nt bbvci  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Nt BbvCI
    Description:
    Nt BbvCI 5 000 units
    Catalog Number:
    r0632l
    Price:
    285
    Size:
    5 000 units
    Category:
    Restriction Enzymes
    Buy from Supplier


    Structured Review

    New England Biolabs nt bbvci
    Nt BbvCI
    Nt BbvCI 5 000 units
    https://www.bioz.com/result/nt bbvci/product/New England Biolabs
    Average 99 stars, based on 78 article reviews
    Price from $9.99 to $1999.99
    nt bbvci - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice"

    Article Title: Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky456

    Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P
    Figure Legend Snippet: Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P

    Techniques Used: Knock-Out, Mouse Assay, Real-time Polymerase Chain Reaction

    2) Product Images from "DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli"

    Article Title: DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli

    Journal: Scientific Reports

    doi: 10.1038/srep19243

    One molecule of LacI tetramer divided a supercoiled DNA molecule plasmid pCB126 into two independent topological domains. ( a ) Plasmid pCB126 carrying two lac O1 operators in two different locations was constructed as detailed in Methods. ( b ) The nicking enzyme Nt.BbvCI was able to rapidly digest pCB126. Time course of enzyme digestion of pCB126 using 16 units of Nt.BbvCI in 1 × NEBuffer 4 at 37 °C. Lane 1 contained the undigested scDNA. ( c ) Time course of DNA supercoiling diffusion in the presence of LacI. The DNA-nicking assays were performed as described under Methods. Each reaction mixture (320 μL) contained 0.156 nM of pCB126, 2.5 nM of LacI, and 16 units of Nt.BbvCI. The reactions were incubated at 37 °C for the time indicated. Then a large excess of a double-stranded oligonucleotide contain an Nt.BbvCI recognition site was added to the reaction mixture to inhibit the restriction enzyme activities. The nicked DNA templates were ligated by T4 DNA ligase in the presence of 1 mM of ATP at 37 °C for 5 min and the reactions were terminated by phenol extraction. The DNA molecules were isolated and subjected to agarose gel electrophoresis. ( d ) Quantification analysis of the time course. The percentage of supercoiled DNA was plotted against the reaction time. The curve was generated by fitting the data to a 1st-order rate equation to yield a rate constant of 0.016 sec −1 and a t 1/2 of 52 sec.
    Figure Legend Snippet: One molecule of LacI tetramer divided a supercoiled DNA molecule plasmid pCB126 into two independent topological domains. ( a ) Plasmid pCB126 carrying two lac O1 operators in two different locations was constructed as detailed in Methods. ( b ) The nicking enzyme Nt.BbvCI was able to rapidly digest pCB126. Time course of enzyme digestion of pCB126 using 16 units of Nt.BbvCI in 1 × NEBuffer 4 at 37 °C. Lane 1 contained the undigested scDNA. ( c ) Time course of DNA supercoiling diffusion in the presence of LacI. The DNA-nicking assays were performed as described under Methods. Each reaction mixture (320 μL) contained 0.156 nM of pCB126, 2.5 nM of LacI, and 16 units of Nt.BbvCI. The reactions were incubated at 37 °C for the time indicated. Then a large excess of a double-stranded oligonucleotide contain an Nt.BbvCI recognition site was added to the reaction mixture to inhibit the restriction enzyme activities. The nicked DNA templates were ligated by T4 DNA ligase in the presence of 1 mM of ATP at 37 °C for 5 min and the reactions were terminated by phenol extraction. The DNA molecules were isolated and subjected to agarose gel electrophoresis. ( d ) Quantification analysis of the time course. The percentage of supercoiled DNA was plotted against the reaction time. The curve was generated by fitting the data to a 1st-order rate equation to yield a rate constant of 0.016 sec −1 and a t 1/2 of 52 sec.

    Techniques Used: Plasmid Preparation, Construct, Diffusion-based Assay, Incubation, Isolation, Agarose Gel Electrophoresis, Generated, Size-exclusion Chromatography

    3) Product Images from "Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments"

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl635

    Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.
    Figure Legend Snippet: Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.

    Techniques Used: Clone Assay, Sequencing, Synthesized, Construct, Plasmid Preparation, Generated

    Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo ® C x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.
    Figure Legend Snippet: Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo ® C x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.

    Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Incubation, Transformation Assay, Ligation, Generated

    4) Product Images from "Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae"

    Article Title: Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky743

    Breakage of S. cerevisiae gDNA and λ DNA at preformed ss nicks upon molecular combing. ( A-C ) Molecular combing of nick-translated gDNA from S. cerevisiae . Biotinylated nucleotides were incorporated by nick-translation conducted either in limiting (L), or non-limiting/standard conditions (N), into agarose-embedded gDNA of unperturbed, non-synchronized (A and C), or G1-synchronized (B) BY4741 cells. Biotin was detected by AlexaFluor 647-conjugated anti-biotin antibody (red) and DNA molecules were stained with YOYO-1 (green). Panel D shows examples of co-localization of nicks labeled with TdT (magenta) and R-loops labeled with the RNA:DNA hybrid specific S9.6 antibody (red), when both entities were visualized in the same sample. The percentage of co-labeled spots was estimated ∼10% of all nick-related DNA associated spots. Arrows indicate examples of co-localization. ( E–G ) Molecular combing of λ DNA. Representative images of YOYO-1 stained (green) control (F) and Nt.BbvCI nickase-treated (G) λ DNA. The size distribution histograms of combed DNA molecules before (blue) and after (orange) nickase treatment are shown in panel E. The full length intact ds λ DNA (48.5 kb) corresponds to 16.2 μm (calculated with 3 bp/nm helical repeat length), i.e. the majority of λ DNA molecules were fragmented after combing alone. Images of DNA fibers were assembled from the fields-of-view analyzed, except for panels F and G which show the original fields-of-view. For statistics see Supplementary Tables S1–S5 .
    Figure Legend Snippet: Breakage of S. cerevisiae gDNA and λ DNA at preformed ss nicks upon molecular combing. ( A-C ) Molecular combing of nick-translated gDNA from S. cerevisiae . Biotinylated nucleotides were incorporated by nick-translation conducted either in limiting (L), or non-limiting/standard conditions (N), into agarose-embedded gDNA of unperturbed, non-synchronized (A and C), or G1-synchronized (B) BY4741 cells. Biotin was detected by AlexaFluor 647-conjugated anti-biotin antibody (red) and DNA molecules were stained with YOYO-1 (green). Panel D shows examples of co-localization of nicks labeled with TdT (magenta) and R-loops labeled with the RNA:DNA hybrid specific S9.6 antibody (red), when both entities were visualized in the same sample. The percentage of co-labeled spots was estimated ∼10% of all nick-related DNA associated spots. Arrows indicate examples of co-localization. ( E–G ) Molecular combing of λ DNA. Representative images of YOYO-1 stained (green) control (F) and Nt.BbvCI nickase-treated (G) λ DNA. The size distribution histograms of combed DNA molecules before (blue) and after (orange) nickase treatment are shown in panel E. The full length intact ds λ DNA (48.5 kb) corresponds to 16.2 μm (calculated with 3 bp/nm helical repeat length), i.e. the majority of λ DNA molecules were fragmented after combing alone. Images of DNA fibers were assembled from the fields-of-view analyzed, except for panels F and G which show the original fields-of-view. For statistics see Supplementary Tables S1–S5 .

