puc19  (New England Biolabs)


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    Structured Review

    New England Biolabs puc19
    Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from <t>pUC19.</t> The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.
    Puc19, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants"

    Article Title: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkj483

    Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.
    Figure Legend Snippet: Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.

    Techniques Used: Variant Assay, Plasmid Preparation, Derivative Assay, Incubation

    2) Product Images from "Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes"

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19920-y

    Synthetic c ysE and cysM gene transformants display recovery of CysE function without cysteine and methionine and CysM function without cysteine. ( A ) E. coli ΔcysE competent cells were transformed with positive control cysE , two cysE variants cysE-C/cysE-CM cloned into the multiple cloning site of pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. ( B ) E. coli ΔcysMΔcysK competent cells transformed with positive control c ysM , two cysM variants cysM-C / cysM-CM in pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. Cells were plated on M9 + glucose medium with 0.4 mM IPTG, 50 μg/ml kanamycin, and 100 μg/ml ampicillin and incubated at 30 °C for 72 h.
    Figure Legend Snippet: Synthetic c ysE and cysM gene transformants display recovery of CysE function without cysteine and methionine and CysM function without cysteine. ( A ) E. coli ΔcysE competent cells were transformed with positive control cysE , two cysE variants cysE-C/cysE-CM cloned into the multiple cloning site of pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. ( B ) E. coli ΔcysMΔcysK competent cells transformed with positive control c ysM , two cysM variants cysM-C / cysM-CM in pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. Cells were plated on M9 + glucose medium with 0.4 mM IPTG, 50 μg/ml kanamycin, and 100 μg/ml ampicillin and incubated at 30 °C for 72 h.

    Techniques Used: Transformation Assay, Positive Control, Clone Assay, Plasmid Preparation, Negative Control, Incubation

    Growth curve of re-transformed auxotrophic E. coli strains in LB and M9 + glucose media. Each panel represents growth of cysteine-dependent E. coli auxotrophs rescued by wild type enzymes: CysE or CysM (blue), cysteine-free enzymes: CysE-C or CysM-C (orange), cysteine- and methionine-free enzymes: CysE-CM or CysM-CM (yellow), and LacZα protein expressed from original pUC19 plasmid (gray). Growth curve was monitored every 10 minutes at OD 600 nm using 96-well plate reader. Standard deviations of the growth curves are displayed as calculated from triplicates.
    Figure Legend Snippet: Growth curve of re-transformed auxotrophic E. coli strains in LB and M9 + glucose media. Each panel represents growth of cysteine-dependent E. coli auxotrophs rescued by wild type enzymes: CysE or CysM (blue), cysteine-free enzymes: CysE-C or CysM-C (orange), cysteine- and methionine-free enzymes: CysE-CM or CysM-CM (yellow), and LacZα protein expressed from original pUC19 plasmid (gray). Growth curve was monitored every 10 minutes at OD 600 nm using 96-well plate reader. Standard deviations of the growth curves are displayed as calculated from triplicates.

    Techniques Used: Transformation Assay, Plasmid Preparation

    3) Product Images from "Translocation of E. coli RecQ Helicase on Single-Stranded DNA"

    Article Title: Translocation of E. coli RecQ Helicase on Single-Stranded DNA

    Journal: Biochemistry

    doi: 10.1021/bi3000676

    The ssDNA-dependent ATP hydrolysis activity of RecQ is cooperative with respect to ATP concentration The rate of ATP hydrolysis by RecQ (100 nM) stimulated by dT 30 (0.3 μM, molecules) is plotted as a function of ATP concentration (black circles). The data were fit to the Hill equation (solid line, R 2 = 0.98). For comparison, a fit to a hyperbolic equation is shown (dashed line, R 2 = 0.95) (B) The rate of ATP hydrolysis by RecQ (100 nM) stimulated by linear pUC19 (2.5 μM, base pairs) is shown (black circles). The data were fit to the Hill equation (solid curve, r-squared value of 0.98). For comparison, a fit to the Hill equation with the Hill coefficient held constant at 3 is shown (dotted curve, r-squared value of 0.95).
    Figure Legend Snippet: The ssDNA-dependent ATP hydrolysis activity of RecQ is cooperative with respect to ATP concentration The rate of ATP hydrolysis by RecQ (100 nM) stimulated by dT 30 (0.3 μM, molecules) is plotted as a function of ATP concentration (black circles). The data were fit to the Hill equation (solid line, R 2 = 0.98). For comparison, a fit to a hyperbolic equation is shown (dashed line, R 2 = 0.95) (B) The rate of ATP hydrolysis by RecQ (100 nM) stimulated by linear pUC19 (2.5 μM, base pairs) is shown (black circles). The data were fit to the Hill equation (solid curve, r-squared value of 0.98). For comparison, a fit to the Hill equation with the Hill coefficient held constant at 3 is shown (dotted curve, r-squared value of 0.95).

    Techniques Used: Activity Assay, Concentration Assay

    4) Product Images from "Hop2/Mnd1 acts on two critical steps in Dmc1-promoted homologous pairing"

    Article Title: Hop2/Mnd1 acts on two critical steps in Dmc1-promoted homologous pairing

    Journal: Genes & Development

    doi: 10.1101/gad.1562907

    The Hop2/Mnd1 heterodimer stimulates the capture of duplex DNA by the Dmc1–ssDNA nucleoprotein complex. ( A ) The experimental outline. Biotinylated DNA, immobilized on streptavidin-agarose beads, was used as a binding substrate for Dmc1 and Hop2/Mnd1 proteins. After incubation with pUC19 dsDNA, nucleoprotein complexes were separated from unbound proteins and dsDNA by centrifugation, and products were deproteinized before analysis by gel electrophoresis. ( B ) The products of dsDNA capture revealed by agarose gel electrophoresis. There is an increase in the homologous (pUC19) and nonhomologous (ϕX174, RFI) dsDNA captured by Dmc1–ssDNA nucleoprotein complexes in the presence of Hop2/Mnd1. In control reactions, the oligonucleotide ssDNA substrate was replaced by standard binding buffer. ( C ) Quantitation of the ethidium-bromide signals from gels similar to those shown in B and plotted as the number of pixels per band.
    Figure Legend Snippet: The Hop2/Mnd1 heterodimer stimulates the capture of duplex DNA by the Dmc1–ssDNA nucleoprotein complex. ( A ) The experimental outline. Biotinylated DNA, immobilized on streptavidin-agarose beads, was used as a binding substrate for Dmc1 and Hop2/Mnd1 proteins. After incubation with pUC19 dsDNA, nucleoprotein complexes were separated from unbound proteins and dsDNA by centrifugation, and products were deproteinized before analysis by gel electrophoresis. ( B ) The products of dsDNA capture revealed by agarose gel electrophoresis. There is an increase in the homologous (pUC19) and nonhomologous (ϕX174, RFI) dsDNA captured by Dmc1–ssDNA nucleoprotein complexes in the presence of Hop2/Mnd1. In control reactions, the oligonucleotide ssDNA substrate was replaced by standard binding buffer. ( C ) Quantitation of the ethidium-bromide signals from gels similar to those shown in B and plotted as the number of pixels per band.

    Techniques Used: Binding Assay, Incubation, Centrifugation, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Quantitation Assay

    Hop2/Mnd1 and Dmc1 proteins are required for the formation of a synaptic complex at sites of DNA homology. ( A ) Formation of joint molecules promoted by Dmc1. (Panel a ) The presynaptic polymerization of Dmc1 protein on ssDNA. (Panel b ) Conjunction of ssDNA and dsDNA without homologous alignment. (Panel c ) Homologous DNA pairing and strand invasion. Deproteinization of the complex in panel c in the context of a supercoiled target duplex DNA results in a stable D-loop. ( B ) Experimental design. ssDNA oligonucleotide was used as a binding substrate for Dmc1 and Hop2/Mnd1 proteins. The nucleoprotein complexes were incubated with pUC19 dsDNA and treated with SspI or NdeI restriction endonucleases, followed by deproteinization and electrophoresis in 1% agarose gel. ( C ) Hop2/Mnd1 and the Dmc1–ssDNA nucleoprotein complex are targeted to sites in which ssDNA and supercoiled dsDNA are homologous, thus protecting dsDNA from endonuclease degradation. ( D ) Both Dmc1 and Hop2/Mnd1 proteins are required for the protection of dsDNA. ( E ) Linear pUC19 dsDNA is a substrate for synaptic complex formation. After Dmc1 and Hop2/Mnd1 were assembled on the ssDNA oligonucleotide, linear pUC19 dsDNA was added to the reaction and the nucleoprotein complexes were treated with SspI or NdeI restriction endonucleases.
    Figure Legend Snippet: Hop2/Mnd1 and Dmc1 proteins are required for the formation of a synaptic complex at sites of DNA homology. ( A ) Formation of joint molecules promoted by Dmc1. (Panel a ) The presynaptic polymerization of Dmc1 protein on ssDNA. (Panel b ) Conjunction of ssDNA and dsDNA without homologous alignment. (Panel c ) Homologous DNA pairing and strand invasion. Deproteinization of the complex in panel c in the context of a supercoiled target duplex DNA results in a stable D-loop. ( B ) Experimental design. ssDNA oligonucleotide was used as a binding substrate for Dmc1 and Hop2/Mnd1 proteins. The nucleoprotein complexes were incubated with pUC19 dsDNA and treated with SspI or NdeI restriction endonucleases, followed by deproteinization and electrophoresis in 1% agarose gel. ( C ) Hop2/Mnd1 and the Dmc1–ssDNA nucleoprotein complex are targeted to sites in which ssDNA and supercoiled dsDNA are homologous, thus protecting dsDNA from endonuclease degradation. ( D ) Both Dmc1 and Hop2/Mnd1 proteins are required for the protection of dsDNA. ( E ) Linear pUC19 dsDNA is a substrate for synaptic complex formation. After Dmc1 and Hop2/Mnd1 were assembled on the ssDNA oligonucleotide, linear pUC19 dsDNA was added to the reaction and the nucleoprotein complexes were treated with SspI or NdeI restriction endonucleases.

