ppumi  (New England Biolabs)


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    Name:
    PpuMI
    Description:
    PpuMI 2 500 units
    Catalog Number:
    R0506L
    Price:
    282
    Category:
    Restriction Enzymes
    Size:
    2 500 units
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    Structured Review

    New England Biolabs ppumi
    PpuMI
    PpuMI 2 500 units
    https://www.bioz.com/result/ppumi/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ppumi - by Bioz Stars, 2021-05
    93/100 stars

    Images

    1) Product Images from "Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition"

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.08.047

    CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .
    Figure Legend Snippet: CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .

    Techniques Used: DNA Synthesis, In Vitro, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Sequencing, Plasmid Preparation, Transfection, Isolation, Nucleic Acid Electrophoresis, Hybridization, Purification, Quantitation Assay

    2) Product Images from "Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition"

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.08.047

    CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .
    Figure Legend Snippet: CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .

    Techniques Used: DNA Synthesis, In Vitro, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Sequencing, Plasmid Preparation, Transfection, Isolation, Nucleic Acid Electrophoresis, Hybridization, Purification, Quantitation Assay

    Related Articles

    Polymerase Chain Reaction:

    Article Title: A Phosphomimetic Mutation at Threonine-57 Abolishes Transactivation Activity and Alters Nuclear Localization Pattern of Human Pregnane X Receptor
    Article Snippet: Polymerase chain reaction (PCR) was performed using pcDNA3-hPXR as the template and specific mutant primers, with forward primers containing a mutation corresponding to either A or D and a PPUMI restriction enzyme cutting site and a reverse primer containing a BsgI restriction enzyme cutting site. .. The PCR product was cloned into a TOPO vector (Zero Blunt TOPO PCR Cloning Kit; Invitrogen), followed by cleaving the sequence-verified TOPO-PCR product for the desired mutations with PPUMI (New England Biolabs, Ipswich, MA) and BsgI (New England Biolabs), and ligating the resulting fragment into PPUMI- and BsgI-cleaved pcDNA3-hPXR or pcDNA3-FLAG-hPXR. .. Only the open reading frame of hPXR, but not pcDNA3 or the FLAG tag, had cleavage sites for PPUMI and BsgI.

    Clone Assay:

    Article Title: A Phosphomimetic Mutation at Threonine-57 Abolishes Transactivation Activity and Alters Nuclear Localization Pattern of Human Pregnane X Receptor
    Article Snippet: Polymerase chain reaction (PCR) was performed using pcDNA3-hPXR as the template and specific mutant primers, with forward primers containing a mutation corresponding to either A or D and a PPUMI restriction enzyme cutting site and a reverse primer containing a BsgI restriction enzyme cutting site. .. The PCR product was cloned into a TOPO vector (Zero Blunt TOPO PCR Cloning Kit; Invitrogen), followed by cleaving the sequence-verified TOPO-PCR product for the desired mutations with PPUMI (New England Biolabs, Ipswich, MA) and BsgI (New England Biolabs), and ligating the resulting fragment into PPUMI- and BsgI-cleaved pcDNA3-hPXR or pcDNA3-FLAG-hPXR. .. Only the open reading frame of hPXR, but not pcDNA3 or the FLAG tag, had cleavage sites for PPUMI and BsgI.

    Plasmid Preparation:

    Article Title: A Phosphomimetic Mutation at Threonine-57 Abolishes Transactivation Activity and Alters Nuclear Localization Pattern of Human Pregnane X Receptor
    Article Snippet: Polymerase chain reaction (PCR) was performed using pcDNA3-hPXR as the template and specific mutant primers, with forward primers containing a mutation corresponding to either A or D and a PPUMI restriction enzyme cutting site and a reverse primer containing a BsgI restriction enzyme cutting site. .. The PCR product was cloned into a TOPO vector (Zero Blunt TOPO PCR Cloning Kit; Invitrogen), followed by cleaving the sequence-verified TOPO-PCR product for the desired mutations with PPUMI (New England Biolabs, Ipswich, MA) and BsgI (New England Biolabs), and ligating the resulting fragment into PPUMI- and BsgI-cleaved pcDNA3-hPXR or pcDNA3-FLAG-hPXR. .. Only the open reading frame of hPXR, but not pcDNA3 or the FLAG tag, had cleavage sites for PPUMI and BsgI.

    Sequencing:

    Article Title: A Phosphomimetic Mutation at Threonine-57 Abolishes Transactivation Activity and Alters Nuclear Localization Pattern of Human Pregnane X Receptor
    Article Snippet: Polymerase chain reaction (PCR) was performed using pcDNA3-hPXR as the template and specific mutant primers, with forward primers containing a mutation corresponding to either A or D and a PPUMI restriction enzyme cutting site and a reverse primer containing a BsgI restriction enzyme cutting site. .. The PCR product was cloned into a TOPO vector (Zero Blunt TOPO PCR Cloning Kit; Invitrogen), followed by cleaving the sequence-verified TOPO-PCR product for the desired mutations with PPUMI (New England Biolabs, Ipswich, MA) and BsgI (New England Biolabs), and ligating the resulting fragment into PPUMI- and BsgI-cleaved pcDNA3-hPXR or pcDNA3-FLAG-hPXR. .. Only the open reading frame of hPXR, but not pcDNA3 or the FLAG tag, had cleavage sites for PPUMI and BsgI.