    Techniques Used: Nick Translation, Staining, Labeling

    5) Product Images from "Competition between the RNA Transcript and the Nontemplate DNA Strand during R-Loop Formation In Vitro: a Nick Can Serve as a Strong R-Loop Initiation Site ▿Competition between the RNA Transcript and the Nontemplate DNA Strand during R-Loop Formation In Vitro: a Nick Can Serve as a Strong R-Loop Initiation Site ▿ †"

    Article Title: Competition between the RNA Transcript and the Nontemplate DNA Strand during R-Loop Formation In Vitro: a Nick Can Serve as a Strong R-Loop Initiation Site ▿Competition between the RNA Transcript and the Nontemplate DNA Strand during R-Loop Formation In Vitro: a Nick Can Serve as a Strong R-Loop Initiation Site ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00897-09

    Presence of a nick on the nontemplate strand downstream of the promoter increases R-loop formation even in the absence of switch repeats. Three substrates, pDR16, pDR89, and pDR90, were mock incubated or incubated with the nicking enzyme Nt.BbvCI, and then linearized with SalI. The position of the linearized fragment is marked “L.” No switch repeats are present in these substrates. pDR16 does not have an Nt.BbvCI recognition site, whereas a nicking site (shown as an inverted triangle) is present upstream of the promoter in pDR88 and downstream of the promoter in pDR90. In each set, the first three lanes are unnicked whereas the next three lanes contain the same substrate treated with Nt.BbvCI. In each subset of lanes, the first lane is mock-transcribed substrate, followed by a lane with T7-transcribed substrate treated with RNase A after transcription. The third lane contains the transcribed substrate treated with RNase A and RNase H. The top panel is the ethidium bromide-stained gel showing the positions of the linearized fragments containing the T7 promoter. No shifts are seen for pDR16, pDR89, or the unnicked version of pDR90. Although small in amount, the nicked version of pDR90 shows a shifted band, indicating an increased ability of nicked pDR90 to form R loops compared to unnicked pDR16, pDR89, and pDR90 or with a promoter-upstream nick in pDR89. The bottom panel is the same gel showing the location of radiolabeled bands (labeled with [α- 32 P]UTP). A distinct radiolabeled band is observed at the shifted position for nicked and transcribed pDR90, compared to the shifted or the linear (“L”) positions for other substrates, where no radiolabel localization is seen. In the same lane (lane 19), an additional radiolabeled band localizes near the linear fragment, indicating an increased propensity for RNA:DNA hybrid formation by the transcript in the presence of the nick.
    Figure Legend Snippet: Presence of a nick on the nontemplate strand downstream of the promoter increases R-loop formation even in the absence of switch repeats. Three substrates, pDR16, pDR89, and pDR90, were mock incubated or incubated with the nicking enzyme Nt.BbvCI, and then linearized with SalI. The position of the linearized fragment is marked “L.” No switch repeats are present in these substrates. pDR16 does not have an Nt.BbvCI recognition site, whereas a nicking site (shown as an inverted triangle) is present upstream of the promoter in pDR88 and downstream of the promoter in pDR90. In each set, the first three lanes are unnicked whereas the next three lanes contain the same substrate treated with Nt.BbvCI. In each subset of lanes, the first lane is mock-transcribed substrate, followed by a lane with T7-transcribed substrate treated with RNase A after transcription. The third lane contains the transcribed substrate treated with RNase A and RNase H. The top panel is the ethidium bromide-stained gel showing the positions of the linearized fragments containing the T7 promoter. No shifts are seen for pDR16, pDR89, or the unnicked version of pDR90. Although small in amount, the nicked version of pDR90 shows a shifted band, indicating an increased ability of nicked pDR90 to form R loops compared to unnicked pDR16, pDR89, and pDR90 or with a promoter-upstream nick in pDR89. The bottom panel is the same gel showing the location of radiolabeled bands (labeled with [α- 32 P]UTP). A distinct radiolabeled band is observed at the shifted position for nicked and transcribed pDR90, compared to the shifted or the linear (“L”) positions for other substrates, where no radiolabel localization is seen. In the same lane (lane 19), an additional radiolabeled band localizes near the linear fragment, indicating an increased propensity for RNA:DNA hybrid formation by the transcript in the presence of the nick.

    Techniques Used: Incubation, Staining, Labeling

    The presence of a nick on the nontemplate strand downstream of the promoter increases R-loop formation at the downstream switch repeats. Three substrates, pDR18, pDR87, and pDR88, were mock incubated or incubated with the nicking enzyme Nt.BbvCI and then linearized with SalI. pDR87 and pDR88 are derived from pDR18, and all have four Sγ3 repeats downstream of the promoter, except that pDR18 does not have a BbvCI recognition site. A nicking site (shown as an inverted triangle) is present upstream of the promoter in pDR87 and between the promoter and the switch repeats in pDR88. The four arrows represent the four Sγ3 repeats. For each substrate, the first three lanes are unnicked substrates, whereas the next three lanes contain the same substrate treated with Nt.BbvCI. The position of the linearized fragment is marked “L.” In each subset of lanes, the first lane is mock-transcribed substrate, followed by a T7-transcribed substrate subsequently treated with RNase A after transcription. The third lane contains the transcribed substrate treated with RNase A and RNase H. All samples were organically extracted before being run on an agarose gel. The top panel is the ethidium bromide-stained profile of the agarose gel. Comparable amounts of shifted nucleic acids are seen for pDR18, pDR87, or the unnicked version of pDR87. The nicked version of pDR88 shows a much larger amount of shift, indicating a highly efficient R-loop formation compared to unnicked substrates or to a substrate with a nick upstream of the promoter. The bottom panel shows the same gel with the location of radiolabeled ([α- 32 P]UTP) bands. While almost no radiolabel is present at the position where the switch repeat-containing linearized fragments run, we observed the presence of a large amount of radiolabel at the shifted position for nicked and transcribed pDR88 (compared to the shifted positions in other substrates and unnicked pDR87). It should be noted that this shifted position, as also observed in the top panel, runs slower than the shifted positions in other substrates, and this mobility change could be because of the presence of the partially displaced nontemplate DNA strand. In the same lane (lane 19), an additional radiolabeled band localizes near the linear fragment (marked “L”), indicating that short RNA:DNA hybrids are formed at or near the nicked position.
    Figure Legend Snippet: The presence of a nick on the nontemplate strand downstream of the promoter increases R-loop formation at the downstream switch repeats. Three substrates, pDR18, pDR87, and pDR88, were mock incubated or incubated with the nicking enzyme Nt.BbvCI and then linearized with SalI. pDR87 and pDR88 are derived from pDR18, and all have four Sγ3 repeats downstream of the promoter, except that pDR18 does not have a BbvCI recognition site. A nicking site (shown as an inverted triangle) is present upstream of the promoter in pDR87 and between the promoter and the switch repeats in pDR88. The four arrows represent the four Sγ3 repeats. For each substrate, the first three lanes are unnicked substrates, whereas the next three lanes contain the same substrate treated with Nt.BbvCI. The position of the linearized fragment is marked “L.” In each subset of lanes, the first lane is mock-transcribed substrate, followed by a T7-transcribed substrate subsequently treated with RNase A after transcription. The third lane contains the transcribed substrate treated with RNase A and RNase H. All samples were organically extracted before being run on an agarose gel. The top panel is the ethidium bromide-stained profile of the agarose gel. Comparable amounts of shifted nucleic acids are seen for pDR18, pDR87, or the unnicked version of pDR87. The nicked version of pDR88 shows a much larger amount of shift, indicating a highly efficient R-loop formation compared to unnicked substrates or to a substrate with a nick upstream of the promoter. The bottom panel shows the same gel with the location of radiolabeled ([α- 32 P]UTP) bands. While almost no radiolabel is present at the position where the switch repeat-containing linearized fragments run, we observed the presence of a large amount of radiolabel at the shifted position for nicked and transcribed pDR88 (compared to the shifted positions in other substrates and unnicked pDR87). It should be noted that this shifted position, as also observed in the top panel, runs slower than the shifted positions in other substrates, and this mobility change could be because of the presence of the partially displaced nontemplate DNA strand. In the same lane (lane 19), an additional radiolabeled band localizes near the linear fragment (marked “L”), indicating that short RNA:DNA hybrids are formed at or near the nicked position.

