sacii  (New England Biolabs)


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    Name:
    SacII
    Description:
    SacII 10 000 units
    Catalog Number:
    r0157l
    Price:
    261
    Size:
    10 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs sacii
    SacII
    SacII 10 000 units
    https://www.bioz.com/result/sacii/product/New England Biolabs
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    sacii - by Bioz Stars, 2020-04
    99/100 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 "Diversity of Proteolytic Clostridium botulinum Strains, Determined by a Pulsed-Field Gel Electrophoresis Approach"

    Article Title: Diversity of Proteolytic Clostridium botulinum Strains, Determined by a Pulsed-Field Gel Electrophoresis Approach

    Journal:

    doi: 10.1128/AEM.71.3.1311-1317.2005

    Digestion patterns of five proteolytic C. botulinum strains, two of type A (ATCC 3502 and ATCC 19397), two of type B (FT 243 and 4B), and one of type F (ATCC 35415), using the rare-cutting restriction enzymes SacII and SmaI. The pulse time ramp was 1
    Figure Legend Snippet: Digestion patterns of five proteolytic C. botulinum strains, two of type A (ATCC 3502 and ATCC 19397), two of type B (FT 243 and 4B), and one of type F (ATCC 35415), using the rare-cutting restriction enzymes SacII and SmaI. The pulse time ramp was 1

    Techniques Used:

    3) Product Images from "Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification"

    Article Title: Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031817

    Colony PCR analysis of the progeny of two primary clones (A.1 and B.1) originating from the Kunkel mutagenesis of CDR-H3 loop. (I) PCR with wild-type-template- specific primer A1 and pAK400rev. 900 bp product is formed, if template is present. (II) SacII digestion analysis of PCR products (940 bp) primed with WO375 and B1 that hybridize outside the CDR-H3 loop. Both wild-type and mutated DNA is amplified, but only the wild-type DNA is cut to 814 bp and 126 bp fragments. (A.2a–d) Daughter colonies of A.1; (B.2a–d) Daughter colonies of B.1; (−) SacII resistant control, (+) SacII sensitive control, (L) 1 kb Fermentas DNA Ladder bands 1500–500 bp.
    Figure Legend Snippet: Colony PCR analysis of the progeny of two primary clones (A.1 and B.1) originating from the Kunkel mutagenesis of CDR-H3 loop. (I) PCR with wild-type-template- specific primer A1 and pAK400rev. 900 bp product is formed, if template is present. (II) SacII digestion analysis of PCR products (940 bp) primed with WO375 and B1 that hybridize outside the CDR-H3 loop. Both wild-type and mutated DNA is amplified, but only the wild-type DNA is cut to 814 bp and 126 bp fragments. (A.2a–d) Daughter colonies of A.1; (B.2a–d) Daughter colonies of B.1; (−) SacII resistant control, (+) SacII sensitive control, (L) 1 kb Fermentas DNA Ladder bands 1500–500 bp.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Mutagenesis, Amplification

    Related Articles

    Transduction:

    Article Title: Characterization of SSR42, a Novel Virulence Factor Regulatory RNA That Contributes to the Pathogenesis of a Staphylococcus aureus USA300 Representative
    Article Snippet: Following PCR amplification, the products were gel purified and digested with the SacII restriction enzyme (New England BioLabs, Ipswich, MA) and then ligated using T4 DNA ligase (Invitrogen). .. The pKOR1::SSR42 vector was subsequently transferred to S. aureus strain UAMS-1 or LAC via φ11-mediated transduction.

    Clone Assay:

    Article Title: In vitro functional assessment of natural HIV-1 group M Vpu sequences using a universal priming approach
    Article Snippet: Paragraph title: 2.3 Cloning and Vpu sequence verification ... Vpu amplicons were verified by gel electrophoresis, gel extracted (GeneJET; ThermoFisher Scientific), digested with AscI and SacII (New England Biolabs) and ligated at a 3:1 molar ratio with linearized pSel-GFP and pSelRRE -GFP vectors.

