ExoIII treatment. This indicates that the linear mtDNA molecules possess an open structure with 5′ overhang or blunt end or covalently closed t-hairpin. ( D ) The C. frijolesensis mtDNA was treated with antarctic phosphatase and labeled with [γ 32 P]ATP and T4 polynucleotide kinase. The mtDNA was then digested with restriction endonuclease EcoRV (lane 1) or BglII (lane 2) and separated in 0.8% (w/v) agarose gel (left panel). The gel was fixed in 10% (v/v) methanol/10% (v/v) acetic acid for 30 min, dried overnight and autoradiographed (right panel). Arrows indicate the position of telomeric fragments containing the open structures accessible to terminal labeling.

    Journal: Nucleic Acids Research

    Article Title: Evolution of linear chromosomes and multipartite genomes in yeast mitochondria

    doi: 10.1093/nar/gkq1345

    Figure Lengend Snippet: Multipartite linear-mapping genomes in C. labiduridarum and C. frijolesensis ( A ) PFGE separated samples of C. labiduridarum NRRL Y-27940 (lane 1) and C. frijolesensis NRRL Y-48060 (lane 2) were blotted onto a nylon membrane and hybridized with the radioactively labeled probes P-668 and H-1030 (regions hybridizing with both probes are shown as dashed lines). Presumed master (I) and two smaller chromosomes (II and III) are indicated. Note that the master chromosome occurs in four isomers (i.e. L III − R III − L II − R II (shown in the scheme), L III − R III − R II − L II , R III − L III − L II − R II and R III − L III − R II − L II . ‘L’ and ‘R’ indicate the left and the right telomere, respectively). The C. frijolesensis mtDNA (∼1 µg) was digested with BAL-31 nuclease ( B ) or exonuclease III (ExoIII) ( C ) as indicated. After nuclease inactivation, the DNA was digested with EcoRV, separated in 0.9% (w/v) agarose gel. The Southern blots were hybridized with the P-668 and EH-1350 probes specific for the left and the right arm of the master chromosome, respectively (see ‘Materials and Methods’ section). Arrows show the positions of the left (L) and right (R) terminal fragments and their fusions (R + R, R + L and L + L). Note that after ExoIII treatment the telomeric fragments form two subpopulations that differ in their sensitivity to the ExoIII treatment. This indicates that the linear mtDNA molecules possess an open structure with 5′ overhang or blunt end or covalently closed t-hairpin. ( D ) The C. frijolesensis mtDNA was treated with antarctic phosphatase and labeled with [γ 32 P]ATP and T4 polynucleotide kinase. The mtDNA was then digested with restriction endonuclease EcoRV (lane 1) or BglII (lane 2) and separated in 0.8% (w/v) agarose gel (left panel). The gel was fixed in 10% (v/v) methanol/10% (v/v) acetic acid for 30 min, dried overnight and autoradiographed (right panel). Arrows indicate the position of telomeric fragments containing the open structures accessible to terminal labeling.

    Article Snippet: Approximately 1 µg mtDNA aliquots were treated with exonuclease III (ExoIII; New England Biolabs ) or BAL-31 nuclease (New England Biolabs ) according to the manufacturer’s instructions, for increasing time periods.

    Techniques: Labeling, Agarose Gel Electrophoresis