    Techniques Used: Incubation, Derivative Assay, Agarose Gel Electrophoresis, Staining

    6) Product Images from "Identification of hemicatenane-specific binding proteins by fractionation of Hela nuclei extracts"

    Article Title: Identification of hemicatenane-specific binding proteins by fractionation of Hela nuclei extracts

    Journal: bioRxiv

    doi: 10.1101/844126

    Interaction between purified SND1 proteins and various DNA constructs. A The organization of SND1 in domains is shown. The double arrow underneath the schematic representation corresponds to the proteins that were expressed in E. coli and purified. The name of the purified protein is indicated on the left side of the double arrow. B Interactions were performed in a final volume of 7.5 μL with 0.1 femtomole of radiolabeled DNA and the indicated amount of purified protein. Species were resolved by electrophoresis under native conditions. Three DNAs were tested for their binding to SND1-64: dsMC09 (lanes 1-5); dsMC10 (lane 6-10); HC (lanes 11-15). Concentrations of protein were as indicated (lanes 1, 6, 11: 0; lanes 2, 7, 12: 0.1 μM; lanes 3, 8, 13: 0.3 μM; lanes 4, 9, 14: 0.9 μM; lanes 5, 10, 15: 2.7 μM). Free DNAs and bound DNAs are indicated. C Interactions were performed in a final volume of 13.25 μL with 0.1 femtomole of radiolabeled DNA and the SDN1-110 at 70 nM. Species were resolved by electrophoresis under native conditions. Three DNAs were tested for their binding to SND1-110: dsMC09 (lanes 1 and 2); dsMC10 (lanes 3 and 4); HC (lanes 5 and 6). Free DNAs and bound DNAs are indicated. D Interactions were as described in (A). The DNA was C10ss, the single stranded circle obtained after nicking of the dsMC10 with Nt.BbvcI. 0.1 femtomole of C10ss was included in the reaction mixture and the concentrations of protein were as indicated (lane 1: 0; lane 2: 0.1 μM; lane 3: 0.3 μM; lane 4: 0.9 μM; lane 5: 2.7 μM). Free DNAs and bound DNAs are indicated. E The two curves (one for HC and one for C10ss) show the percentage of (SDN1-64)-DNA complexes as a function of protein concentration. Error bars correspond to the standard deviation. Percentages are the mean of three independent experiments. F Interactions were as described in (C). C10ss is the single stranded circle obtained after nicking of the dsMC10 with Nt.BbvcI. 0.1 femtomole of C10ss or HC was included in the reaction mixture and the concentration of protein was 70 nM. Free DNAs and bound DNAs are indicated. G The plot shows the percentage of (SDN1-110)-DNA complexes assembled at 70 nM SND1-110. Error bars correspond to the standard deviation. Percentages are the mean of three independent experiments. H The interaction between radiolabeled HC and SND1-64 was tested in the presence of OL21, an oligonucleotide long of 21 nucleotides. SDN1-64 was at 2.7 μM and HC at 14 pM. HC was premixed with increasing amount of OL21 (lane 1: no OL21, no SND1-64; lane 2: no OL21; lane 3: 0.2 nM OL21; lane 4: 0.7 nM OL21; lane 5: 2 nM OL21; lane 6: 7 nM OL21) before adding SND1-64. Free DNAs and bound DNAs are indicated. I The interaction between radiolabeled HC and SND1-110 was tested in the presence of OL21, an oligonucleotide long of 21 nucleotides. SDN1-110 was at 70 nM and HC at 7.5 pM. HC was premixed with increasing amount of OL21 (lane 1: no OL21, no SND1-110; lane 2: no OL21; lane 3: 0.2 nM OL21; lane 4: 0.7 nM OL21; lane 5: 2 nM OL21; lane 6: 7 nM OL21) before adding SND1-110. Species are separated by electrophoresis on a polyacrylamide gel. Free DNAs and bound DNAs are indicated. The plot shows the percentage of (SND1-110)-HC complexes as function of concentration of OL21. The standard deviation is calculated from two independent experiments.
    Figure Legend Snippet: Interaction between purified SND1 proteins and various DNA constructs. A The organization of SND1 in domains is shown. The double arrow underneath the schematic representation corresponds to the proteins that were expressed in E. coli and purified. The name of the purified protein is indicated on the left side of the double arrow. B Interactions were performed in a final volume of 7.5 μL with 0.1 femtomole of radiolabeled DNA and the indicated amount of purified protein. Species were resolved by electrophoresis under native conditions. Three DNAs were tested for their binding to SND1-64: dsMC09 (lanes 1-5); dsMC10 (lane 6-10); HC (lanes 11-15). Concentrations of protein were as indicated (lanes 1, 6, 11: 0; lanes 2, 7, 12: 0.1 μM; lanes 3, 8, 13: 0.3 μM; lanes 4, 9, 14: 0.9 μM; lanes 5, 10, 15: 2.7 μM). Free DNAs and bound DNAs are indicated. C Interactions were performed in a final volume of 13.25 μL with 0.1 femtomole of radiolabeled DNA and the SDN1-110 at 70 nM. Species were resolved by electrophoresis under native conditions. Three DNAs were tested for their binding to SND1-110: dsMC09 (lanes 1 and 2); dsMC10 (lanes 3 and 4); HC (lanes 5 and 6). Free DNAs and bound DNAs are indicated. D Interactions were as described in (A). The DNA was C10ss, the single stranded circle obtained after nicking of the dsMC10 with Nt.BbvcI. 0.1 femtomole of C10ss was included in the reaction mixture and the concentrations of protein were as indicated (lane 1: 0; lane 2: 0.1 μM; lane 3: 0.3 μM; lane 4: 0.9 μM; lane 5: 2.7 μM). Free DNAs and bound DNAs are indicated. E The two curves (one for HC and one for C10ss) show the percentage of (SDN1-64)-DNA complexes as a function of protein concentration. Error bars correspond to the standard deviation. Percentages are the mean of three independent experiments. F Interactions were as described in (C). C10ss is the single stranded circle obtained after nicking of the dsMC10 with Nt.BbvcI. 0.1 femtomole of C10ss or HC was included in the reaction mixture and the concentration of protein was 70 nM. Free DNAs and bound DNAs are indicated. G The plot shows the percentage of (SDN1-110)-DNA complexes assembled at 70 nM SND1-110. Error bars correspond to the standard deviation. Percentages are the mean of three independent experiments. H The interaction between radiolabeled HC and SND1-64 was tested in the presence of OL21, an oligonucleotide long of 21 nucleotides. SDN1-64 was at 2.7 μM and HC at 14 pM. HC was premixed with increasing amount of OL21 (lane 1: no OL21, no SND1-64; lane 2: no OL21; lane 3: 0.2 nM OL21; lane 4: 0.7 nM OL21; lane 5: 2 nM OL21; lane 6: 7 nM OL21) before adding SND1-64. Free DNAs and bound DNAs are indicated. I The interaction between radiolabeled HC and SND1-110 was tested in the presence of OL21, an oligonucleotide long of 21 nucleotides. SDN1-110 was at 70 nM and HC at 7.5 pM. HC was premixed with increasing amount of OL21 (lane 1: no OL21, no SND1-110; lane 2: no OL21; lane 3: 0.2 nM OL21; lane 4: 0.7 nM OL21; lane 5: 2 nM OL21; lane 6: 7 nM OL21) before adding SND1-110. Species are separated by electrophoresis on a polyacrylamide gel. Free DNAs and bound DNAs are indicated. The plot shows the percentage of (SND1-110)-HC complexes as function of concentration of OL21. The standard deviation is calculated from two independent experiments.

    Techniques Used: Purification, Construct, Electrophoresis, Binding Assay, Protein Concentration, Standard Deviation, Concentration Assay

    7) Product Images from "Mapping the nicking efficiencies of nickase R.BbvCI for side-specific LNA-substituted substrates using rolling circle amplification"

    Article Title: Mapping the nicking efficiencies of nickase R.BbvCI for side-specific LNA-substituted substrates using rolling circle amplification

    Journal: Scientific Reports

    doi: 10.1038/srep32560

    Cleavage efficiency of Nb.BbvCI or Nt.BbvCI affected by BS or TS modifications. ( a ) Nb.BbvCI cleavage activities affected by LNA substitutions on the same strand to be nicked (BS). ( b ) Nb.BbvCI cleavage activities affected by the LNA substitutions on the complementary strand (TS). ( c ) Nt.BbvCI cleavage activities affected by the LNA substitutions on the same strand to be nicked (TS). ( d ) Nt.BbvCI cleavage activities affected by the LNA substitutions on the complementary strand. DNA represents the unmodified strand. G1, C2, T3, G4, A5, G6 and G7 represent the LNA modified bottom strands. C1, C2, T3, C4, A5, G6 and C7 represent the LNA modified top strands. RCE represents the relative cleavage efficiency.
    Figure Legend Snippet: Cleavage efficiency of Nb.BbvCI or Nt.BbvCI affected by BS or TS modifications. ( a ) Nb.BbvCI cleavage activities affected by LNA substitutions on the same strand to be nicked (BS). ( b ) Nb.BbvCI cleavage activities affected by the LNA substitutions on the complementary strand (TS). ( c ) Nt.BbvCI cleavage activities affected by the LNA substitutions on the same strand to be nicked (TS). ( d ) Nt.BbvCI cleavage activities affected by the LNA substitutions on the complementary strand. DNA represents the unmodified strand. G1, C2, T3, G4, A5, G6 and G7 represent the LNA modified bottom strands. C1, C2, T3, C4, A5, G6 and C7 represent the LNA modified top strands. RCE represents the relative cleavage efficiency.