    Techniques Used: Binding Assay, Incubation, Electrophoresis, Agarose Gel Electrophoresis

    The stabilization of the Dmc1–ssDNA presynaptic filament is not sufficient to stimulate Dmc1-promoted strand invasion. ( A ) The real-time dissociation phase of Dmc1 from ssDNA in the presence of AMPPNP plus Hop2/Mnd1, Hop2/Mnd1, AMPPNP, and buffer with or without ATP was analyzed using surface plasmon resonance. ( B ) Hop2/Mnd1 and Dmc1–ssDNA complexes protect DNA against exonuclease I degradation in the presence of AMPPNP. ( C ) D-loop formation carried out with Dmc1 (1.5 μM) allowed to bind a 57-mer oligonucleotide (#3, ssDNA, 4.5 μM nucleotide), in a buffer containing 2.5 mM MgCl 2 , before the addition of Hop2/Mnd1 (0.2 μM) and supercoiled pUC19 (10 μM bp). ( D ) The extent of synaptic complex formation by the Dmc1 filament with and without Hop2/Mnd1 in the presence of ATP or AMPPNP. ( E ) Bars represent the quantitation of the ethidium-bromide signal corresponding to the dsDNA captured by the Dmc1–ssDNA filament alone or in the presence of Hop2/Mnd1.
    Figure Legend Snippet: The stabilization of the Dmc1–ssDNA presynaptic filament is not sufficient to stimulate Dmc1-promoted strand invasion. ( A ) The real-time dissociation phase of Dmc1 from ssDNA in the presence of AMPPNP plus Hop2/Mnd1, Hop2/Mnd1, AMPPNP, and buffer with or without ATP was analyzed using surface plasmon resonance. ( B ) Hop2/Mnd1 and Dmc1–ssDNA complexes protect DNA against exonuclease I degradation in the presence of AMPPNP. ( C ) D-loop formation carried out with Dmc1 (1.5 μM) allowed to bind a 57-mer oligonucleotide (#3, ssDNA, 4.5 μM nucleotide), in a buffer containing 2.5 mM MgCl 2 , before the addition of Hop2/Mnd1 (0.2 μM) and supercoiled pUC19 (10 μM bp). ( D ) The extent of synaptic complex formation by the Dmc1 filament with and without Hop2/Mnd1 in the presence of ATP or AMPPNP. ( E ) Bars represent the quantitation of the ethidium-bromide signal corresponding to the dsDNA captured by the Dmc1–ssDNA filament alone or in the presence of Hop2/Mnd1.

    Techniques Used: SPR Assay, Quantitation Assay

    5) Product Images from "Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer"

    Article Title: Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00787-16

    In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.
    Figure Legend Snippet: In vitro analysis of RpnA endonuclease activity. (A) WT RpnA cleaves pUC19, RpnA-D63A does not cleave pUC19, and RpnA-D165A is more active on pUC19. The pUC19 DNA (29 nM, 50 μg/ml) is initially supercoiled but can be relaxed by nicks, linearized by double-strand cleavage, or cleaved further. The supercoiled (control), relaxed (Nb.BtsI), and linear (HindIII) forms are indicated. pUC19 was treated with RpnA-inactive RpnA-D63A or hyperactive RpnA-D165A (15 μM, 45 min). (B) Time course of an RpnA (7.5 μM)-pUC19 (29 nM) digest. Band intensity was compared to determine the relative amounts of supercoiled, nicked, and linear pUC19 at each time point. Over 90% of the supercoiled pUC19 was digested within 180 min. (C) RpnA endonuclease activity depends on divalent cation and is stimulated by Ca 2+ . The reaction buffer was 50 mM NaCl and 10 mM Tris, pH 8.0; the indicated additives were at 10 mM each. RpnA at 3.8 μM was added for 18 h. (D) RpnA cleavage products provide a DNA polymerase primer. pUC19 was digested with RpnA, DNase I, or micrococcal nuclease (MNase) to produce similar smears and then incubated with fluorescein-labeled dNTPs and the Klenow fragment of DNA polymerase. DNA was visualized by ethidium bromide (EtBr; left) or fluorescein (middle), with the two signals being merged at the right. RpnA- and DNase I-digested DNAs were effectively labeled, but micrococcal nuclease-digested DNA was not.

    Techniques Used: In Vitro, Activity Assay, Incubation, Labeling

    6) Product Images from "Specificities and Functions of the recA and pps1 Intein Genes of Mycobacterium tuberculosis and Application for Diagnosis of Tuberculosis"

    Article Title: Specificities and Functions of the recA and pps1 Intein Genes of Mycobacterium tuberculosis and Application for Diagnosis of Tuberculosis

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.40.3.943-950.2002

    Endonuclease assays. (A) DNA substrates for MtuPps1 and MtuRecA. The sequences of MtuPps1 and MtuRecA homing sites (HS) inserted in pUC19 (the sequence of which appears in boldface) are underlined. (B) Cleavage assay for MtuPps1. One hundred nanograms of linearized substrate 2 was incubated with either 2.5 μg of the crude extract of MtuPps1 (+) or 2.5 μg of a crude extract of nontransformed E. coli BL21(De3)(pLysS) (−), for 1 h at 37°C, in 10 mM Tris-HCl (pH 8) buffer containing 10 mM MgCl 2 and 25 mM KCl. Substrate (S2; 2,730 bp) and products (P; 940 and 1,790 bp) were separated on a 1% agarose gel in TBE buffer. (C) Cleavage assay for MtuRecA. One hundred nanograms of linearized substrate 1 was incubated with either 2.5 μg of the crude extract of MtuRecA (+) or 2.5 μg of a crude extract of nontransformed E. coli BL21(De3)(pLysS) (−), for 1 h at 37°C, in 10 mM Tris-HCl (pH 8) buffer containing 10 mM MgCl 2 and 25 mM KCl. The reaction mixture was analyzed on a 1% agarose gel in TBE buffer.
    Figure Legend Snippet: Endonuclease assays. (A) DNA substrates for MtuPps1 and MtuRecA. The sequences of MtuPps1 and MtuRecA homing sites (HS) inserted in pUC19 (the sequence of which appears in boldface) are underlined. (B) Cleavage assay for MtuPps1. One hundred nanograms of linearized substrate 2 was incubated with either 2.5 μg of the crude extract of MtuPps1 (+) or 2.5 μg of a crude extract of nontransformed E. coli BL21(De3)(pLysS) (−), for 1 h at 37°C, in 10 mM Tris-HCl (pH 8) buffer containing 10 mM MgCl 2 and 25 mM KCl. Substrate (S2; 2,730 bp) and products (P; 940 and 1,790 bp) were separated on a 1% agarose gel in TBE buffer. (C) Cleavage assay for MtuRecA. One hundred nanograms of linearized substrate 1 was incubated with either 2.5 μg of the crude extract of MtuRecA (+) or 2.5 μg of a crude extract of nontransformed E. coli BL21(De3)(pLysS) (−), for 1 h at 37°C, in 10 mM Tris-HCl (pH 8) buffer containing 10 mM MgCl 2 and 25 mM KCl. The reaction mixture was analyzed on a 1% agarose gel in TBE buffer.

    Techniques Used: Sequencing, Cleavage Assay, Incubation, Agarose Gel Electrophoresis

    7) Product Images from "The dual role of HOP2 in mammalian meiotic homologous recombination"

    Article Title: The dual role of HOP2 in mammalian meiotic homologous recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1234

    The mechanism of recombination mediated by HOP2. ( A ) Formation of joint molecules promoted by HOP2. (Panel a) The presynaptic polymerization of HOP2 protein on ssDNA. (Panel b) Conjunction of ssDNA and dsDNA without homologous alignment. (Panel c) Homologous DNA pairing and strand invasion. Deproteinization of the complex in panel (c) in the context of a supercoiled target duplex DNA results in a stable D-loop. ( B ) HOP2 unstacks bases upon binding to ssDNA. Unstacking is manifested as increased reactivity of the thymine residues to potassium permanganate (arrows). Lane 1 contains a ladder generated by cleavage at purine bases ( C ) Experimental outline for DNA capture assay. Biotinylated DNA, immobilized on streptavidin–agarose beads, was used as a binding substrate for HOP2 protein. After incubation with pUC19 dsDNA, nucleoprotein complexes were separated from unbound proteins and dsDNA by centrifugation, and products were deproteinized before analysis by gel electrophoresis. ( D ) Quantitation of captured DNA as determined by intensity of ethidium bromide fluorescence. ( E ) Schematic of the synaptic complex assay. Fluorescein and rhodamine are represented in green and red, respectively. ( F ) Synaptic complex formation promoted by HOP2 and DMC1 with homologous and heterologous DNA. Synaptic complexes for homologous dsDNA in the absence of protein and DMC1 with homologous DNA are also shown.
    Figure Legend Snippet: The mechanism of recombination mediated by HOP2. ( A ) Formation of joint molecules promoted by HOP2. (Panel a) The presynaptic polymerization of HOP2 protein on ssDNA. (Panel b) Conjunction of ssDNA and dsDNA without homologous alignment. (Panel c) Homologous DNA pairing and strand invasion. Deproteinization of the complex in panel (c) in the context of a supercoiled target duplex DNA results in a stable D-loop. ( B ) HOP2 unstacks bases upon binding to ssDNA. Unstacking is manifested as increased reactivity of the thymine residues to potassium permanganate (arrows). Lane 1 contains a ladder generated by cleavage at purine bases ( C ) Experimental outline for DNA capture assay. Biotinylated DNA, immobilized on streptavidin–agarose beads, was used as a binding substrate for HOP2 protein. After incubation with pUC19 dsDNA, nucleoprotein complexes were separated from unbound proteins and dsDNA by centrifugation, and products were deproteinized before analysis by gel electrophoresis. ( D ) Quantitation of captured DNA as determined by intensity of ethidium bromide fluorescence. ( E ) Schematic of the synaptic complex assay. Fluorescein and rhodamine are represented in green and red, respectively. ( F ) Synaptic complex formation promoted by HOP2 and DMC1 with homologous and heterologous DNA. Synaptic complexes for homologous dsDNA in the absence of protein and DMC1 with homologous DNA are also shown.