    Purification:

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition
    Article Snippet: DNA was extracted by phenol-chloroform extraction and precipitated in an equal volume of isopropanol with 0.5 μL polyacryl carrier (Molecular Research Center, Cincinatti, OH). .. To analyze replication intermediates, the purified DNA was digested with DpnI, EcoRI, and EcoN1 (ori-proximal vectors; New England Biolabs) or DpnI, PpuMI, and SacII (ori-distal vectors; New England Biolabs) for 3 hours, followed by ethanol precipitation. .. DNA products were resuspended in Tris-EDTA and separated first through a 0.4% TBE agarose gel (1V/cm, 14 hr, room temperature, -EtBr) and second through a 1% TBE agarose gel (4V/cm, 6–8hr, 4˚C, +EtBr) ( ).

    Article Title: Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs
    Article Snippet: Total DNA was isolated from lung samples powdered with a mortar and pestle, by previously described methods ( , ). .. Purified DNA samples were digested with Ppu MI and Ahd I restriction enzymes (New England Biolabs, Beverly, MA) and used for further analyses. .. To search for oxidative damage to the mitochondrial genome, Southern blot analysis was performed as published previously with minor modifications ( ).

    Article Title: 6S RNA mimics B-form DNA to regulate Escherichia coli RNA polymerase
    Article Snippet: Oligos used for mutagenesis are available upon request. .. Plasmids were maxi-prepped (Qiagen), digested with PpuMI (NEB) and gel purified (Qiagen) for in vitro T7 RNAP transcription, or digested with SmaI (NEB) or EcoRV (NEB) and ethanol precipitated for in vitro T3 RNAP transcription. .. For cryoEM, footprinting, and transcription assays, a mutant T7 RNAP that dissociates more rapidly from the 3′-end of run-off transcription templates and therefore minimizes non-templated addition of nucleotides to the RNA transcript 3′-end (T7 RNAP*) was purified from plasmid pRC9 (a generous gift from W.T.

    Article Title: Mitochondrial DNA Damage Initiates Acute Lung Injury and Multi-Organ System Failure Evoked in Rats by Intra-tracheal Pseudomonas aeruginosa
    Article Snippet: At termination of ex vivo perfusion, lungs were snap-frozen in liquid N2 and stored at −80° C. Frozen lungs were then powdered with a mortar and pestle, total DNA was isolated, and oxidative mtDNA damage assessed using previously described methods ( , ). .. In brief, purified DNA samples were digested with the restriction enzymes PpuMI and AhdI (New England Biolabs). .. Digested samples were precipitated, dissolved in TE buffer, and quantified using a VersaFluor fluorometer (Bio-Rad) with a Quant-IT Picogreen dsDNA Assay kit (Molecular Probe).

    Ethanol Precipitation:

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition
    Article Snippet: DNA was extracted by phenol-chloroform extraction and precipitated in an equal volume of isopropanol with 0.5 μL polyacryl carrier (Molecular Research Center, Cincinatti, OH). .. To analyze replication intermediates, the purified DNA was digested with DpnI, EcoRI, and EcoN1 (ori-proximal vectors; New England Biolabs) or DpnI, PpuMI, and SacII (ori-distal vectors; New England Biolabs) for 3 hours, followed by ethanol precipitation. .. DNA products were resuspended in Tris-EDTA and separated first through a 0.4% TBE agarose gel (1V/cm, 14 hr, room temperature, -EtBr) and second through a 1% TBE agarose gel (4V/cm, 6–8hr, 4˚C, +EtBr) ( ).

    In Vitro:

    Article Title: 6S RNA mimics B-form DNA to regulate Escherichia coli RNA polymerase
    Article Snippet: Oligos used for mutagenesis are available upon request. .. Plasmids were maxi-prepped (Qiagen), digested with PpuMI (NEB) and gel purified (Qiagen) for in vitro T7 RNAP transcription, or digested with SmaI (NEB) or EcoRV (NEB) and ethanol precipitated for in vitro T3 RNAP transcription. .. For cryoEM, footprinting, and transcription assays, a mutant T7 RNAP that dissociates more rapidly from the 3′-end of run-off transcription templates and therefore minimizes non-templated addition of nucleotides to the RNA transcript 3′-end (T7 RNAP*) was purified from plasmid pRC9 (a generous gift from W.T.

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    New England Biolabs ppumi
    CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with <t>DpnI,</t> EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, <t>PpuMI,</t> and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .
    Ppumi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ppumi/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ppumi - by Bioz Stars, 2021-05
    93/100 stars
      Buy from Supplier

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    CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .

    Journal: Molecular cell

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition

    doi: 10.1016/j.molcel.2018.08.047

    Figure Lengend Snippet: CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .

    Article Snippet: To analyze replication intermediates, the purified DNA was digested with DpnI, EcoRI, and EcoN1 (ori-proximal vectors; New England Biolabs) or DpnI, PpuMI, and SacII (ori-distal vectors; New England Biolabs) for 3 hours, followed by ethanol precipitation.

    Techniques: DNA Synthesis, In Vitro, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Sequencing, Plasmid Preparation, Transfection, Isolation, Nucleic Acid Electrophoresis, Hybridization, Purification, Quantitation Assay