    Techniques Used: Modification

    8) Product Images from "Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments"

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl635

    Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.
    Figure Legend Snippet: Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.

    Techniques Used: Clone Assay, Sequencing, Synthesized, Construct, Plasmid Preparation, Generated

    Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo ® C x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.
    Figure Legend Snippet: Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo ® C x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.

    Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Incubation, Transformation Assay, Ligation, Generated

    9) Product Images from "High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments"

    Article Title: High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz851

    Experimental strategies to assemble long DNA and RNA hairpins. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled. ( A ) DNA hairpin construct using LNC: linear or plasmid DNA is used as template for PCR reactions; amplified fragments are purified and digested; fragments are then submitted to three rounds of purification and ligation (L1, L2, L3) to obtain the desired final product. ( B ) DNA hairpin construct, annealing method (ANC): template DNA is amplified by PCR and purified (pur.); one strand of the amplified fragments is nicked with enzymes Nb.BbvCI or Nt.BbvCI, gel purified and annealed (ann.) to obtain the final construct. ( C ) RNA hairpin construct: template DNA is amplified by PCR and purified, stem is amplified in three separate parts; RNA products are obtained by IVTR, purified and monophosphorylated (mP); products are then annealed and ligated to obtained the final construct.
    Figure Legend Snippet: Experimental strategies to assemble long DNA and RNA hairpins. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled. ( A ) DNA hairpin construct using LNC: linear or plasmid DNA is used as template for PCR reactions; amplified fragments are purified and digested; fragments are then submitted to three rounds of purification and ligation (L1, L2, L3) to obtain the desired final product. ( B ) DNA hairpin construct, annealing method (ANC): template DNA is amplified by PCR and purified (pur.); one strand of the amplified fragments is nicked with enzymes Nb.BbvCI or Nt.BbvCI, gel purified and annealed (ann.) to obtain the final construct. ( C ) RNA hairpin construct: template DNA is amplified by PCR and purified, stem is amplified in three separate parts; RNA products are obtained by IVTR, purified and monophosphorylated (mP); products are then annealed and ligated to obtained the final construct.

    Techniques Used: Labeling, Construct, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Purification, Ligation

    10) Product Images from "Novel approach reveals genomic landscapes of single-strand DNA breaks with nucleotide resolution in human cells"

    Article Title: Novel approach reveals genomic landscapes of single-strand DNA breaks with nucleotide resolution in human cells

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13602-7

    Validation of SSiNGLe-SMS and SSiNGLe-ILM. a Agarose gel electrophoresis analysis of genomic DNA isolated from cells treated for 48 h with indicated drugs and then digested (right) or not digested (left) with MNase. Signal at the bottom of the gel with un-digested material represents residual degraded RNA. b Fraction of uniquely aligned reads that survives filtration for the adjacent endogenous polyA stretches in different tailed and untailed samples. c Results of three replicas (“Rep 1” – “Rep 3”) of in situ digestion of nuclei from H 2 O 2 -treated K562 cells using Nt.BbvCI with or without SAP. Fraction of SSBs ( Y -axis) found by SSiNGLe-ILM (relative to total SSBs detected in each sample) mapping to each of the indicated bases of the Nt.BbvCI sites and flanking sequences ( X -axis) for the top and bottom strands of the site. The cleavage site is shown with the red arrow. d Overlap between SSBs or DSB found by SSiNGLe-ILM in HeLa cells with DSBs found by the BLESS protocol 9 . The odds ratios and numbers of overlapping breaks are shown for the different distances between the breaks in the two datasets. e Fraction of detected AsiSI and Nt.BbvCI sites ( Y -axis) at different numbers of filtered reads ( X -axis). Each experiment was done in two replicas. f Median fraction of SSBs ( Y -axes) mapping to each base ( X -axes) of the minus or H strand of the chrM genome is shown for each of the three sample types. g Zoom-in on the region of the major peak in the panel ( f ). The red arrow represents the previously reported 3′ end of 7S DNA 21 , 22 . Only background-level signal can be observed on the opposite strand (Supplementary Table 3 ). h Box plots of normalized scores of SSBs at each position around AsiSI cut sites (±10 bp) for 28 samples (14+Dox and 14 −Dox). Normalized score = \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\rm{SSBs}}\;{\rm{detected}}\;{\rm{at}}\;{\rm{each}}\;{\rm{position}} \ast 10^9}}{{{\rm{Total}}\;{\rm{number}}\;{\rm{of}}\;{\rm{SSBs}}}}$$\end{document} SSBs detected at each position * 1 0 9 Total number of SSBs .
    Figure Legend Snippet: Validation of SSiNGLe-SMS and SSiNGLe-ILM. a Agarose gel electrophoresis analysis of genomic DNA isolated from cells treated for 48 h with indicated drugs and then digested (right) or not digested (left) with MNase. Signal at the bottom of the gel with un-digested material represents residual degraded RNA. b Fraction of uniquely aligned reads that survives filtration for the adjacent endogenous polyA stretches in different tailed and untailed samples. c Results of three replicas (“Rep 1” – “Rep 3”) of in situ digestion of nuclei from H 2 O 2 -treated K562 cells using Nt.BbvCI with or without SAP. Fraction of SSBs ( Y -axis) found by SSiNGLe-ILM (relative to total SSBs detected in each sample) mapping to each of the indicated bases of the Nt.BbvCI sites and flanking sequences ( X -axis) for the top and bottom strands of the site. The cleavage site is shown with the red arrow. d Overlap between SSBs or DSB found by SSiNGLe-ILM in HeLa cells with DSBs found by the BLESS protocol 9 . The odds ratios and numbers of overlapping breaks are shown for the different distances between the breaks in the two datasets. e Fraction of detected AsiSI and Nt.BbvCI sites ( Y -axis) at different numbers of filtered reads ( X -axis). Each experiment was done in two replicas. f Median fraction of SSBs ( Y -axes) mapping to each base ( X -axes) of the minus or H strand of the chrM genome is shown for each of the three sample types. g Zoom-in on the region of the major peak in the panel ( f ). The red arrow represents the previously reported 3′ end of 7S DNA 21 , 22 . Only background-level signal can be observed on the opposite strand (Supplementary Table 3 ). h Box plots of normalized scores of SSBs at each position around AsiSI cut sites (±10 bp) for 28 samples (14+Dox and 14 −Dox). Normalized score = \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{{{\rm{SSBs}}\;{\rm{detected}}\;{\rm{at}}\;{\rm{each}}\;{\rm{position}} \ast 10^9}}{{{\rm{Total}}\;{\rm{number}}\;{\rm{of}}\;{\rm{SSBs}}}}$$\end{document} SSBs detected at each position * 1 0 9 Total number of SSBs .