    Techniques Used: Binding Assay, Generated, Incubation, Centrifugation, Nucleic Acid Electrophoresis, Quantitation Assay, Fluorescence

    8) Product Images from "Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori"

    Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw730

    ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and DNase I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.
    Figure Legend Snippet: ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and DNase I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.

    Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Electrophoresis, Clear Native PAGE, Autoradiography, Activity Assay, Incubation, Agarose Gel Electrophoresis, Negative Control, Lambda DNA Preparation

    9) Product Images from "Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel"

    Article Title: Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel

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

    doi: 10.1073/pnas.1117549109

    An epigenetic analysis workflow using fluorescence-activated, single molecule sorting. ( Top ) DNA preparation. Linearized DNA plasmids, unmethylated pUC19, and methylated pML4.2 were fluorescently labeled with a red stain and then mixed with green-labeled
    Figure Legend Snippet: An epigenetic analysis workflow using fluorescence-activated, single molecule sorting. ( Top ) DNA preparation. Linearized DNA plasmids, unmethylated pUC19, and methylated pML4.2 were fluorescently labeled with a red stain and then mixed with green-labeled

    Techniques Used: Fluorescence, Methylation, Labeling, Staining

    Single molecule detection and sorting of methylated DNA. DNA methylation state was identified using a green-labeled methyl binding domain-1 (MBD1) protein incubated in a mixture of red-labeled DNAs including unmethylated-pUC19 and methylated-pML4.2. Methylation-specific
    Figure Legend Snippet: Single molecule detection and sorting of methylated DNA. DNA methylation state was identified using a green-labeled methyl binding domain-1 (MBD1) protein incubated in a mixture of red-labeled DNAs including unmethylated-pUC19 and methylated-pML4.2. Methylation-specific

    Techniques Used: Methylation, DNA Methylation Assay, Labeling, Binding Assay, Incubation

    Optimization of molecule sorting efficiency. A 1∶4 molar ratio mixture of red-intercalated pML4.2 (15.2 Kb) and pUC19 (2.7 Kb) DNA was loaded into the nanofluidic device and sorted on differences in fluorescence intensity to collect
    Figure Legend Snippet: Optimization of molecule sorting efficiency. A 1∶4 molar ratio mixture of red-intercalated pML4.2 (15.2 Kb) and pUC19 (2.7 Kb) DNA was loaded into the nanofluidic device and sorted on differences in fluorescence intensity to collect

    Techniques Used: Fluorescence

    10) Product Images from "Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density"

    Article Title: Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01070-14

    ISWI, ACF, and Chd1 have clamping activity, i.e., they generate very similar and constant nucleosomal repeat lengths regardless of nucleosome density. (A) Limited digests with the indicated MNase concentrations of SGD chromatin (plasmid pUC19-PHO8) at the indicated assembly degrees after incubation with (+ISWI) or without (−ISWI) ISWI remodeler. Asterisks denote the trinucleosomal fragment band. MNase digestion fragments were visualized by Southern blotting and probing against the yeast PHO8 gene. M, DNA marker (2-log; NEB). (B and C) As for panel A but for the ACF or Chd1 remodeler, respectively.
    Figure Legend Snippet: ISWI, ACF, and Chd1 have clamping activity, i.e., they generate very similar and constant nucleosomal repeat lengths regardless of nucleosome density. (A) Limited digests with the indicated MNase concentrations of SGD chromatin (plasmid pUC19-PHO8) at the indicated assembly degrees after incubation with (+ISWI) or without (−ISWI) ISWI remodeler. Asterisks denote the trinucleosomal fragment band. MNase digestion fragments were visualized by Southern blotting and probing against the yeast PHO8 gene. M, DNA marker (2-log; NEB). (B and C) As for panel A but for the ACF or Chd1 remodeler, respectively.

    Techniques Used: Activity Assay, Plasmid Preparation, Incubation, Southern Blot, Marker

    11) Product Images from "Investigating the endonuclease activity of four Pyrococcus abyssi inteins"

    Article Title: Investigating the endonuclease activity of four Pyrococcus abyssi inteins

    Journal: Nucleic Acids Research

    doi:

    Standard cleavage assays for PI- Pab I ( Pab RIR1-2) and PI- Pab II ( Pab RIR1-3). Cleavage assays for PI- Pab I on its circular substrate ( A ), linear substrate ( B ) and on linear plasmid pUC19 ( C ). 100 ng of purified pSiteC plasmid was incubated with 0.10 ng (A), 0.40 ng (B) or 15 ng (C) of PI- Pab I for different times at 70°C, in a 5 mM Tris–acetate pH 9.5 buffer containing 5 mM KCl and 5 mM MgCl 2 . Cleavage assays for PI- Pab II on its circular substrate ( D ), linear substrate ( E ) and on linear plasmid pUC19 ( F ). 100 ng of purified pSiteD plasmid were incubated with 1 ng [(D) and (E)] or 15 ng (F) of PI- Pab II for different times at 70°C, in a 10 mM Tris–HCl pH 8 buffer containing 25 mM NH 4 OAc and 2 mM MgCl 2 . Either supercoiled (sc), open circular (oc) and linear (lin) forms of DNA or linear substrate (S, 2710 bp) and fragment products (P, 960 and 1750 bp) were separated on a 1% agarose gel in 0.5× TBE buffer.
    Figure Legend Snippet: Standard cleavage assays for PI- Pab I ( Pab RIR1-2) and PI- Pab II ( Pab RIR1-3). Cleavage assays for PI- Pab I on its circular substrate ( A ), linear substrate ( B ) and on linear plasmid pUC19 ( C ). 100 ng of purified pSiteC plasmid was incubated with 0.10 ng (A), 0.40 ng (B) or 15 ng (C) of PI- Pab I for different times at 70°C, in a 5 mM Tris–acetate pH 9.5 buffer containing 5 mM KCl and 5 mM MgCl 2 . Cleavage assays for PI- Pab II on its circular substrate ( D ), linear substrate ( E ) and on linear plasmid pUC19 ( F ). 100 ng of purified pSiteD plasmid were incubated with 1 ng [(D) and (E)] or 15 ng (F) of PI- Pab II for different times at 70°C, in a 10 mM Tris–HCl pH 8 buffer containing 25 mM NH 4 OAc and 2 mM MgCl 2 . Either supercoiled (sc), open circular (oc) and linear (lin) forms of DNA or linear substrate (S, 2710 bp) and fragment products (P, 960 and 1750 bp) were separated on a 1% agarose gel in 0.5× TBE buffer.

    Techniques Used: Plasmid Preparation, Purification, Incubation, Agarose Gel Electrophoresis

    12) Product Images from "Engineering Nt.BtsCI and Nb.BtsCI nicking enzymes and applications in generating long overhangs"

    Article Title: Engineering Nt.BtsCI and Nb.BtsCI nicking enzymes and applications in generating long overhangs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp1092

    DNA nicking activity of top-strand nicking BtsCI variant. Two-fold serial dilutions of the cell extract of BtsCI nicking variant E128F were incubated with pUC19 as described in ‘Materials and Methods’ section. The cleavage products were analyzed on a 1% agarose gel. OC, open circle; SC, supercoiled.
    Figure Legend Snippet: DNA nicking activity of top-strand nicking BtsCI variant. Two-fold serial dilutions of the cell extract of BtsCI nicking variant E128F were incubated with pUC19 as described in ‘Materials and Methods’ section. The cleavage products were analyzed on a 1% agarose gel. OC, open circle; SC, supercoiled.

    Techniques Used: Activity Assay, Variant Assay, Incubation, Agarose Gel Electrophoresis

    DNA nicking activity of the bottom-strand nicking BtsCI variant. Two-fold serial dilutions of the crude extract of BtsCI nicking variant (D388A/E403A/E405A) were incubated with pUC19 as described in ‘Materials and Methods’ section. The cleavage products were analyzed on a 1% agarose gel. OC, open circle; SC, supercoiled.
    Figure Legend Snippet: DNA nicking activity of the bottom-strand nicking BtsCI variant. Two-fold serial dilutions of the crude extract of BtsCI nicking variant (D388A/E403A/E405A) were incubated with pUC19 as described in ‘Materials and Methods’ section. The cleavage products were analyzed on a 1% agarose gel. OC, open circle; SC, supercoiled.

    Techniques Used: Activity Assay, Variant Assay, Incubation, Agarose Gel Electrophoresis

    DNA nicking activity of BtsCI mutants. Two-fold serial dilutions of the clarified cell extracts of E. coli cultures that expressed the indicated BtsCI mutants were incubated with 0.5 µg of pUC19 as described in ‘Materials and Methods’ section. The cleavage products were analyzed on a 1% agarose gel. OC, open circle; SC, supercoiled; −, no cleavage; +, pUC19 nicked by Nt.BsmAI.
    Figure Legend Snippet: DNA nicking activity of BtsCI mutants. Two-fold serial dilutions of the clarified cell extracts of E. coli cultures that expressed the indicated BtsCI mutants were incubated with 0.5 µg of pUC19 as described in ‘Materials and Methods’ section. The cleavage products were analyzed on a 1% agarose gel. OC, open circle; SC, supercoiled; −, no cleavage; +, pUC19 nicked by Nt.BsmAI.

