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    Nb BbvCI 5 000 units
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    New England Biolabs nb bbvci
    Nb BbvCI
    Nb BbvCI 5 000 units
    https://www.bioz.com/result/nb bbvci/product/New England Biolabs
    Average 95 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    nb bbvci - by Bioz Stars, 2020-09
    95/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) Product Images from "An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity †Electronic supplementary information (ESI) available: Fig. S1 to S7 and Table S1. See DOI: 10.1039/c8sc05102j"

    Article Title: An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity †Electronic supplementary information (ESI) available: Fig. S1 to S7 and Table S1. See DOI: 10.1039/c8sc05102j

    Journal: Chemical Science

    doi: 10.1039/c8sc05102j

    (A) PAGE result of the proposed sensing platform. Line M: DNA marker, line 1: SDA proceeding on shortened MB without Nb.BbvcI, line 2: SDA proceeding on shortened MB with primer 0, line 3: SDA proceeding on shortened MB without primers, line 4: complete SDA proceeding on shortened MB only, line 5: DpnI treated MB without Dam MTase, line 6: shortened MB cut by DpnI, line 7: MB only. (B) Fluorescent spectrum in response to the addition of different components.
    Figure Legend Snippet: (A) PAGE result of the proposed sensing platform. Line M: DNA marker, line 1: SDA proceeding on shortened MB without Nb.BbvcI, line 2: SDA proceeding on shortened MB with primer 0, line 3: SDA proceeding on shortened MB without primers, line 4: complete SDA proceeding on shortened MB only, line 5: DpnI treated MB without Dam MTase, line 6: shortened MB cut by DpnI, line 7: MB only. (B) Fluorescent spectrum in response to the addition of different components.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Marker

    5) Product Images from "Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules"

    Article Title: Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw689

    RepC–DNA interaction during nicking and religation activities. ( A ) Characteristic supercoils removal time course taken at 0.34 pN. Approximately 30 turns were released in 0.3 s. Data were acquired at 500 Hz. ( B ) Histogram of velocities of plectoneme release for RepC, hTopIB and Nb.BbvCI. Frequencies are fitted to a Gaussian function and the mean and s.e.m values are quoted in the figure. RepC showed an intermediate velocity between the topoisomerase and the nicking enzyme. ( C ) Nicking and religation experiment with RepC. RepC occasionally religated the nicked DNA. This is shown as a dip in the extension while the magnets continuously rotate in a negative direction. ( D ) RepC cannot nick positively supercoiled DNA. ( E and F ) Nicking and religation experiment with hTopIB. hTopIB nicks and religates efficiently while the magnets continuously rotate in either negative or positive direction.
    Figure Legend Snippet: RepC–DNA interaction during nicking and religation activities. ( A ) Characteristic supercoils removal time course taken at 0.34 pN. Approximately 30 turns were released in 0.3 s. Data were acquired at 500 Hz. ( B ) Histogram of velocities of plectoneme release for RepC, hTopIB and Nb.BbvCI. Frequencies are fitted to a Gaussian function and the mean and s.e.m values are quoted in the figure. RepC showed an intermediate velocity between the topoisomerase and the nicking enzyme. ( C ) Nicking and religation experiment with RepC. RepC occasionally religated the nicked DNA. This is shown as a dip in the extension while the magnets continuously rotate in a negative direction. ( D ) RepC cannot nick positively supercoiled DNA. ( E and F ) Nicking and religation experiment with hTopIB. hTopIB nicks and religates efficiently while the magnets continuously rotate in either negative or positive direction.

    Techniques Used:

    6) Product Images from "A novel dinuclear iridium(III) complex as a G-quadruplex-selective probe for the luminescent switch-on detection of transcription factor HIF-1α"

    Article Title: A novel dinuclear iridium(III) complex as a G-quadruplex-selective probe for the luminescent switch-on detection of transcription factor HIF-1α