    Techniques Used: Agarose Gel Electrophoresis, Isolation, Filtration, In Situ, AST Assay

    11) Product Images from "UCE: A uracil excision (USER(TM))-based toolbox for transformation of cereals"

    Article Title: UCE: A uracil excision (USER(TM))-based toolbox for transformation of cereals

    Journal: Plant Methods

    doi: 10.1186/1746-4811-6-15

    Overview of the USER™ reaction . A . The vector carrying the USER™ cassette is digested simultaneously with the restriction enzyme PacI and the nicking enzyme Nt.BbvCI , which creates 9 nt long 3' overhangs. The insert, which is to be fused with the vector, is amplified by PCR using standard PCR primers for the insert, but with the addition of 9 nt complementary to the USER™ site, including a uracil base. In the USER™ reaction USER ™ enzymes remove the uracil creating 9 nt long 5' overhangs complementary to the overhangs of the digested vector. Spontaneous annealing will then result in the desired fusion of the PCR product and vector. Bases in bold indicates the recognition sites of the relevant enzymes, and the arrowheads indicates the site of cleavage. B . The two USER™ cassettes designed and used in this study, the USER TC-TG and the USER TC-CC .
    Figure Legend Snippet: Overview of the USER™ reaction . A . The vector carrying the USER™ cassette is digested simultaneously with the restriction enzyme PacI and the nicking enzyme Nt.BbvCI , which creates 9 nt long 3' overhangs. The insert, which is to be fused with the vector, is amplified by PCR using standard PCR primers for the insert, but with the addition of 9 nt complementary to the USER™ site, including a uracil base. In the USER™ reaction USER ™ enzymes remove the uracil creating 9 nt long 5' overhangs complementary to the overhangs of the digested vector. Spontaneous annealing will then result in the desired fusion of the PCR product and vector. Bases in bold indicates the recognition sites of the relevant enzymes, and the arrowheads indicates the site of cleavage. B . The two USER™ cassettes designed and used in this study, the USER TC-TG and the USER TC-CC .

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction

    12) Product Images from "Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice"

    Article Title: Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky456

    Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P
    Figure Legend Snippet: Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P

    Techniques Used: Knock-Out, Mouse Assay, Real-time Polymerase Chain Reaction

    13) Product Images from "Cleavage of a model DNA replication fork by a methyl-specific endonuclease"

    Article Title: Cleavage of a model DNA replication fork by a methyl-specific endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr153

    Preparation of a long-branched DNA with methylation. The two plasmids were modified in vivo by M.FnuDII to generate 5′-m5 C GCG, a McrBC recognition sequence {(i), (ii)}. Potential unmethylated plasmids were eliminated by cleavage with BstUI (5′-CGCG). pME63 was cleaved with PvuII and then with nicking endonuclease Nb.BbvCI (iii), while pMap63 was treated with nicking endonuclease Nt.BbvCI (iv). The resulting short single strands were dissociated by heating and removed by annealing with a complementary single-strand oligo DNA. The 5′-ends of intermediate (iv) were labeled with 32 P (v), followed by BspHI cleavage for removal of one of the radio-labels (vii). The two DNAs with complementary single-strand regions {(vi), (vii)} were annealed to form a branched structure {viii, eM63(++)} as detailed in ‘Materials and Methods’ section. Open circle, 32 P label at 5′-end; filled diamond, DNA methylation.
    Figure Legend Snippet: Preparation of a long-branched DNA with methylation. The two plasmids were modified in vivo by M.FnuDII to generate 5′-m5 C GCG, a McrBC recognition sequence {(i), (ii)}. Potential unmethylated plasmids were eliminated by cleavage with BstUI (5′-CGCG). pME63 was cleaved with PvuII and then with nicking endonuclease Nb.BbvCI (iii), while pMap63 was treated with nicking endonuclease Nt.BbvCI (iv). The resulting short single strands were dissociated by heating and removed by annealing with a complementary single-strand oligo DNA. The 5′-ends of intermediate (iv) were labeled with 32 P (v), followed by BspHI cleavage for removal of one of the radio-labels (vii). The two DNAs with complementary single-strand regions {(vi), (vii)} were annealed to form a branched structure {viii, eM63(++)} as detailed in ‘Materials and Methods’ section. Open circle, 32 P label at 5′-end; filled diamond, DNA methylation.

    Techniques Used: DNA Methylation Assay, Modification, In Vivo, Sequencing, Labeling

    14) Product Images from "Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases"

    Article Title: Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

    Journal: Scientific Reports

    doi: 10.1038/srep36006

    An experimental strategy to construct relaxed (rx) or supercoiled (sc) pAB1_FL905. ( A ) Oligomer FL905 that contains the 42 nt. AT sequence is ligated between the two Nt.BbvCI sites of plasmid pAB1 to yield rx pAB1_FL905. ( B ) Sc pAB1_FL905 can be generated through the treatment of rx pAB1_FL905 by E. coli DNA gyrase. The fluorescence intensity of fluorescein is dependent on the supercoiling status of pAB1_FL905.
    Figure Legend Snippet: An experimental strategy to construct relaxed (rx) or supercoiled (sc) pAB1_FL905. ( A ) Oligomer FL905 that contains the 42 nt. AT sequence is ligated between the two Nt.BbvCI sites of plasmid pAB1 to yield rx pAB1_FL905. ( B ) Sc pAB1_FL905 can be generated through the treatment of rx pAB1_FL905 by E. coli DNA gyrase. The fluorescence intensity of fluorescein is dependent on the supercoiling status of pAB1_FL905.

    Techniques Used: Construct, Sequencing, Plasmid Preparation, Generated, Fluorescence

    ( A ) Fluorescence spectra of sc (red line), rx (black line), and nk (blue line) pAB1_FL509. λex = 470 nm. ( B ) Kinetics of the nicking reaction by Nt.BbvCI. Briefly, 60 μL of 1 × CutSmart buffer containing 500 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of Nt.BbvCI were added to initiate the nicking reaction. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( C ) Kinetics of the relaxation reaction by E. coli DNA topoisomerase I. For the relaxation reaction, 90 μL of 1 × NEBuffer 4 (50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(AC) 2 , 1 mM DTT, pH 7.9) containing 270 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 0.67 μM of E. coli DNA topoisomerase I was used to relax the sc pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( D ) Kinetics of the supercoiling reaction by E. coli DNA gyrase. For the supercoiling reaction, 90 μL of 1 × gyrase buffer containing 1 μg of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 30 units of E. coli DNA gyrase was used to supercoil the rx pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm.
    Figure Legend Snippet: ( A ) Fluorescence spectra of sc (red line), rx (black line), and nk (blue line) pAB1_FL509. λex = 470 nm. ( B ) Kinetics of the nicking reaction by Nt.BbvCI. Briefly, 60 μL of 1 × CutSmart buffer containing 500 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of Nt.BbvCI were added to initiate the nicking reaction. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( C ) Kinetics of the relaxation reaction by E. coli DNA topoisomerase I. For the relaxation reaction, 90 μL of 1 × NEBuffer 4 (50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(AC) 2 , 1 mM DTT, pH 7.9) containing 270 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 0.67 μM of E. coli DNA topoisomerase I was used to relax the sc pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( D ) Kinetics of the supercoiling reaction by E. coli DNA gyrase. For the supercoiling reaction, 90 μL of 1 × gyrase buffer containing 1 μg of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 30 units of E. coli DNA gyrase was used to supercoil the rx pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm.

    Techniques Used: Fluorescence

    15) Product Images from "Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae"