    Techniques Used: Activity Assay, Incubation, Agarose Gel Electrophoresis

    ( A ) Generation of overhangs. Cleavage sites for BtsCI, FokI and a combination of FokI/Nt.BtsCI and FokI/Nb.BtsCI are indicated. For BtsCI, a 2-nt 5′ recessive end is generated. For FokI, a 4-nt 3′ recessive end is generated. For FokI/Nt.BtsCI, an 11-nt 3′ recessive end is generated. For FokI/Nb.BtsCI, a 9-nt 5′ recessive end is generated. Grey arrows, BtsCI top-strand cleavage site; white arrows, BtsCI bottom-strand cleavage site; black arrows, FokI cleavage sites. ( B ) Annealing of oligonucleotides to the long overhangs for PCR. Plasmid pUC19 was cleaved by the indicated enzyme(s) and then ligated to a 100-bp long oligonucleotide as described in ‘Materials and Methods’ section. The ligation products were used as template for PCR that specifically detect the ligated DNA. Only the cleavage product of FokI/Nt.BtsCI can be annealed to the 11-nt 3′ recessive end oligonucleotide (left panel), whereas only the cleavage product of FokI/Nb.BtsCI can be annealed to the 9-nt 5′ recessive end oligonucleotide (right panel).
    Figure Legend Snippet: ( A ) Generation of overhangs. Cleavage sites for BtsCI, FokI and a combination of FokI/Nt.BtsCI and FokI/Nb.BtsCI are indicated. For BtsCI, a 2-nt 5′ recessive end is generated. For FokI, a 4-nt 3′ recessive end is generated. For FokI/Nt.BtsCI, an 11-nt 3′ recessive end is generated. For FokI/Nb.BtsCI, a 9-nt 5′ recessive end is generated. Grey arrows, BtsCI top-strand cleavage site; white arrows, BtsCI bottom-strand cleavage site; black arrows, FokI cleavage sites. ( B ) Annealing of oligonucleotides to the long overhangs for PCR. Plasmid pUC19 was cleaved by the indicated enzyme(s) and then ligated to a 100-bp long oligonucleotide as described in ‘Materials and Methods’ section. The ligation products were used as template for PCR that specifically detect the ligated DNA. Only the cleavage product of FokI/Nt.BtsCI can be annealed to the 11-nt 3′ recessive end oligonucleotide (left panel), whereas only the cleavage product of FokI/Nb.BtsCI can be annealed to the 9-nt 5′ recessive end oligonucleotide (right panel).

    Techniques Used: Generated, Polymerase Chain Reaction, Plasmid Preparation, Ligation

    13) Product Images from "MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection"

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn711

    MmeI cleaves the two DNA strands at one site simultaneously. ( A ) Time course of MmeI digestion of supercoiled pUC19 DNA (2 U/μg) for 10 s, 20 s, 30 s, 1, 3 10, 20, 30 and 60 min. Supercoiled plasmid (sc) is converted directly to linear (lin) DNA, with no accumulation of open circular DNA (oc). ‘A+B cut’ indicates the 184-bp product of MmeI cleavage at both pUC19 sites. ( B ) MmeI digestion of linear pUC19 DNA (previously cut with PstI), in a 2-fold serial dilution from 8 to 0.03 U/μg. MmeI cuts at a one site, forming products from either site A or site B, before forming product from cleavage at both sites (A+B). ‘lin’ indicates linear pUC19, ‘A-R’ and ‘A-L’ indicate the cleavage products from MmeI cutting at the 996 site, ‘B-R’ and ‘B-L’ indicate the cleavage products from MmeI cutting at the 1180 site, while ‘A+B’ indicates the cleavage product from MmeI cutting at both sites.
    Figure Legend Snippet: MmeI cleaves the two DNA strands at one site simultaneously. ( A ) Time course of MmeI digestion of supercoiled pUC19 DNA (2 U/μg) for 10 s, 20 s, 30 s, 1, 3 10, 20, 30 and 60 min. Supercoiled plasmid (sc) is converted directly to linear (lin) DNA, with no accumulation of open circular DNA (oc). ‘A+B cut’ indicates the 184-bp product of MmeI cleavage at both pUC19 sites. ( B ) MmeI digestion of linear pUC19 DNA (previously cut with PstI), in a 2-fold serial dilution from 8 to 0.03 U/μg. MmeI cuts at a one site, forming products from either site A or site B, before forming product from cleavage at both sites (A+B). ‘lin’ indicates linear pUC19, ‘A-R’ and ‘A-L’ indicate the cleavage products from MmeI cutting at the 996 site, ‘B-R’ and ‘B-L’ indicate the cleavage products from MmeI cutting at the 1180 site, while ‘A+B’ indicates the cleavage product from MmeI cutting at both sites.

    Techniques Used: Plasmid Preparation, Serial Dilution

    Detection of MmeI modification using antibodies specific for N6-methyladenine (m6A) and N4-methylcytosine (m4C). (Top row) unmethylated DNA; (second row) MmeI in vitro modified DNA; (third row) M.EcoRI in vitro modified DNA; (fourth row) M.BamHI in vitro modified DNA. Positive controls: m6A antibody panel; (fifth row) dam (m6A) in vivo methylated pUC19 DNA (400–25 ng dilution), m4C antibody panel; (fifth row) M.EsaBC3I (m4C) in vivo methylated plasmid DNA (400–25 ng dilution).
    Figure Legend Snippet: Detection of MmeI modification using antibodies specific for N6-methyladenine (m6A) and N4-methylcytosine (m4C). (Top row) unmethylated DNA; (second row) MmeI in vitro modified DNA; (third row) M.EcoRI in vitro modified DNA; (fourth row) M.BamHI in vitro modified DNA. Positive controls: m6A antibody panel; (fifth row) dam (m6A) in vivo methylated pUC19 DNA (400–25 ng dilution), m4C antibody panel; (fifth row) M.EsaBC3I (m4C) in vivo methylated plasmid DNA (400–25 ng dilution).

    Techniques Used: Modification, In Vitro, In Vivo, Methylation, Plasmid Preparation

    14) Product Images from "Relationship of DNA degradation by Saccharomyces cerevisiae Exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection"

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

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

    doi: 10.1073/pnas.1305166110

    Exo1 does not stimulate DNA end resection by Dna2, Sgs1, and Top3-Rmi1. ( A ) Nuclease assays with Exo1 (7 nM), Dna2 (7 nM), Sgs1 (7 nM), Top3-Rmi1 (14 nM), and unlabeled pUC19 dsDNA containing 4-nt ssDNA overhangs at the 5′ ends (3.8 nM) in the presence of RPA (3 μM) for 4, 8, or 12 min. The gel is an inverted image of ethidium bromide-stained DNA. ( B ) Quantification of experiments as shown in A . Error bars show SE. ( C ) Nuclease assays with Exo1 (1 nM), Sgs1 (1 nM), Top3-Rmi1 (3 nM), Dna2 (1 nM), Mre11-Rad50-Xrs2 (25 nM), as indicated, for 1, 2, 4, 8, and 16 min in the presence of RPA (0.4 μM). Blunt-ended pUC19 dsDNA (1 nM), 32 P labeled at the 3′ end, was used. ( D ) Quantification of experiments as shown in C . Error bars show SE.
    Figure Legend Snippet: Exo1 does not stimulate DNA end resection by Dna2, Sgs1, and Top3-Rmi1. ( A ) Nuclease assays with Exo1 (7 nM), Dna2 (7 nM), Sgs1 (7 nM), Top3-Rmi1 (14 nM), and unlabeled pUC19 dsDNA containing 4-nt ssDNA overhangs at the 5′ ends (3.8 nM) in the presence of RPA (3 μM) for 4, 8, or 12 min. The gel is an inverted image of ethidium bromide-stained DNA. ( B ) Quantification of experiments as shown in A . Error bars show SE. ( C ) Nuclease assays with Exo1 (1 nM), Sgs1 (1 nM), Top3-Rmi1 (3 nM), Dna2 (1 nM), Mre11-Rad50-Xrs2 (25 nM), as indicated, for 1, 2, 4, 8, and 16 min in the presence of RPA (0.4 μM). Blunt-ended pUC19 dsDNA (1 nM), 32 P labeled at the 3′ end, was used. ( D ) Quantification of experiments as shown in C . Error bars show SE.

    Techniques Used: Recombinase Polymerase Amplification, Staining, Labeling

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

    Techniques Used: Recombinase Polymerase Amplification, Labeling, Nuclease Assay

    In the presence of RPA, resection of linear plasmid-length dsDNA by Exo1 produces kilobase-sized ssDNA. ( A ) Nuclease activity of Exo1 (2.7 nM) as a function of time (0.5, 1, 2, 4, 6, 10, 15, 20, and 30 min). pUC19 dsDNA (blunt, 1 nM), 32 P labeled at the 3′ end, was the substrate. “Heat” refers to ssDNA produced by heat denaturation of the pUC19 dsDNA. ( B ) Reaction as in A carried out in the presence of RPA (0.4 μM). ( C ) Illustration summarizing results from A and B , showing the intermediates and products of dsDNA degradation by Exo1 in the presence or absence of RPA.
    Figure Legend Snippet: In the presence of RPA, resection of linear plasmid-length dsDNA by Exo1 produces kilobase-sized ssDNA. ( A ) Nuclease activity of Exo1 (2.7 nM) as a function of time (0.5, 1, 2, 4, 6, 10, 15, 20, and 30 min). pUC19 dsDNA (blunt, 1 nM), 32 P labeled at the 3′ end, was the substrate. “Heat” refers to ssDNA produced by heat denaturation of the pUC19 dsDNA. ( B ) Reaction as in A carried out in the presence of RPA (0.4 μM). ( C ) Illustration summarizing results from A and B , showing the intermediates and products of dsDNA degradation by Exo1 in the presence or absence of RPA.