    Journal: Scientific Reports

    doi: 10.1038/srep22458

    ( a ) Luminescence response of 5 to various concentrations of HIF-1α: 0, 5, 10, 20, 40, 80, 120, 160, 200, 240 and 300 nM in buffered solution. ( b ) The relationship of signal intensity (λ Em = 580 nm) and the concentration of HIF-1α. ( c ) Linear plot of the signal change in luminescence intensity (λ Em = 580 nm) vs . HIF-1α concentration. Error bars represent the standard deviations (SD) of the results from three independent experiments. ( d ) Luminescence spectra of 5 in response to various concentrations of HIF-1α: 0, 5, 10, 20, 40, 80, 120, 160, 200, 240 and 300 nM in a detection system without Nt.BbvCI.
    Figure Legend Snippet: ( a ) Luminescence response of 5 to various concentrations of HIF-1α: 0, 5, 10, 20, 40, 80, 120, 160, 200, 240 and 300 nM in buffered solution. ( b ) The relationship of signal intensity (λ Em = 580 nm) and the concentration of HIF-1α. ( c ) Linear plot of the signal change in luminescence intensity (λ Em = 580 nm) vs . HIF-1α concentration. Error bars represent the standard deviations (SD) of the results from three independent experiments. ( d ) Luminescence spectra of 5 in response to various concentrations of HIF-1α: 0, 5, 10, 20, 40, 80, 120, 160, 200, 240 and 300 nM in a detection system without Nt.BbvCI.

    Techniques Used: Concentration Assay

    7) Product Images from "Acetylation regulates DNA repair mechanisms in human cells"

    Article Title: Acetylation regulates DNA repair mechanisms in human cells

    Journal: Cell Cycle

    doi: 10.1080/15384101.2016.1176815

    Two restriction sites recognized by the nicking enzyme Nb.BbvCI were inserted into the ORF of EGFP to efficiently remove the native single-stranded DNA fragment (i). The single-stranded DNA fragments of the transcribed template excised by the nicking endonuclease were sequentially exchanged for the matching synthetic oligonucleotides containing the modification, 8-oxoG and/or single base alteration creating a stop codon in N-terminal EGFP (ii). The new oligonucleotide annealed to a gapped plasmid was ligated or left unsealed (iii). (D) Generation of gapped plasmids by Nb.BbvCI (left panel, lane 3). Nicking of plasmid results in formation of an open circular (oc) form, which migrates in the ethidium bromide agarose gel slower than covalently closed (ccc) untreated plasmid (lane 1), and linear form (lane 2) of plasmid from treatment with HindIII. Presence of 180-fold molar excess of the competitor oligonucleotides efficiently replaces native single-stranded DNA fragment (right panel). Exchange of the 5′-phosphorylated native DNA fragments with unphosphorylated synthetic oligonucleotides is detected by the inhibition of ligation in the absence of PNK. Reactions performed in the presence of PNK prior ligations result in covalently closed (ccc) plasmid.
    Figure Legend Snippet: Two restriction sites recognized by the nicking enzyme Nb.BbvCI were inserted into the ORF of EGFP to efficiently remove the native single-stranded DNA fragment (i). The single-stranded DNA fragments of the transcribed template excised by the nicking endonuclease were sequentially exchanged for the matching synthetic oligonucleotides containing the modification, 8-oxoG and/or single base alteration creating a stop codon in N-terminal EGFP (ii). The new oligonucleotide annealed to a gapped plasmid was ligated or left unsealed (iii). (D) Generation of gapped plasmids by Nb.BbvCI (left panel, lane 3). Nicking of plasmid results in formation of an open circular (oc) form, which migrates in the ethidium bromide agarose gel slower than covalently closed (ccc) untreated plasmid (lane 1), and linear form (lane 2) of plasmid from treatment with HindIII. Presence of 180-fold molar excess of the competitor oligonucleotides efficiently replaces native single-stranded DNA fragment (right panel). Exchange of the 5′-phosphorylated native DNA fragments with unphosphorylated synthetic oligonucleotides is detected by the inhibition of ligation in the absence of PNK. Reactions performed in the presence of PNK prior ligations result in covalently closed (ccc) plasmid.

    Techniques Used: Modification, Plasmid Preparation, Agarose Gel Electrophoresis, Countercurrent Chromatography, Inhibition, Ligation

    8) Product Images from "A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection"