    Article Title: Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky743

    Breakage of S. cerevisiae gDNA and λ DNA at preformed ss nicks upon molecular combing. ( A-C ) Molecular combing of nick-translated gDNA from S. cerevisiae . Biotinylated nucleotides were incorporated by nick-translation conducted either in limiting (L), or non-limiting/standard conditions (N), into agarose-embedded gDNA of unperturbed, non-synchronized (A and C), or G1-synchronized (B) BY4741 cells. Biotin was detected by AlexaFluor 647-conjugated anti-biotin antibody (red) and DNA molecules were stained with YOYO-1 (green). Panel D shows examples of co-localization of nicks labeled with TdT (magenta) and R-loops labeled with the RNA:DNA hybrid specific S9.6 antibody (red), when both entities were visualized in the same sample. The percentage of co-labeled spots was estimated ∼10% of all nick-related DNA associated spots. Arrows indicate examples of co-localization. ( E–G ) Molecular combing of λ DNA. Representative images of YOYO-1 stained (green) control (F) and Nt.BbvCI nickase-treated (G) λ DNA. The size distribution histograms of combed DNA molecules before (blue) and after (orange) nickase treatment are shown in panel E. The full length intact ds λ DNA (48.5 kb) corresponds to 16.2 μm (calculated with 3 bp/nm helical repeat length), i.e. the majority of λ DNA molecules were fragmented after combing alone. Images of DNA fibers were assembled from the fields-of-view analyzed, except for panels F and G which show the original fields-of-view. For statistics see Supplementary Tables S1–S5 .
    Figure Legend Snippet: Breakage of S. cerevisiae gDNA and λ DNA at preformed ss nicks upon molecular combing. ( A-C ) Molecular combing of nick-translated gDNA from S. cerevisiae . Biotinylated nucleotides were incorporated by nick-translation conducted either in limiting (L), or non-limiting/standard conditions (N), into agarose-embedded gDNA of unperturbed, non-synchronized (A and C), or G1-synchronized (B) BY4741 cells. Biotin was detected by AlexaFluor 647-conjugated anti-biotin antibody (red) and DNA molecules were stained with YOYO-1 (green). Panel D shows examples of co-localization of nicks labeled with TdT (magenta) and R-loops labeled with the RNA:DNA hybrid specific S9.6 antibody (red), when both entities were visualized in the same sample. The percentage of co-labeled spots was estimated ∼10% of all nick-related DNA associated spots. Arrows indicate examples of co-localization. ( E–G ) Molecular combing of λ DNA. Representative images of YOYO-1 stained (green) control (F) and Nt.BbvCI nickase-treated (G) λ DNA. The size distribution histograms of combed DNA molecules before (blue) and after (orange) nickase treatment are shown in panel E. The full length intact ds λ DNA (48.5 kb) corresponds to 16.2 μm (calculated with 3 bp/nm helical repeat length), i.e. the majority of λ DNA molecules were fragmented after combing alone. Images of DNA fibers were assembled from the fields-of-view analyzed, except for panels F and G which show the original fields-of-view. For statistics see Supplementary Tables S1–S5 .

    Techniques Used: Nick Translation, Staining, Labeling

    16) Product Images from "Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice"

    Article Title: Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky456

    Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P
    Figure Legend Snippet: Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P

    Techniques Used: Knock-Out, Mouse Assay, Real-time Polymerase Chain Reaction

    17) Product Images from "A Rapid, Simple DNA Mismatch Repair Substrate Construction Method"

    Article Title: A Rapid, Simple DNA Mismatch Repair Substrate Construction Method

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2011.00008

    Illustration of the whole procedure of this new method . (1) the original pWDAH1A/SH1B plasmid; (2) Nt. BstNBI digestion to produce two nicks followed by addition of 20× biotinylated supplementary oligo; (3) two rounds of treatment with streptavidin beads and column to remove the biotinylated oligo to generate gap DNA; (4) the mismatch-containing DNA oligo added to anneal with the gap DNA, followed by a ligation reaction; (5) CsCl density ultracentrifuge, DNA recovery, and nick generation using Nt.BbvCI or Nb.BbvCI; (6) mismatch-containing DNA substrate (G/T mismatch or G/IU).
    Figure Legend Snippet: Illustration of the whole procedure of this new method . (1) the original pWDAH1A/SH1B plasmid; (2) Nt. BstNBI digestion to produce two nicks followed by addition of 20× biotinylated supplementary oligo; (3) two rounds of treatment with streptavidin beads and column to remove the biotinylated oligo to generate gap DNA; (4) the mismatch-containing DNA oligo added to anneal with the gap DNA, followed by a ligation reaction; (5) CsCl density ultracentrifuge, DNA recovery, and nick generation using Nt.BbvCI or Nb.BbvCI; (6) mismatch-containing DNA substrate (G/T mismatch or G/IU).

    Techniques Used: Plasmid Preparation, Ligation

    Generation and identification of the mismatch DNA substrate . Lane 1: pWDAH1A plasmid (supercoiled DNA); Lane 2, 3: gap DNA obtained after CsCl gradient ultracentrifuge + oligo (containing “T” or “IUdR”) heteroduplex, without ligation; Lane 4, 5: ligation of lane 2 and 3, gap DNA + mismatch-introducing oligo to produce G/T mismatch; Lane 6: NheI digestion of pWDAH1A plasmid; Lane 7, 8: NheI digestion of G/T- or G/IU-containing DNA; Lane 9: CsCl-method recovered mismatch DNA (G/T); Lane 10, 11: Nt. BbvCI-digested nick G/T or G/IU mismatch DNA as a DNA substrate.
    Figure Legend Snippet: Generation and identification of the mismatch DNA substrate . Lane 1: pWDAH1A plasmid (supercoiled DNA); Lane 2, 3: gap DNA obtained after CsCl gradient ultracentrifuge + oligo (containing “T” or “IUdR”) heteroduplex, without ligation; Lane 4, 5: ligation of lane 2 and 3, gap DNA + mismatch-introducing oligo to produce G/T mismatch; Lane 6: NheI digestion of pWDAH1A plasmid; Lane 7, 8: NheI digestion of G/T- or G/IU-containing DNA; Lane 9: CsCl-method recovered mismatch DNA (G/T); Lane 10, 11: Nt. BbvCI-digested nick G/T or G/IU mismatch DNA as a DNA substrate.

    Techniques Used: Plasmid Preparation, Ligation

    Confirmation of the mismatch DNA substrate . Lane 1: supercoiled DNA; Lane 2: Nick DNA control (Nt. BbvCI-digested); Lane 3, 4: G/T and G/IU nick DNA substrate; Lane 5, 6: NheI digestion of lane 3, 4; Lane 7: NheI digestion of pWDAH1A plasmid, linear; Lane 8: G/T nick DNA substrate + NE + proteinase K digestion, DNA extraction using ethanol + NheI digestion; Lane 9: G/IU nick DNA substrate + NE + proteinase K digestion, DNA extraction using ethanol + NheI digestion.
    Figure Legend Snippet: Confirmation of the mismatch DNA substrate . Lane 1: supercoiled DNA; Lane 2: Nick DNA control (Nt. BbvCI-digested); Lane 3, 4: G/T and G/IU nick DNA substrate; Lane 5, 6: NheI digestion of lane 3, 4; Lane 7: NheI digestion of pWDAH1A plasmid, linear; Lane 8: G/T nick DNA substrate + NE + proteinase K digestion, DNA extraction using ethanol + NheI digestion; Lane 9: G/IU nick DNA substrate + NE + proteinase K digestion, DNA extraction using ethanol + NheI digestion.

    Techniques Used: Plasmid Preparation, DNA Extraction

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

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

    Journal: eLife

    doi: 10.7554/eLife.51605

    Shuttle vector-based assay of repair-induced mutations and A3 deaminase activity in breast cancer cell lines. ( A ) Nicking and ligation controls. The shuttle vector pSP189-SnA contains 2 KpnI restriction sites (marked as ‘K’ on the mismatch plasmid in Figure 1A ), one of which is in the mismatch region (MM). Removal of the top strand after nicking by Nt.BbvCI generates a gapped plasmid that migrates as a single band after KpnI treatment. Insertion of either the original (control, 0 MM) or a U-containing oligonucleotide (U/G MM) restores the KpnI site and results in two fragments upon KpnI digestion. Klenow treatment (see Materials and methods part) eliminates residual gapped plasmids, which otherwise are highly mutagenic. ( B ) In vitro deamination assay by nuclear extracts from four breast cancer cell lines shows specificity on a 39 nt -TCT-containing single strand substrate. An -A C T-containing substrate was used as a negative control. Whole cell extract from HEK293T expressing A3B-3HA (A3B OE) was used as a positive control. S, substrate; P, product. ( C ) Time course of deamination by nuclear extracts from Hs578T and MDA-MB-453 cells using the -T C T-containing substrate. Whole cell extract from HEK293T expressing A3B-3HA (A3B OE) was used as a positive control. The right panel shows the deamination percentage. S, substrate; P, product. ( D ) Generation of APOBEC3-mediated mutations. Downstream processing of BER hijacked by MMR exposes a C in a 5’-T C -3’ context on the bottom strand, which is a substrate for A3 deaminase converting C to U. U is copied to A by replication, or if it is removed by a glycosylase to generate an AP site, then copied to an A (the A rule) but also to T or C. Thus, the original G on the top strand will be mutated to T, C, or A. In this scenario the originally introduced U/G is restored to C/G.
    Figure Legend Snippet: Shuttle vector-based assay of repair-induced mutations and A3 deaminase activity in breast cancer cell lines. ( A ) Nicking and ligation controls. The shuttle vector pSP189-SnA contains 2 KpnI restriction sites (marked as ‘K’ on the mismatch plasmid in Figure 1A ), one of which is in the mismatch region (MM). Removal of the top strand after nicking by Nt.BbvCI generates a gapped plasmid that migrates as a single band after KpnI treatment. Insertion of either the original (control, 0 MM) or a U-containing oligonucleotide (U/G MM) restores the KpnI site and results in two fragments upon KpnI digestion. Klenow treatment (see Materials and methods part) eliminates residual gapped plasmids, which otherwise are highly mutagenic. ( B ) In vitro deamination assay by nuclear extracts from four breast cancer cell lines shows specificity on a 39 nt -TCT-containing single strand substrate. An -A C T-containing substrate was used as a negative control. Whole cell extract from HEK293T expressing A3B-3HA (A3B OE) was used as a positive control. S, substrate; P, product. ( C ) Time course of deamination by nuclear extracts from Hs578T and MDA-MB-453 cells using the -T C T-containing substrate. Whole cell extract from HEK293T expressing A3B-3HA (A3B OE) was used as a positive control. The right panel shows the deamination percentage. S, substrate; P, product. ( D ) Generation of APOBEC3-mediated mutations. Downstream processing of BER hijacked by MMR exposes a C in a 5’-T C -3’ context on the bottom strand, which is a substrate for A3 deaminase converting C to U. U is copied to A by replication, or if it is removed by a glycosylase to generate an AP site, then copied to an A (the A rule) but also to T or C. Thus, the original G on the top strand will be mutated to T, C, or A. In this scenario the originally introduced U/G is restored to C/G.