    Techniques Used: Recombinase Polymerase Amplification, Plasmid Preparation, Activity Assay, Labeling, Produced

    Mre11-Rad50-Xrs2 complex stimulates resection of dsDNA by Exo1. ( A ) A representative experiment with blunt-ended pUC19 dsDNA (1 nM, 32 P labeled at the 3′ end) showing degradation by Exo1 (0.4 nM, where indicated) and its stimulation by MRX [1, 3, 10, 30, and 100 nM (lanes 2–6) and 1, 3, 10, and 30 nM (lanes 8–11), respectively]. ( B ) Reaction as in A carried out in the presence of RPA (0.4 μM). ( C ) Quantitation of experiments as in A and B . Error bars show SE.
    Figure Legend Snippet: Mre11-Rad50-Xrs2 complex stimulates resection of dsDNA by Exo1. ( A ) A representative experiment with blunt-ended pUC19 dsDNA (1 nM, 32 P labeled at the 3′ end) showing degradation by Exo1 (0.4 nM, where indicated) and its stimulation by MRX [1, 3, 10, 30, and 100 nM (lanes 2–6) and 1, 3, 10, and 30 nM (lanes 8–11), respectively]. ( B ) Reaction as in A carried out in the presence of RPA (0.4 μM). ( C ) Quantitation of experiments as in A and B . Error bars show SE.

    Techniques Used: Labeling, Recombinase Polymerase Amplification, Quantitation Assay

    Exo1 preferentially degrades dsDNA resected at the 5′ end to produce an ssDNA tail at the 3′ end. ( A ) Quantification of Exo1 nuclease activity, in the presence of RPA (3 μM), using unlabeled pUC19 dsDNA (7.6 nM) that either was blunt or contained an ssDNA overhang (4 nt) at either 3′ or 5′ ends. Error bars indicate SE. ( B ) A representative experiment showing degradation of pUC19 dsDNA with a 5′-ssDNA overhang of 3 nt (7 nM, 32 P labeled at the 3′ end) by Exo1 (0.05, 0.15, 0.45, 1.3, 4, and 12 nM, respectively) in the presence of RPA (2.8 μM). “D173A”: The nuclease-deficient Exo1 D173A mutant was used instead of wild-type Exo1 (12 nM). “Heat”: Heat-denatured substrate. ( C ) Illustration summarizing results from B showing degradation by Exo1 of a dsDNA substrate with 5′-end ssDNA overhangs. ( D ) Quantification of Exo1 nuclease activity on 3.0 kb unlabeled dsDNA (7.6 nM) containing an ssDNA overhang of 4 nt at both 5′ ends (squares) vs. dsDNA with a 4-nt 5′ overhang at one end and a 3′ (circles) or 5′ (triangles) overhang of ∼100 nt at the other end. The reactions were carried out in the presence of RPA (3 μM). Error bars show SE.
    Figure Legend Snippet: Exo1 preferentially degrades dsDNA resected at the 5′ end to produce an ssDNA tail at the 3′ end. ( A ) Quantification of Exo1 nuclease activity, in the presence of RPA (3 μM), using unlabeled pUC19 dsDNA (7.6 nM) that either was blunt or contained an ssDNA overhang (4 nt) at either 3′ or 5′ ends. Error bars indicate SE. ( B ) A representative experiment showing degradation of pUC19 dsDNA with a 5′-ssDNA overhang of 3 nt (7 nM, 32 P labeled at the 3′ end) by Exo1 (0.05, 0.15, 0.45, 1.3, 4, and 12 nM, respectively) in the presence of RPA (2.8 μM). “D173A”: The nuclease-deficient Exo1 D173A mutant was used instead of wild-type Exo1 (12 nM). “Heat”: Heat-denatured substrate. ( C ) Illustration summarizing results from B showing degradation by Exo1 of a dsDNA substrate with 5′-end ssDNA overhangs. ( D ) Quantification of Exo1 nuclease activity on 3.0 kb unlabeled dsDNA (7.6 nM) containing an ssDNA overhang of 4 nt at both 5′ ends (squares) vs. dsDNA with a 4-nt 5′ overhang at one end and a 3′ (circles) or 5′ (triangles) overhang of ∼100 nt at the other end. The reactions were carried out in the presence of RPA (3 μM). Error bars show SE.

    Techniques Used: Activity Assay, Recombinase Polymerase Amplification, Labeling, Mutagenesis

    15) Product Images from "CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides"

    Article Title: CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2019.12.038

    Time Course of Degradation of DNA Substrates by Cas12a (A and B) Representative gel images of Cas12a activity toward M13mp18 ssDNA (A) and pUC19 dsDNA (B) at indicated time points (min) were shown in the upper panel. Positions of M13, supercoiled (SC), nicked (N), and linear (L) DNA were indicated. The vertical dotted line indicates the border between two separate gels. The fraction cleaved (%) from three independent experiments is plotted against the time points (min) and fitted with a nonlinear regression in the lower panel. Error bars are presented as mean ± SEM.
    Figure Legend Snippet: Time Course of Degradation of DNA Substrates by Cas12a (A and B) Representative gel images of Cas12a activity toward M13mp18 ssDNA (A) and pUC19 dsDNA (B) at indicated time points (min) were shown in the upper panel. Positions of M13, supercoiled (SC), nicked (N), and linear (L) DNA were indicated. The vertical dotted line indicates the border between two separate gels. The fraction cleaved (%) from three independent experiments is plotted against the time points (min) and fitted with a nonlinear regression in the lower panel. Error bars are presented as mean ± SEM.

    Techniques Used: Activity Assay

    Inhibitory Effects of Anti-Cas12a psDNA on Cas12a-Mediated DNase Activity toward Linear DNA (A–C) The effects of anti-Cas12a psDNA on metal-dependent ssDNase activity of Cas12a orthologs (AsCas12a, LbCas12a, and FnCas12a) toward linear ssDNA (A), dsDNA (B), and linearized pUC19 (C).
    Figure Legend Snippet: Inhibitory Effects of Anti-Cas12a psDNA on Cas12a-Mediated DNase Activity toward Linear DNA (A–C) The effects of anti-Cas12a psDNA on metal-dependent ssDNase activity of Cas12a orthologs (AsCas12a, LbCas12a, and FnCas12a) toward linear ssDNA (A), dsDNA (B), and linearized pUC19 (C).

    Techniques Used: Activity Assay

    crRNA-Free DNase Activities of Cas12a Orthologs toward Circular DNA (A) Representative in vitro cleavage of circular phage M13mp18 ssDNA by AsCas12a and LbCas12a in the presence of various divalent metal ions. FnCas12a was used as a positive group. Control (Ctrl), M13mp18 ssDNA alone. (B) Metal ion concentration dependence for cleavage of M13mp18 ssDNA substrates by AsCas12a and LbCas12a. Different concentrations of Mg 2+ or Mn 2+ ranging from 0.5 to 10 mM were used. Ctrl, M13mp18 ssDNA alone. (C) Representative in vitro cleavage of circular plasmid pUC19 dsDNA by AsCas12a and LbCas12a in the presence of various divalent metal ions. FnCas12a was used as a positive group. Ctrl, pUC19 dsDNA alone. (D) Metal ion concentration dependence for cleavage of pUC19 dsDNA substrates by AsCas12a and LbCas12a. Different concentrations of Mg 2+ or Mn 2+ ranging from 0.5 to 10 mM were used. Ctrl, pUC19 dsDNA alone. Positions of M13mp18, supercoiled (SC, green arrow), nicked (N, red arrow), and linear (L, red arrow) DNA were indicated.
    Figure Legend Snippet: crRNA-Free DNase Activities of Cas12a Orthologs toward Circular DNA (A) Representative in vitro cleavage of circular phage M13mp18 ssDNA by AsCas12a and LbCas12a in the presence of various divalent metal ions. FnCas12a was used as a positive group. Control (Ctrl), M13mp18 ssDNA alone. (B) Metal ion concentration dependence for cleavage of M13mp18 ssDNA substrates by AsCas12a and LbCas12a. Different concentrations of Mg 2+ or Mn 2+ ranging from 0.5 to 10 mM were used. Ctrl, M13mp18 ssDNA alone. (C) Representative in vitro cleavage of circular plasmid pUC19 dsDNA by AsCas12a and LbCas12a in the presence of various divalent metal ions. FnCas12a was used as a positive group. Ctrl, pUC19 dsDNA alone. (D) Metal ion concentration dependence for cleavage of pUC19 dsDNA substrates by AsCas12a and LbCas12a. Different concentrations of Mg 2+ or Mn 2+ ranging from 0.5 to 10 mM were used. Ctrl, pUC19 dsDNA alone. Positions of M13mp18, supercoiled (SC, green arrow), nicked (N, red arrow), and linear (L, red arrow) DNA were indicated.

    Techniques Used: In Vitro, Concentration Assay, Plasmid Preparation

    Inhibitory Effects of Anti-Cas12a psDNA on Cas12a-Mediated DNase Activity toward Circular DNA (A) Dose-dependent inhibitory effects of anti-Cas12a psDNA on Mg 2+ -promoted AsCas12a activity. Control (Ctrl), M13mp18 ssDNA alone. ssDNA1 was parallelly used to rule out the possibility of oligonucleotides interference. The concentration of AsCas12a was 200 nM. (B–D) The effects of anti-Cas12a psDNA on metal-dependent ssDNase activity of Cas12a orthologs (AsCas12a, LbCas12a, and FnCas12a) on circular ssDNA M13mp18 (B), ΦX174 (C), and dsDNA pUC19 (D). The concentration of anti-Cas12a psDNA was 200 nM. Ctrl, M13mp18 ssDNA alone. The vertical dotted line indicates the border between two separate gels. Ctrl, pUC19 dsDNA alone. Positions of M13, ΦX174, supercoiled (SC), nicked (N), and linear (L) DNA were indicated.
    Figure Legend Snippet: Inhibitory Effects of Anti-Cas12a psDNA on Cas12a-Mediated DNase Activity toward Circular DNA (A) Dose-dependent inhibitory effects of anti-Cas12a psDNA on Mg 2+ -promoted AsCas12a activity. Control (Ctrl), M13mp18 ssDNA alone. ssDNA1 was parallelly used to rule out the possibility of oligonucleotides interference. The concentration of AsCas12a was 200 nM. (B–D) The effects of anti-Cas12a psDNA on metal-dependent ssDNase activity of Cas12a orthologs (AsCas12a, LbCas12a, and FnCas12a) on circular ssDNA M13mp18 (B), ΦX174 (C), and dsDNA pUC19 (D). The concentration of anti-Cas12a psDNA was 200 nM. Ctrl, M13mp18 ssDNA alone. The vertical dotted line indicates the border between two separate gels. Ctrl, pUC19 dsDNA alone. Positions of M13, ΦX174, supercoiled (SC), nicked (N), and linear (L) DNA were indicated.