    Article Title: A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07324-5

    Schematic reaction mechanism of CRISDA. Step 1: A pair of Cas9 ribonucleoproteins is programmed to recognize each border of the target DNA and to induce a pair of nicks in both non-target strands. Step 2: A pair of IP primers is introduced and hybridized to the exposed non-target strands. Step 3: After adding SDA mixtures containing KF polymerase (3′– > 5′ exo − ), Nb.BbvCI nikase, and single-stranded DNA binding protein TP32 (SSB), linear SDA is initiated from the binding sites of IP primers, giving linearly replaced single strands, the Strand-for and Strand-rev. Step 4: The products, Strand-For and Strand-Rev, are annealed again to the IP primers, which further induce exponential SDA of the selected target sequence. Step 5: The amplicons are quantitatively determined by a PNA invasion-mediated endpoint measurement via magnetic pull-down and fluorescence measurements. The well-characterized S. pyogenes Cas9 with a mutation of HNH catalytic residue (spyCas9H840A nickase) is used as a model. *Two bands will be observed in PAGE analyses, where one corresponds to the final products 1 and 2 with the same length and the other one is product 3
    Figure Legend Snippet: Schematic reaction mechanism of CRISDA. Step 1: A pair of Cas9 ribonucleoproteins is programmed to recognize each border of the target DNA and to induce a pair of nicks in both non-target strands. Step 2: A pair of IP primers is introduced and hybridized to the exposed non-target strands. Step 3: After adding SDA mixtures containing KF polymerase (3′– > 5′ exo − ), Nb.BbvCI nikase, and single-stranded DNA binding protein TP32 (SSB), linear SDA is initiated from the binding sites of IP primers, giving linearly replaced single strands, the Strand-for and Strand-rev. Step 4: The products, Strand-For and Strand-Rev, are annealed again to the IP primers, which further induce exponential SDA of the selected target sequence. Step 5: The amplicons are quantitatively determined by a PNA invasion-mediated endpoint measurement via magnetic pull-down and fluorescence measurements. The well-characterized S. pyogenes Cas9 with a mutation of HNH catalytic residue (spyCas9H840A nickase) is used as a model. *Two bands will be observed in PAGE analyses, where one corresponds to the final products 1 and 2 with the same length and the other one is product 3

    Techniques Used: Binding Assay, Sequencing, Fluorescence, Mutagenesis, Polyacrylamide Gel Electrophoresis

    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 "A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection"

    Article Title: A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07324-5

    Schematic reaction mechanism of CRISDA. Step 1: A pair of Cas9 ribonucleoproteins is programmed to recognize each border of the target DNA and to induce a pair of nicks in both non-target strands. Step 2: A pair of IP primers is introduced and hybridized to the exposed non-target strands. Step 3: After adding SDA mixtures containing KF polymerase (3′– > 5′ exo − ), Nb.BbvCI nikase, and single-stranded DNA binding protein TP32 (SSB), linear SDA is initiated from the binding sites of IP primers, giving linearly replaced single strands, the Strand-for and Strand-rev. Step 4: The products, Strand-For and Strand-Rev, are annealed again to the IP primers, which further induce exponential SDA of the selected target sequence. Step 5: The amplicons are quantitatively determined by a PNA invasion-mediated endpoint measurement via magnetic pull-down and fluorescence measurements. The well-characterized S. pyogenes Cas9 with a mutation of HNH catalytic residue (spyCas9H840A nickase) is used as a model. *Two bands will be observed in PAGE analyses, where one corresponds to the final products 1 and 2 with the same length and the other one is product 3
    Figure Legend Snippet: Schematic reaction mechanism of CRISDA. Step 1: A pair of Cas9 ribonucleoproteins is programmed to recognize each border of the target DNA and to induce a pair of nicks in both non-target strands. Step 2: A pair of IP primers is introduced and hybridized to the exposed non-target strands. Step 3: After adding SDA mixtures containing KF polymerase (3′– > 5′ exo − ), Nb.BbvCI nikase, and single-stranded DNA binding protein TP32 (SSB), linear SDA is initiated from the binding sites of IP primers, giving linearly replaced single strands, the Strand-for and Strand-rev. Step 4: The products, Strand-For and Strand-Rev, are annealed again to the IP primers, which further induce exponential SDA of the selected target sequence. Step 5: The amplicons are quantitatively determined by a PNA invasion-mediated endpoint measurement via magnetic pull-down and fluorescence measurements. The well-characterized S. pyogenes Cas9 with a mutation of HNH catalytic residue (spyCas9H840A nickase) is used as a model. *Two bands will be observed in PAGE analyses, where one corresponds to the final products 1 and 2 with the same length and the other one is product 3

    Techniques Used: Binding Assay, Sequencing, Fluorescence, Mutagenesis, Polyacrylamide Gel Electrophoresis

    11) 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

    12) Product Images from "Structural diversity of supercoiled DNA"