    Techniques Used: Plasmid Preparation, Activity Assay, Ligation, In Vitro, Negative Control, Expressing, Positive Control, Multiple Displacement Amplification

    19) Product Images from "A Novel Ultrasensitive ECL Sensor for DNA Detection Based on Nicking Endonuclease-Assisted Target Recycling Amplification, Rolling Circle Amplification and Hemin/G-Quadruplex"

    Article Title: A Novel Ultrasensitive ECL Sensor for DNA Detection Based on Nicking Endonuclease-Assisted Target Recycling Amplification, Rolling Circle Amplification and Hemin/G-Quadruplex

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s150202629

    Illustration of the dual signal amplification strategy for specific DNA detection based on the Nt.BbvCI-assisted recycling amplification (TRA), rolling circle amplification (RCA) reaction and the formation of hemin/G-quadruplex.
    Figure Legend Snippet: Illustration of the dual signal amplification strategy for specific DNA detection based on the Nt.BbvCI-assisted recycling amplification (TRA), rolling circle amplification (RCA) reaction and the formation of hemin/G-quadruplex.

    Techniques Used: Amplification

    Verification of Nt.BbvCI-assisted catalyzed TRA. ( A ) Polyacrylamide gel electrophoresis: 1. SH-CP; 2. AP; 3. target DNA; 4. SH-CP/AP; 5. SH-CP/AP/target DNA (“Y” junction is observed, which is closer to the notch); 6. SH-CP/ AP/ Nt.BbvCI; 7. SH-CP/AP/target DNA/Nt.BbvCI (most of the “Y” junction is digested by Nt.BbvCI compared with lane 5). The concentrations of SH-CP, AP, target DNA and Nt.BbvCI were 5.0 μM, 5.0 μM, 5.0 μM and 5.0 U, respectively, for the above samples, which were incubated for 2 h before transferring into the gel; ( B ) DPV curves for (a) HT/SH-CP/AuE; (b) Nt.BbvCI/target DNA/AP/HT/SH-CP/AuE; (c) target DNA/AP/HT/SH-CP/AuE, the concentrations are 1.0 μM AP, 10.0 fM target DNA and 5.0 U Nt.BbvCI, incubation in 10.0 mM Tris-HCl containing 10.0 mM RuHex (pH 7.4) with a pulse amplitude of 50 mV and a pulse width of 0.05 s, respectively.
    Figure Legend Snippet: Verification of Nt.BbvCI-assisted catalyzed TRA. ( A ) Polyacrylamide gel electrophoresis: 1. SH-CP; 2. AP; 3. target DNA; 4. SH-CP/AP; 5. SH-CP/AP/target DNA (“Y” junction is observed, which is closer to the notch); 6. SH-CP/ AP/ Nt.BbvCI; 7. SH-CP/AP/target DNA/Nt.BbvCI (most of the “Y” junction is digested by Nt.BbvCI compared with lane 5). The concentrations of SH-CP, AP, target DNA and Nt.BbvCI were 5.0 μM, 5.0 μM, 5.0 μM and 5.0 U, respectively, for the above samples, which were incubated for 2 h before transferring into the gel; ( B ) DPV curves for (a) HT/SH-CP/AuE; (b) Nt.BbvCI/target DNA/AP/HT/SH-CP/AuE; (c) target DNA/AP/HT/SH-CP/AuE, the concentrations are 1.0 μM AP, 10.0 fM target DNA and 5.0 U Nt.BbvCI, incubation in 10.0 mM Tris-HCl containing 10.0 mM RuHex (pH 7.4) with a pulse amplitude of 50 mV and a pulse width of 0.05 s, respectively.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Incubation, Transferring

    Related Articles

    Incubation:

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments
    Article Snippet: .. Additional 20 U of PacI were added the next day together with 20 U Nt.BbvCI (New England Biolabs), and the digestion was incubated for 2 h at 37°C. .. The linearized vector was purified using the Qiagen PCR purification kit.

    Article Title: Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae
    Article Snippet: .. In the case of combing of λ phage DNA, site-specific nicks were introduced by Nt.BbvCI nickase cutting 7 times in the phage genome (delimiting 306, 318, 614, 3977, 8013 and 12 451 bp fragments): 1.5 μg λ DNA was incubated with 50 U/ml Nt.BbvCI nickase (New England Biolabs) in 20 μl CutSmart buffer for 30 min at 37°C. ..

    Article Title: Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice
    Article Snippet: .. To visualize different topological isomers of the mtDNA, 400 ng of control total DNA was additionally incubated at 37°C for 30 min with only buffer (no treatment), SacI (linear) (New England Biolabs; 20 U), Nt.BbvCI (nicked circles) (New England Biolabs; 10 U), Topo I (relaxes the closed circles) (New England Biolabs; 5 U), DNA gyrase (compacts the closed circles) (New England Biolabs; 5 U). ..

    other:

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments
    Article Snippet: In our PacI cassette, only one variable nucleotide lies between the Nt.BbvCI and PacI recognition sites ( ).

    Article Title: Identification of hemicatenane-specific binding proteins by fractionation of Hela nuclei extracts
    Article Snippet: T4 polynucleotide kinase, T4 DNA ligase, Nt.BbvcI and Nb.BbvcI nicking enzymes were from New England Biolabs.

    Introduce:

    Article Title: Competition between the RNA Transcript and the Nontemplate DNA Strand during R-Loop Formation In Vitro: a Nick Can Serve as a Strong R-Loop Initiation Site ▿Competition between the RNA Transcript and the Nontemplate DNA Strand during R-Loop Formation In Vitro: a Nick Can Serve as a Strong R-Loop Initiation Site ▿ †
    Article Snippet: .. For these experiments, the supercoiled forms of pDR18, pDR87, and pDR88 were treated with Nt.BbvCI to introduce the nick. pDR18 has no recognition site for the enzyme. .. After confirming that pDR87 and pDR88 have been nicked (most of the DNA in the nicked form of the plasmid runs at the “nicked circular” position as opposed to the “supercoiled” position), we linearized them and performed in vitro transcription, followed by agarose gel electrophoresis (Fig. ).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs nt bbvci
    Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. <t>SacI</t> cuts both strands of mtDNA once (linear), Nt. <t>BbvCI</t> cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P
    Nt Bbvci, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nt bbvci/product/New England Biolabs
    Average 99 stars, based on 43 article reviews
    Price from $9.99 to $1999.99
    nt bbvci - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P

    Journal: Nucleic Acids Research

    Article Title: Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice

    doi: 10.1093/nar/gky456

    Figure Lengend Snippet: Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P

    Article Snippet: To visualize different topological isomers of the mtDNA, 400 ng of control total DNA was additionally incubated at 37°C for 30 min with only buffer (no treatment), SacI (linear) (New England Biolabs; 20 U), Nt.BbvCI (nicked circles) (New England Biolabs; 10 U), Topo I (relaxes the closed circles) (New England Biolabs; 5 U), DNA gyrase (compacts the closed circles) (New England Biolabs; 5 U).