    Techniques Used: Activity Assay, Concentration Assay

    16) Product Images from "A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage"

    Article Title: A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky806

    ( A ) BDNPP hydrolytic cleavage mechanism in the presence of Cu 2 TPNap; ( B ) Lineweaver–Burk plot; ( C ) rate-pH profile for the cleavage of BDNPP in the presence of Cu 2 TPNap at 40°C; ( D ) DNA cleavage reactions by Cu 2 TPNap on pUC19 plasmid DNA over 1 h at 37°C in the absence of added reductant; and ( E ) T4 DNA ligase experiments with Cu 2 TPNap and restriction enzymes EcoRI and Nt.BspQI.
    Figure Legend Snippet: ( A ) BDNPP hydrolytic cleavage mechanism in the presence of Cu 2 TPNap; ( B ) Lineweaver–Burk plot; ( C ) rate-pH profile for the cleavage of BDNPP in the presence of Cu 2 TPNap at 40°C; ( D ) DNA cleavage reactions by Cu 2 TPNap on pUC19 plasmid DNA over 1 h at 37°C in the absence of added reductant; and ( E ) T4 DNA ligase experiments with Cu 2 TPNap and restriction enzymes EcoRI and Nt.BspQI.

    Techniques Used: Plasmid Preparation

    ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 TPNap for 4 h at 37°C and compared directly to Cu-Phen and Cu-Terph (reported elsewhere, ( 44 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.
    Figure Legend Snippet: ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 TPNap for 4 h at 37°C and compared directly to Cu-Phen and Cu-Terph (reported elsewhere, ( 44 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.

    Techniques Used: Plasmid Preparation, Incubation

    17) Product Images from "Identification of the first eubacterial endonuclease coded by an intein allele in the pps1 gene of mycobacteria"

    Article Title: Identification of the first eubacterial endonuclease coded by an intein allele in the pps1 gene of mycobacteria

    Journal: Nucleic Acids Research

    doi:

    Cleavage assay for Mga Pps1. Aliquots of 100 ng Sca I-linearized p Mga Site substrate ( A ) or linearized pUC19 ( B ) were incubated with 250 ng of a crude extract of (lane 1) non-transformed BL21(DE3)[pLysS], (lane 2) untagged Mga Pps1 or (lane 3) tagged Mga Pps1, or with (lane 4) 100, (lane 5) 50 or (lane 6) 25 ng of a crude extract of untagged Mga Pps1 for 10 min at 37°C, or with 25 ng of a crude extract of untagged Mga Pps1 for (lanes 7–12) 0, 10, 30, 40, 100 or 150 min, in 10 mM Tris–HCl, pH 8, buffer containing 10 mM MgCl 2 and 25 mM KCl.
    Figure Legend Snippet: Cleavage assay for Mga Pps1. Aliquots of 100 ng Sca I-linearized p Mga Site substrate ( A ) or linearized pUC19 ( B ) were incubated with 250 ng of a crude extract of (lane 1) non-transformed BL21(DE3)[pLysS], (lane 2) untagged Mga Pps1 or (lane 3) tagged Mga Pps1, or with (lane 4) 100, (lane 5) 50 or (lane 6) 25 ng of a crude extract of untagged Mga Pps1 for 10 min at 37°C, or with 25 ng of a crude extract of untagged Mga Pps1 for (lanes 7–12) 0, 10, 30, 40, 100 or 150 min, in 10 mM Tris–HCl, pH 8, buffer containing 10 mM MgCl 2 and 25 mM KCl.

    Techniques Used: Cleavage Assay, Incubation, Transformation Assay

    18) Product Images from "A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage"

    Article Title: A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky806

    ( A ) BDNPP hydrolytic cleavage mechanism in the presence of Cu 2 TPNap; ( B ) Lineweaver–Burk plot; ( C ) rate-pH profile for the cleavage of BDNPP in the presence of Cu 2 TPNap at 40°C; ( D ) DNA cleavage reactions by Cu 2 TPNap on pUC19 plasmid DNA over 1 h at 37°C in the absence of added reductant; and ( E ) T4 DNA ligase experiments with Cu 2 TPNap and restriction enzymes EcoRI and Nt.BspQI.
    Figure Legend Snippet: ( A ) BDNPP hydrolytic cleavage mechanism in the presence of Cu 2 TPNap; ( B ) Lineweaver–Burk plot; ( C ) rate-pH profile for the cleavage of BDNPP in the presence of Cu 2 TPNap at 40°C; ( D ) DNA cleavage reactions by Cu 2 TPNap on pUC19 plasmid DNA over 1 h at 37°C in the absence of added reductant; and ( E ) T4 DNA ligase experiments with Cu 2 TPNap and restriction enzymes EcoRI and Nt.BspQI.

    Techniques Used: Plasmid Preparation

    ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.
    Figure Legend Snippet: ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.

    Techniques Used: Plasmid Preparation, Incubation

    19) Product Images from "Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes"

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19920-y

    Synthetic c ysE and cysM gene transformants display recovery of CysE function without cysteine and methionine and CysM function without cysteine. ( A ) E. coli ΔcysE competent cells were transformed with positive control cysE , two cysE variants cysE-C/cysE-CM cloned into the multiple cloning site of pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. ( B ) E. coli ΔcysMΔcysK competent cells transformed with positive control c ysM , two cysM variants cysM-C / cysM-CM in pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. Cells were plated on M9 + glucose medium with 0.4 mM IPTG, 50 μg/ml kanamycin, and 100 μg/ml ampicillin and incubated at 30 °C for 72 h.
    Figure Legend Snippet: Synthetic c ysE and cysM gene transformants display recovery of CysE function without cysteine and methionine and CysM function without cysteine. ( A ) E. coli ΔcysE competent cells were transformed with positive control cysE , two cysE variants cysE-C/cysE-CM cloned into the multiple cloning site of pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. ( B ) E. coli ΔcysMΔcysK competent cells transformed with positive control c ysM , two cysM variants cysM-C / cysM-CM in pUC19 plasmid, and original pUC19 encoding N-terminal fragment of lacZ α as a negative control. Cells were plated on M9 + glucose medium with 0.4 mM IPTG, 50 μg/ml kanamycin, and 100 μg/ml ampicillin and incubated at 30 °C for 72 h.

    Techniques Used: Transformation Assay, Positive Control, Clone Assay, Plasmid Preparation, Negative Control, Incubation

    20) Product Images from "A bacterial DNA repair pathway specific to a natural antibiotic"

    Article Title: A bacterial DNA repair pathway specific to a natural antibiotic

    Journal: Molecular microbiology

    doi: 10.1111/mmi.14158

    MrfB is a metal-dependent exonuclease. (A) A schematic of MrfB depicting putative catalytic residues and C-terminal tetratrichopeptide repeat (TPR) domain. (B) Spot titer assay using strains with the indicated genotypes spotted on the indicated media. (C) 1 μg of purified MrfB stained with Coomassie brilliant blue. (D) Exonuclease assay using pUC19 linearized with BamHI (lanes 3–7). Reactions were incubated at 37°C for 15 minutes with or without MrfB, MgCl 2 , or EDTA as indicated, and separated on an agarose gel stained with ethidium bromide. Lane 1 is a 1 kb plus molecular weight marker (M) and lane 2 is undigested pUC19 plasmid. (E) Exonuclease assay testing substrate preference. The indicated exonucleases were incubated with a closed covalent circular plasmid (CCC), a nicked plasmid (Nicked) or a linear plasmid (Linear) in the presence of Mg 2+ at 37°C for 10 minutes. Reaction products were separated on an agarose gel stained with ethidium bromide. Lane 1 is a 1 kb plus molecular weight marker (M).
    Figure Legend Snippet: MrfB is a metal-dependent exonuclease. (A) A schematic of MrfB depicting putative catalytic residues and C-terminal tetratrichopeptide repeat (TPR) domain. (B) Spot titer assay using strains with the indicated genotypes spotted on the indicated media. (C) 1 μg of purified MrfB stained with Coomassie brilliant blue. (D) Exonuclease assay using pUC19 linearized with BamHI (lanes 3–7). Reactions were incubated at 37°C for 15 minutes with or without MrfB, MgCl 2 , or EDTA as indicated, and separated on an agarose gel stained with ethidium bromide. Lane 1 is a 1 kb plus molecular weight marker (M) and lane 2 is undigested pUC19 plasmid. (E) Exonuclease assay testing substrate preference. The indicated exonucleases were incubated with a closed covalent circular plasmid (CCC), a nicked plasmid (Nicked) or a linear plasmid (Linear) in the presence of Mg 2+ at 37°C for 10 minutes. Reaction products were separated on an agarose gel stained with ethidium bromide. Lane 1 is a 1 kb plus molecular weight marker (M).

    Techniques Used: Titer Assay, Purification, Staining, Incubation, Agarose Gel Electrophoresis, Molecular Weight, Marker, Plasmid Preparation, Countercurrent Chromatography

    21) Product Images from "Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4"

    Article Title: Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1191

    gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.
    Figure Legend Snippet: gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.