    Article Title: Structural diversity of supercoiled DNA

    Journal: Nature Communications

    doi: 10.1038/ncomms9440

    Effect of supercoiling on the structure of minicircle DNA. ( a ) Individual 336 bp minicircle topoisomers were isolated and analysed by polyacrylamide gel electrophoresis in the presence of 10 mM CaCl 2 . Mr: 100 bp DNA ladder, L: minicircle linearized by EcoRV, N: minicircle nicked by Nb.BbvCI. ( b ) Projections of cryo-ET subtomograms of hydrated 336 bp DNA minicircles of the Lk =34 topoisomer. ( c ) Commonly observed shapes were open circle, open figure-8, figure-8, racquet, handcuffs, needle, and rod, each of which are shown in orthogonal views. ( d ) Other shapes observed, especially in the more highly supercoiled topoisomers. ( e ) Shape frequency distribution plot for each topoisomer population (n=number of minicircles analysed). A weighted average for each topoisomer, approximating the average degree of compactness, is denoted by the black triangle. The weighted average was calculated by assigning each conformation a value that increased in line with compactness. Open circles were given a value of 1, open figure-8 s a value of 2, figure-8 s as a value of 3, and so on. The relative fraction of each was subsequently used to determine the average degree of compactness. Lk , Δ Lk and superhelical density (σ) for each topoisomer are shown (see Supplementary Note 1 ).
    Figure Legend Snippet: Effect of supercoiling on the structure of minicircle DNA. ( a ) Individual 336 bp minicircle topoisomers were isolated and analysed by polyacrylamide gel electrophoresis in the presence of 10 mM CaCl 2 . Mr: 100 bp DNA ladder, L: minicircle linearized by EcoRV, N: minicircle nicked by Nb.BbvCI. ( b ) Projections of cryo-ET subtomograms of hydrated 336 bp DNA minicircles of the Lk =34 topoisomer. ( c ) Commonly observed shapes were open circle, open figure-8, figure-8, racquet, handcuffs, needle, and rod, each of which are shown in orthogonal views. ( d ) Other shapes observed, especially in the more highly supercoiled topoisomers. ( e ) Shape frequency distribution plot for each topoisomer population (n=number of minicircles analysed). A weighted average for each topoisomer, approximating the average degree of compactness, is denoted by the black triangle. The weighted average was calculated by assigning each conformation a value that increased in line with compactness. Open circles were given a value of 1, open figure-8 s a value of 2, figure-8 s as a value of 3, and so on. The relative fraction of each was subsequently used to determine the average degree of compactness. Lk , Δ Lk and superhelical density (σ) for each topoisomer are shown (see Supplementary Note 1 ).

    Techniques Used: Isolation, Polyacrylamide Gel Electrophoresis

    13) Product Images from "Microfluidic Exponential Rolling Circle Amplification for Sensitive microRNA Detection Directly from Biological Samples"

    Article Title: Microfluidic Exponential Rolling Circle Amplification for Sensitive microRNA Detection Directly from Biological Samples

    Journal: Sensors and actuators. B, Chemical

    doi: 10.1016/j.snb.2018.09.121

    Optimization of eRCA conditions using the adapter sequence. ( A, B ) Plots of fluorescence signals normalized to the maximum signal obtained under different concentrations of ( A ) the padlock probe and ( B ) T4 DNA ligase. ( C ) Comparison of the fluorescent signals of RCA with and without adding Nb.BbvCI (1 U per 20 μL reaction). ( D, E ) Bar graphs showing the effects of ( D ) reaction time and ( E ) the SYBR Green on the assay response. The 20 μL reaction solution contained 10 −8 M padlock probe and 50 nM let-7a adapter. The ligation reactions were performed at 37 °C for 2 h, and RCA reactions were performed at 30 °C. Error bars represent one standard deviation from three replicates.
    Figure Legend Snippet: Optimization of eRCA conditions using the adapter sequence. ( A, B ) Plots of fluorescence signals normalized to the maximum signal obtained under different concentrations of ( A ) the padlock probe and ( B ) T4 DNA ligase. ( C ) Comparison of the fluorescent signals of RCA with and without adding Nb.BbvCI (1 U per 20 μL reaction). ( D, E ) Bar graphs showing the effects of ( D ) reaction time and ( E ) the SYBR Green on the assay response. The 20 μL reaction solution contained 10 −8 M padlock probe and 50 nM let-7a adapter. The ligation reactions were performed at 37 °C for 2 h, and RCA reactions were performed at 30 °C. Error bars represent one standard deviation from three replicates.