    Techniques: Knock-Out, Mouse Assay, Real-time Polymerase Chain Reaction

    One molecule of LacI tetramer divided a supercoiled DNA molecule plasmid pCB126 into two independent topological domains. ( a ) Plasmid pCB126 carrying two lac O1 operators in two different locations was constructed as detailed in Methods. ( b ) The nicking enzyme Nt.BbvCI was able to rapidly digest pCB126. Time course of enzyme digestion of pCB126 using 16 units of Nt.BbvCI in 1 × NEBuffer 4 at 37 °C. Lane 1 contained the undigested scDNA. ( c ) Time course of DNA supercoiling diffusion in the presence of LacI. The DNA-nicking assays were performed as described under Methods. Each reaction mixture (320 μL) contained 0.156 nM of pCB126, 2.5 nM of LacI, and 16 units of Nt.BbvCI. The reactions were incubated at 37 °C for the time indicated. Then a large excess of a double-stranded oligonucleotide contain an Nt.BbvCI recognition site was added to the reaction mixture to inhibit the restriction enzyme activities. The nicked DNA templates were ligated by T4 DNA ligase in the presence of 1 mM of ATP at 37 °C for 5 min and the reactions were terminated by phenol extraction. The DNA molecules were isolated and subjected to agarose gel electrophoresis. ( d ) Quantification analysis of the time course. The percentage of supercoiled DNA was plotted against the reaction time. The curve was generated by fitting the data to a 1st-order rate equation to yield a rate constant of 0.016 sec −1 and a t 1/2 of 52 sec.

    Journal: Scientific Reports

    Article Title: DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli

    doi: 10.1038/srep19243

    Figure Lengend Snippet: One molecule of LacI tetramer divided a supercoiled DNA molecule plasmid pCB126 into two independent topological domains. ( a ) Plasmid pCB126 carrying two lac O1 operators in two different locations was constructed as detailed in Methods. ( b ) The nicking enzyme Nt.BbvCI was able to rapidly digest pCB126. Time course of enzyme digestion of pCB126 using 16 units of Nt.BbvCI in 1 × NEBuffer 4 at 37 °C. Lane 1 contained the undigested scDNA. ( c ) Time course of DNA supercoiling diffusion in the presence of LacI. The DNA-nicking assays were performed as described under Methods. Each reaction mixture (320 μL) contained 0.156 nM of pCB126, 2.5 nM of LacI, and 16 units of Nt.BbvCI. The reactions were incubated at 37 °C for the time indicated. Then a large excess of a double-stranded oligonucleotide contain an Nt.BbvCI recognition site was added to the reaction mixture to inhibit the restriction enzyme activities. The nicked DNA templates were ligated by T4 DNA ligase in the presence of 1 mM of ATP at 37 °C for 5 min and the reactions were terminated by phenol extraction. The DNA molecules were isolated and subjected to agarose gel electrophoresis. ( d ) Quantification analysis of the time course. The percentage of supercoiled DNA was plotted against the reaction time. The curve was generated by fitting the data to a 1st-order rate equation to yield a rate constant of 0.016 sec −1 and a t 1/2 of 52 sec.

    Article Snippet: Restriction enzymes Nt.BbvCI, Nb.BbvCI, Nb.BtsI, T4 DNA ligase, and E. coli DNA gyrase were purchased from New England Biolabs (Beverly, MA, USA).

    Techniques: Plasmid Preparation, Construct, Diffusion-based Assay, Incubation, Isolation, Agarose Gel Electrophoresis, Generated, Size-exclusion Chromatography

    Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.

    Journal: Nucleic Acids Research

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

    doi: 10.1093/nar/gkl635

    Figure Lengend Snippet: Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.

    Article Snippet: In our PacI cassette, only one variable nucleotide lies between the Nt.BbvCI and PacI recognition sites ( ).

    Techniques: Clone Assay, Sequencing, Synthesized, Construct, Plasmid Preparation, Generated

    Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo ® C x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.

    Journal: Nucleic Acids Research

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

    doi: 10.1093/nar/gkl635

    Figure Lengend Snippet: Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo ® C x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.

    Article Snippet: In our PacI cassette, only one variable nucleotide lies between the Nt.BbvCI and PacI recognition sites ( ).

    Techniques: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Incubation, Transformation Assay, Ligation, Generated

    Breakage of S. cerevisiae gDNA and λ DNA at preformed ss nicks upon molecular combing. ( A-C ) Molecular combing of nick-translated gDNA from S. cerevisiae . Biotinylated nucleotides were incorporated by nick-translation conducted either in limiting (L), or non-limiting/standard conditions (N), into agarose-embedded gDNA of unperturbed, non-synchronized (A and C), or G1-synchronized (B) BY4741 cells. Biotin was detected by AlexaFluor 647-conjugated anti-biotin antibody (red) and DNA molecules were stained with YOYO-1 (green). Panel D shows examples of co-localization of nicks labeled with TdT (magenta) and R-loops labeled with the RNA:DNA hybrid specific S9.6 antibody (red), when both entities were visualized in the same sample. The percentage of co-labeled spots was estimated ∼10% of all nick-related DNA associated spots. Arrows indicate examples of co-localization. ( E–G ) Molecular combing of λ DNA. Representative images of YOYO-1 stained (green) control (F) and Nt.BbvCI nickase-treated (G) λ DNA. The size distribution histograms of combed DNA molecules before (blue) and after (orange) nickase treatment are shown in panel E. The full length intact ds λ DNA (48.5 kb) corresponds to 16.2 μm (calculated with 3 bp/nm helical repeat length), i.e. the majority of λ DNA molecules were fragmented after combing alone. Images of DNA fibers were assembled from the fields-of-view analyzed, except for panels F and G which show the original fields-of-view. For statistics see Supplementary Tables S1–S5 .

    Journal: Nucleic Acids Research

    Article Title: Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae

    doi: 10.1093/nar/gky743

    Figure Lengend Snippet: Breakage of S. cerevisiae gDNA and λ DNA at preformed ss nicks upon molecular combing. ( A-C ) Molecular combing of nick-translated gDNA from S. cerevisiae . Biotinylated nucleotides were incorporated by nick-translation conducted either in limiting (L), or non-limiting/standard conditions (N), into agarose-embedded gDNA of unperturbed, non-synchronized (A and C), or G1-synchronized (B) BY4741 cells. Biotin was detected by AlexaFluor 647-conjugated anti-biotin antibody (red) and DNA molecules were stained with YOYO-1 (green). Panel D shows examples of co-localization of nicks labeled with TdT (magenta) and R-loops labeled with the RNA:DNA hybrid specific S9.6 antibody (red), when both entities were visualized in the same sample. The percentage of co-labeled spots was estimated ∼10% of all nick-related DNA associated spots. Arrows indicate examples of co-localization. ( E–G ) Molecular combing of λ DNA. Representative images of YOYO-1 stained (green) control (F) and Nt.BbvCI nickase-treated (G) λ DNA. The size distribution histograms of combed DNA molecules before (blue) and after (orange) nickase treatment are shown in panel E. The full length intact ds λ DNA (48.5 kb) corresponds to 16.2 μm (calculated with 3 bp/nm helical repeat length), i.e. the majority of λ DNA molecules were fragmented after combing alone. Images of DNA fibers were assembled from the fields-of-view analyzed, except for panels F and G which show the original fields-of-view. For statistics see Supplementary Tables S1–S5 .

    Article Snippet: In the case of combing of λ phage DNA, site-specific nicks were introduced by Nt.BbvCI nickase cutting 7 times in the phage genome (delimiting 306, 318, 614, 3977, 8013 and 12 451 bp fragments): 1.5 μg λ DNA was incubated with 50 U/ml Nt.BbvCI nickase (New England Biolabs) in 20 μl CutSmart buffer for 30 min at 37°C.

    Techniques: Nick Translation, Staining, Labeling