    Techniques Used: Incubation, Plasmid Preparation, Labeling

    22) Product Images from "Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density"

    Article Title: Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01070-14

    ISWI, ACF, and Chd1 have clamping activity, i.e., they generate very similar and constant nucleosomal repeat lengths regardless of nucleosome density. (A) Limited digests with the indicated MNase concentrations of SGD chromatin (plasmid pUC19-PHO8) at the indicated assembly degrees after incubation with (+ISWI) or without (−ISWI) ISWI remodeler. Asterisks denote the trinucleosomal fragment band. MNase digestion fragments were visualized by Southern blotting and probing against the yeast PHO8 gene. M, DNA marker (2-log; NEB). (B and C) As for panel A but for the ACF or Chd1 remodeler, respectively.
    Figure Legend Snippet: ISWI, ACF, and Chd1 have clamping activity, i.e., they generate very similar and constant nucleosomal repeat lengths regardless of nucleosome density. (A) Limited digests with the indicated MNase concentrations of SGD chromatin (plasmid pUC19-PHO8) at the indicated assembly degrees after incubation with (+ISWI) or without (−ISWI) ISWI remodeler. Asterisks denote the trinucleosomal fragment band. MNase digestion fragments were visualized by Southern blotting and probing against the yeast PHO8 gene. M, DNA marker (2-log; NEB). (B and C) As for panel A but for the ACF or Chd1 remodeler, respectively.

    Techniques Used: Activity Assay, Plasmid Preparation, Incubation, Southern Blot, Marker

    Related Articles

    Sequencing:

    Article Title: Global remodeling of nucleosome positions in C. elegans
    Article Snippet: .. We identified well-positioned nucleosomes in each sequence read profile using the following cutoffs: Rsa I, nucleosomes marked by reads with height 4 and above (1.3% of all reads); Hinc II, nucleosomes marked by reads with height 3 and above (1.1% of all reads); Gu & Fire [ ], nucleosomes marked by reads with height 3 and above (0.9% of all reads); Valouev et al. [ ], nucleosomes marked by reads with height 9 and above (1.5% of all reads). ..

    Concentration Assay:

    Article Title: Nucleoprotein complex intermediates in HIV-1 integration
    Article Snippet: .. Digest 500 μg of the plasmid DNA with 500 U each of ScaI and HincII (New England Biolabs) at a DNA concentration of 0.3 mg/ml. .. Incubate the reaction mixture at 37 °C for 2 h. Purification of the 1513 bp DNA fragment.

    other:

    Article Title: Global remodeling of nucleosome positions in C. elegans
    Article Snippet: The filter was applied to all cut sites, which on average occur once per 490 bp for Rsa I (GTAC) and once per 2109 bp for Hinc II (GTYRAC).

    Article Title: Replication-coupled DNA Interstrand Crosslink repair in Xenopus egg extracts
    Article Snippet: HincII and SapI restriction enzymes (NEB).

    Polyacrylamide Gel Electrophoresis:

    Article Title: An Intronic Sequence Element Mediates Both Activation and Repression of Rat Fibroblast Growth Factor Receptor 2 Pre-mRNA Splicing
    Article Snippet: .. In order to analyze the splicing products of pI-11-FL and pI-11-FS, we digested the RT-PCR products with Ava I and Hin cII and performed polyacrylamide gel electrophoresis. .. When stable transfections with pI-11-FL and pI-11-FS were analyzed as shown in Fig. C and D, the primary product was 380 or 377 bp, and this product contained almost exclusively exon IIIb in DT3 cells and IIIc in AT3 cells.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: An Intronic Sequence Element Mediates Both Activation and Repression of Rat Fibroblast Growth Factor Receptor 2 Pre-mRNA Splicing
    Article Snippet: .. In order to analyze the splicing products of pI-11-FL and pI-11-FS, we digested the RT-PCR products with Ava I and Hin cII and performed polyacrylamide gel electrophoresis. .. When stable transfections with pI-11-FL and pI-11-FS were analyzed as shown in Fig. C and D, the primary product was 380 or 377 bp, and this product contained almost exclusively exon IIIb in DT3 cells and IIIc in AT3 cells.

    Antiviral Assay:

    Article Title: An Intronic Sequence Element Mediates Both Activation and Repression of Rat Fibroblast Growth Factor Receptor 2 Pre-mRNA Splicing
    Article Snippet: .. Because exon IIIb contains an Ava I site not present in IIIc, and exon IIIc contains two Hin cII sites not present in IIIb, we expected the inclusion of IIIb to result in a 367-bp product which is cut with Ava I but not Hin cII, and we expected the inclusion of IIIc to result in a 364-bp product which is cut only by Hin cII. ..

    Article Title: An Intronic Sequence Element Mediates Both Activation and Repression of Rat Fibroblast Growth Factor Receptor 2 Pre-mRNA Splicing
    Article Snippet: .. In order to analyze the splicing products of pI-11-FL and pI-11-FS, we digested the RT-PCR products with Ava I and Hin cII and performed polyacrylamide gel electrophoresis. .. When stable transfections with pI-11-FL and pI-11-FS were analyzed as shown in Fig. C and D, the primary product was 380 or 377 bp, and this product contained almost exclusively exon IIIb in DT3 cells and IIIc in AT3 cells.

    Plasmid Preparation:

    Article Title: Nucleoprotein complex intermediates in HIV-1 integration
    Article Snippet: .. Digest 500 μg of the plasmid DNA with 500 U each of ScaI and HincII (New England Biolabs) at a DNA concentration of 0.3 mg/ml. .. Incubate the reaction mixture at 37 °C for 2 h. Purification of the 1513 bp DNA fragment.

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  • 89
    New England Biolabs puc19 dna
    Differences between G signals complementary to unmethylated C residues, N 4 -methylcytosine residues and 5-methylcytosine residues using the DYEnamic ET Terminator Kit. A1: untreated <t>pUC19</t> <t>DNA;</t> A2: M.BamHI N 4 -cytosine methylated pUC19DNA; A3: trace difference between A2 and A1. B1: untreated pUC19DNA; B2: M.HaeIII 5-cytosine methylated pUC19 DNA; B3: trace difference between B2 and B1. The G residues complementary to the methylated cytosines are boxed. N 4 -methylcytosine results in an increase in the complementary G signal, whereas 5-methylcytosine results in a decrease in the complementary G signal.
    Puc19 Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs puc19 construct
    Schematic diagram of the 199-bp fragment, constructs with inserts at various positions, control fragments with sequences offset from the apparent bend center, and a 345-bp fragment with 75 bp of unbent DNA from <t>pUC19</t> replacing 70 bp containing the curvature
    Puc19 Construct, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    New England Biolabs plasmids puc19
    Analyses of the yield and purity for <t>pUC19</t> purified with IEXM or anion exchange beads. Four replicate preparations were performed. A: Yields of pUC19 were determined by PicoGreen dsDNA assay ( gray bars ) and NanoDrop spectrophotometer ( white bars ). Results
    Plasmids Puc19, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Differences between G signals complementary to unmethylated C residues, N 4 -methylcytosine residues and 5-methylcytosine residues using the DYEnamic ET Terminator Kit. A1: untreated pUC19 DNA; A2: M.BamHI N 4 -cytosine methylated pUC19DNA; A3: trace difference between A2 and A1. B1: untreated pUC19DNA; B2: M.HaeIII 5-cytosine methylated pUC19 DNA; B3: trace difference between B2 and B1. The G residues complementary to the methylated cytosines are boxed. N 4 -methylcytosine results in an increase in the complementary G signal, whereas 5-methylcytosine results in a decrease in the complementary G signal.

    Journal: Nucleic Acids Research

    Article Title: Direct detection of methylation in genomic DNA

    doi: 10.1093/nar/gni121

    Figure Lengend Snippet: Differences between G signals complementary to unmethylated C residues, N 4 -methylcytosine residues and 5-methylcytosine residues using the DYEnamic ET Terminator Kit. A1: untreated pUC19 DNA; A2: M.BamHI N 4 -cytosine methylated pUC19DNA; A3: trace difference between A2 and A1. B1: untreated pUC19DNA; B2: M.HaeIII 5-cytosine methylated pUC19 DNA; B3: trace difference between B2 and B1. The G residues complementary to the methylated cytosines are boxed. N 4 -methylcytosine results in an increase in the complementary G signal, whereas 5-methylcytosine results in a decrease in the complementary G signal.

    Article Snippet: In vitro methylation of pUC19 DNA was performed with M.EcoRI (N 6 -methyladenine), M.HaeIII (5-methylcytosine) or M.BamHI (N 4 -methylcytosine) obtained from New England Biolabs according to the manufacturer's instructions.

    Techniques: Methylation

    Schematic diagram of the 199-bp fragment, constructs with inserts at various positions, control fragments with sequences offset from the apparent bend center, and a 345-bp fragment with 75 bp of unbent DNA from pUC19 replacing 70 bp containing the curvature

    Journal: Biophysical Journal

    Article Title: Intrinsic Curvature in the VP1 Gene of SV40: Comparison of Solution and Gel Results

    doi: 10.1529/biophysj.104.039834

    Figure Lengend Snippet: Schematic diagram of the 199-bp fragment, constructs with inserts at various positions, control fragments with sequences offset from the apparent bend center, and a 345-bp fragment with 75 bp of unbent DNA from pUC19 replacing 70 bp containing the curvature

    Article Snippet: As an example of the procedure used, the pUC19 construct containing the 199-bp SV40 fragment was cut at the unique Bsm FI site, the sticky ends were filled in, Cla I linkers (New England Biolabs, Beverly, MA) were added, and the plasmid was recircularized by ligase, using standard procedures ( ).

    Techniques: Construct

    PNA-DNA association obeys pseudo-first-order kinetics but it is affected by the total concentration of nontarget DNA present in the reaction mixture. ( A ) Gel-electrophoresis shift assay is used to monitor time course of pcPNAs 1690 binding to DNA target site located within a 350-bp fragment of p8/PvuII digest. Both experiments were performed at 37°C in 10 mM TE (pH 7.4), which contains 2 mM Na + . The concentration of pcPNAs and DNA target sites was 625 nM and 23 nM, respectively. The amount of nonspecific DNA (in bp) in the reaction mixture was varied from 60 μ M ( upper panel ) to 131 μ M ( lower panel ) by addition of appropriate amounts of linearized pUC19. ( B ) The yield of the PNA/DNA complex, C , fits linear regression; total concentrations of nonspecific DNA (in bp): 60 μ M (•) and 131 μ M (○). Pseudo-first-order rate constants given by the slopes of linear regression are 0.0297 and 0.0100 min −1 , respectively.