    Techniques Used: Sequencing, Fluorescence, SYBR Green Assay, Ligation, Standard Deviation

    Related Articles

    other:

    Article Title: An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity †<
    Article Snippet: Dam and M. SssI methyltransferase (MTase), endonuclease DpnI, Klenow Fragment Polymerase (3′–5′ exo-) (KFP), nicking enzyme Nb.BbvcI, S -adenyl methionine (SAM), EcoRI enzyme and the corresponding buffer solution were obtained from New England Biolabs (Beijing, China).

    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.

    Amplification:

    Article Title: A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection
    Article Snippet: .. Proteins and other reagents such as single-strand binding protein (T4 gene 32 protein), Nb.BbvCI endonuclease, DNA polymerases (Klenow Fragment 3′– > 5′ exo− , KF), and dNTPs used in CRISDA amplification reactions were obtained from New England Biolabs (NEB). .. Bovine serum albumin (BSA, 20 mg mL−1 ), and nuclease free water were purchased from TAKARA.

    Binding Assay:

    Article Title: A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection
    Article Snippet: .. Proteins and other reagents such as single-strand binding protein (T4 gene 32 protein), Nb.BbvCI endonuclease, DNA polymerases (Klenow Fragment 3′– > 5′ exo− , KF), and dNTPs used in CRISDA amplification reactions were obtained from New England Biolabs (NEB). .. Bovine serum albumin (BSA, 20 mg mL−1 ), and nuclease free water were purchased from TAKARA.

    Plasmid Preparation:

    Article Title: Acetylation regulates DNA repair mechanisms in human cells
    Article Snippet: .. The bottom DNA strand of the gene (“top” refers to the protein-coding strand or untranscribed strand, and “bottom” refers to the transcribed strand) was selectively nicked by incubating 60 μg of plasmid DNA with 13U of Nb.BbvCI (New England BioLabs) for 1.5 h at 37°C in 120 μl of CutSmart Buffer (supplied by the manufacturer). ..

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    New England Biolabs nb bbvci
    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 <t>Nb.BbvCI</t> (iii), while <t>pMap63</t> 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.
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    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkr153

    Figure Lengend 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.

    Article Snippet: Then it was treated with a nicking enzyme, Nb.BbvCI (New England Biolabs), while pMap63 was nicked with Nt.BbvCI (New England Biolabs).

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

    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.

    Journal: bioRxiv

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

    doi: 10.1101/844126

    Figure Lengend 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.

    Article Snippet: T4 polynucleotide kinase, T4 DNA ligase, Nt.BbvcI and Nb.BbvcI nicking enzymes were from New England Biolabs.

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

    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

    (A) PAGE result of the proposed sensing platform. Line M: DNA marker, line 1: SDA proceeding on shortened MB without Nb.BbvcI, line 2: SDA proceeding on shortened MB with primer 0, line 3: SDA proceeding on shortened MB without primers, line 4: complete SDA proceeding on shortened MB only, line 5: DpnI treated MB without Dam MTase, line 6: shortened MB cut by DpnI, line 7: MB only. (B) Fluorescent spectrum in response to the addition of different components.

    Journal: Chemical Science

    Article Title: An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity †Electronic supplementary information (ESI) available: Fig. S1 to S7 and Table S1. See DOI: 10.1039/c8sc05102j

    doi: 10.1039/c8sc05102j

    Figure Lengend Snippet: (A) PAGE result of the proposed sensing platform. Line M: DNA marker, line 1: SDA proceeding on shortened MB without Nb.BbvcI, line 2: SDA proceeding on shortened MB with primer 0, line 3: SDA proceeding on shortened MB without primers, line 4: complete SDA proceeding on shortened MB only, line 5: DpnI treated MB without Dam MTase, line 6: shortened MB cut by DpnI, line 7: MB only. (B) Fluorescent spectrum in response to the addition of different components.

    Article Snippet: Dam and M. SssI methyltransferase (MTase), endonuclease DpnI, Klenow Fragment Polymerase (3′–5′ exo-) (KFP), nicking enzyme Nb.BbvcI, S -adenyl methionine (SAM), EcoRI enzyme and the corresponding buffer solution were obtained from New England Biolabs (Beijing, China).

    Techniques: Polyacrylamide Gel Electrophoresis, Marker