    Journal: Biophysical Journal

    Article Title: Specific versus Nonspecific Binding of Cationic PNAs to Duplex DNA

    doi:

    Figure Lengend Snippet: PNA-DNA association obeys pseudo-first-order kinetics but it is affected by the total concentration of nontarget DNA present in the reaction mixture. ( A ) Gel-electrophoresis shift assay is used to monitor time course of pcPNAs 1690 binding to DNA target site located within a 350-bp fragment of p8/PvuII digest. Both experiments were performed at 37°C in 10 mM TE (pH 7.4), which contains 2 mM Na + . The concentration of pcPNAs and DNA target sites was 625 nM and 23 nM, respectively. The amount of nonspecific DNA (in bp) in the reaction mixture was varied from 60 μ M ( upper panel ) to 131 μ M ( lower panel ) by addition of appropriate amounts of linearized pUC19. ( B ) The yield of the PNA/DNA complex, C , fits linear regression; total concentrations of nonspecific DNA (in bp): 60 μ M (•) and 131 μ M (○). Pseudo-first-order rate constants given by the slopes of linear regression are 0.0297 and 0.0100 min −1 , respectively.

    Article Snippet: Recombinant plasmids, p8, p10, and p10bis, carrying PNA-binding sites, are pUC19 derivatives with corresponding inserts cloned into BamHI (all restriction endonucleases used herein were purchased from New England Biolabs, Beverly, MA) site of the polylinker.

    Techniques: Concentration Assay, Nucleic Acid Electrophoresis, Shift Assay, Binding Assay

    Salt-dependence of the pcPNA 1690 binding to DNA target sites. The extent of complex formation was assessed after 40-min incubation at 37°C in 10 mM TE, pH 7.4 buffer with addition of desirable amounts of NaCl. PNA concentration (571 nM) and DNA target concentration (17 nM) were kept constant. Total concentration of DNA (in bp) changed from 45 μ M (•) to 90 μ M (○) by addition of linearized pUC19.

    Journal: Biophysical Journal

    Article Title: Specific versus Nonspecific Binding of Cationic PNAs to Duplex DNA

    doi:

    Figure Lengend Snippet: Salt-dependence of the pcPNA 1690 binding to DNA target sites. The extent of complex formation was assessed after 40-min incubation at 37°C in 10 mM TE, pH 7.4 buffer with addition of desirable amounts of NaCl. PNA concentration (571 nM) and DNA target concentration (17 nM) were kept constant. Total concentration of DNA (in bp) changed from 45 μ M (•) to 90 μ M (○) by addition of linearized pUC19.

    Article Snippet: Recombinant plasmids, p8, p10, and p10bis, carrying PNA-binding sites, are pUC19 derivatives with corresponding inserts cloned into BamHI (all restriction endonucleases used herein were purchased from New England Biolabs, Beverly, MA) site of the polylinker.

    Techniques: Binding Assay, Incubation, Concentration Assay

    Dependence of k ps on the total concentration of nontarget DNA, [ N ] 0 . Pseudo-first-order rate constants for pcPNA 1690 ( circles ) and bisPNA 522 ( triangles ) binding to corresponding DNA targets. Concentration of PNA was 625 nM in both cases. DNA concentration was varied by changing the amounts of the PCR-amplified, 265-bp fragment (⊙), PvuII restriction digest of corresponding plasmid (○ and ▵), and addition of nontarget linearized pUC19 (•). Experiments were performed in 10 mM TE, pH 7.4 (pcPNA) and pH 7.0 (bisPNA) buffer at 37°C. The lines are drawn to guide the eye.

    Journal: Biophysical Journal

    Article Title: Specific versus Nonspecific Binding of Cationic PNAs to Duplex DNA

    doi:

    Figure Lengend Snippet: Dependence of k ps on the total concentration of nontarget DNA, [ N ] 0 . Pseudo-first-order rate constants for pcPNA 1690 ( circles ) and bisPNA 522 ( triangles ) binding to corresponding DNA targets. Concentration of PNA was 625 nM in both cases. DNA concentration was varied by changing the amounts of the PCR-amplified, 265-bp fragment (⊙), PvuII restriction digest of corresponding plasmid (○ and ▵), and addition of nontarget linearized pUC19 (•). Experiments were performed in 10 mM TE, pH 7.4 (pcPNA) and pH 7.0 (bisPNA) buffer at 37°C. The lines are drawn to guide the eye.

    Article Snippet: Recombinant plasmids, p8, p10, and p10bis, carrying PNA-binding sites, are pUC19 derivatives with corresponding inserts cloned into BamHI (all restriction endonucleases used herein were purchased from New England Biolabs, Beverly, MA) site of the polylinker.

    Techniques: Concentration Assay, Binding Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    Analyses of the yield and purity for pUC19 purified with IEXM or anion exchange beads. Four replicate preparations were performed. A: Yields of pUC19 were determined by PicoGreen dsDNA assay ( gray bars ) and NanoDrop spectrophotometer ( white bars ). Results

    Journal: Journal of Biomolecular Techniques : JBT

    Article Title: High Performance DNA Purification using a Novel Ion Exchange Matrix

    doi:

    Figure Lengend Snippet: Analyses of the yield and purity for pUC19 purified with IEXM or anion exchange beads. Four replicate preparations were performed. A: Yields of pUC19 were determined by PicoGreen dsDNA assay ( gray bars ) and NanoDrop spectrophotometer ( white bars ). Results

    Article Snippet: Plasmids pUC19 and pBR322 (New England Biolabs, Beverly, MA) were transformed into ElectroEB10B competent cells (EdgeBio, Gaithersburg, MD) with a MicroPulser Electroporator (BioRad, Hercules, CA).

    Techniques: Purification, Picogreen Assay, Spectrophotometry

    Isolation of plasmids with anion exchange membranes. A: Purification of high-copy plasmid pUC19 (lanes 1–4) and low-copy plasmid pBR322 (lanes 5–8) using IEXM. Fractions were precipitated by isopropanol to remove excess salts and redissolved

    Journal: Journal of Biomolecular Techniques : JBT

    Article Title: High Performance DNA Purification using a Novel Ion Exchange Matrix

    doi:

    Figure Lengend Snippet: Isolation of plasmids with anion exchange membranes. A: Purification of high-copy plasmid pUC19 (lanes 1–4) and low-copy plasmid pBR322 (lanes 5–8) using IEXM. Fractions were precipitated by isopropanol to remove excess salts and redissolved

    Article Snippet: Plasmids pUC19 and pBR322 (New England Biolabs, Beverly, MA) were transformed into ElectroEB10B competent cells (EdgeBio, Gaithersburg, MD) with a MicroPulser Electroporator (BioRad, Hercules, CA).

    Techniques: Isolation, Purification, Plasmid Preparation

    DNA sequencing using pUC19 purified with IEXM. Purified pUC19 was serially diluted and sequenced. Results shown are average from duplicate sequencing reactions. A: Phred20 scores. B: Intensity of G-signal. The line shows linear regression of G-signal

    Journal: Journal of Biomolecular Techniques : JBT

    Article Title: High Performance DNA Purification using a Novel Ion Exchange Matrix

    doi:

    Figure Lengend Snippet: DNA sequencing using pUC19 purified with IEXM. Purified pUC19 was serially diluted and sequenced. Results shown are average from duplicate sequencing reactions. A: Phred20 scores. B: Intensity of G-signal. The line shows linear regression of G-signal

    Article Snippet: Plasmids pUC19 and pBR322 (New England Biolabs, Beverly, MA) were transformed into ElectroEB10B competent cells (EdgeBio, Gaithersburg, MD) with a MicroPulser Electroporator (BioRad, Hercules, CA).

    Techniques: DNA Sequencing, Purification, Sequencing

    Binding capacity of IEXM for pUC19 plasmid. Cleared lysate from 1 L LB culture was loaded repeatedly on one IEXM maxi column which had a bed volume of 0.58 ml. M, 1-kb DNA ladder. Lane 1: cleared lysate; lanes 2–12: flow-through fractions for

    Journal: Journal of Biomolecular Techniques : JBT

    Article Title: High Performance DNA Purification using a Novel Ion Exchange Matrix

    doi:

    Figure Lengend Snippet: Binding capacity of IEXM for pUC19 plasmid. Cleared lysate from 1 L LB culture was loaded repeatedly on one IEXM maxi column which had a bed volume of 0.58 ml. M, 1-kb DNA ladder. Lane 1: cleared lysate; lanes 2–12: flow-through fractions for

    Article Snippet: Plasmids pUC19 and pBR322 (New England Biolabs, Beverly, MA) were transformed into ElectroEB10B competent cells (EdgeBio, Gaithersburg, MD) with a MicroPulser Electroporator (BioRad, Hercules, CA).

    Techniques: Binding Assay, Plasmid Preparation, Flow Cytometry

    Endotoxin levels in plasmids purified with different methods. 1: Qiagen Plasmid Maxi kit. 2: Two rounds of CsCl-ethidium bromide gradient centrifugation. 3: Qiagen EndoFree Plasmid Maxi kit. IEXM: pUC19 maxi preparation from 100 ml of LB culture using

    Journal: Journal of Biomolecular Techniques : JBT

    Article Title: High Performance DNA Purification using a Novel Ion Exchange Matrix

    doi:

    Figure Lengend Snippet: Endotoxin levels in plasmids purified with different methods. 1: Qiagen Plasmid Maxi kit. 2: Two rounds of CsCl-ethidium bromide gradient centrifugation. 3: Qiagen EndoFree Plasmid Maxi kit. IEXM: pUC19 maxi preparation from 100 ml of LB culture using

    Article Snippet: Plasmids pUC19 and pBR322 (New England Biolabs, Beverly, MA) were transformed into ElectroEB10B competent cells (EdgeBio, Gaithersburg, MD) with a MicroPulser Electroporator (BioRad, Hercules, CA).

    Techniques: Purification, Plasmid Preparation, Gradient Centrifugation