    Article Title: Cloning of a functional mannose-6-phosphate reductase (M6PR) gene homolog from Egyptian celery plants (Apium graveolens): overexpression in non-mannitol producing plants resulted in mannitol accumulation in transgenic individuals
    Article Snippet: .. Plasmid DNA isolated from the selected clones was digested with Sac ΙΙ (NEB, Cat #R0157S) enzyme, and Klenow fragment enzyme (NEB, Cat #M0210S) was employed to create blunt ends, then the linear plasmids were digested with Sal Ι (NEB, Cat #R0138S) enzyme to release the gene from the pGEM T-easy vector. .. The product was run on 1% agarose gel and the full-length gene band was purified.

    Article Title: A Metalloprotease Homolog Venom Protein From a Parasitoid Wasp Suppresses the Toll Pathway in Host Hemocytes
    Article Snippet: .. SacI (NEB) and SacII (NEB) restriction sites were incorporated into the primers for directional cloning of the gene. .. PCR-amplified gene fragments were first cloned into PGEM-Teasy (Promega) and then cloned into the expression vector pIZT/V5-His (Invitrogen).

    Article Title: Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification
    Article Snippet: .. SacII digestion analysis of CDR-H3 region mutants The presence of unmutated DNA was also analysed from cm-plate clones by digesting 2 µl of the WO375-B1 amplicons with 1 U SacII (NEB) for 4 h at 37°C in the manufacturer's recommended buffer. .. In addition, a phagemid pEB07-scFv[STOP] containing the SacII site was amplified with the same WO375-B1 primers and used as a positive control to assess the completeness of the SacII-digestions. pEB07-scFv[NONSTOP] lacking the SacII site was used as a negative control.

    Centrifugation:

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition
    Article Snippet: Chromosomal DNA was precipitated by incubation of lysate overnight in 5M NaCl followed by centrifugation at 27, 200 xg for 50 minutes at 4°C. .. 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.

    Amplification:

    Article Title: A Metalloprotease Homolog Venom Protein From a Parasitoid Wasp Suppresses the Toll Pathway in Host Hemocytes
    Article Snippet: Transfection of IOZCAS-Ha-I and cell extracts The VRF1151−483 was PCR amplified with venom apparatus cDNA as the template and the primers listed in Supplementary Table . .. SacI (NEB) and SacII (NEB) restriction sites were incorporated into the primers for directional cloning of the gene.

    Article Title: Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection
    Article Snippet: The amplified fragments were purified from PCR mixtures with the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA), following the manufacturer's instructions. .. The purified downstream homology region and the pSFS2 plasmid were digested with the combination of SacII/SacI restriction enzymes (New England Biolabs, Ipswich, MA, USA) and then ligated together with T4 DNA ligase (Promega) in order to create plasmid p3ALS7 ( Table S3 ).

    Article Title: Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification
    Article Snippet: SacII digestion analysis of CDR-H3 region mutants The presence of unmutated DNA was also analysed from cm-plate clones by digesting 2 µl of the WO375-B1 amplicons with 1 U SacII (NEB) for 4 h at 37°C in the manufacturer's recommended buffer. .. In addition, a phagemid pEB07-scFv[STOP] containing the SacII site was amplified with the same WO375-B1 primers and used as a positive control to assess the completeness of the SacII-digestions. pEB07-scFv[NONSTOP] lacking the SacII site was used as a negative control.

    Article Title: Characterization of SSR42, a Novel Virulence Factor Regulatory RNA That Contributes to the Pathogenesis of a Staphylococcus aureus USA300 Representative
    Article Snippet: .. Following PCR amplification, the products were gel purified and digested with the SacII restriction enzyme (New England BioLabs, Ipswich, MA) and then ligated using T4 DNA ligase (Invitrogen). .. The ligated products containing a deletion of SSR42 were then inserted into the pKOR1 vector using in vitro recombination via BP Clonase (Invitrogen), propagated in Escherichia coli strain DH5α, and purified using Plasmid Miniprep kits (Qiagen, Valencia, CA).

    Binding Assay:

    Article Title: Basement membrane collagen IV: Isolation of functional domains
    Article Snippet: Application of this technology has led to the discovery of the target antigen for human autoantibodies in Goodpasture’s disease, mapping of pathogenic epitopes within the α3NC1 and α5NC1 domains and identification of critical residues for antibody binding ( ; ; ; ). .. Phusion DNA polymerase; ApaI, NheI, and SacII restriction enzymes; T4 DNA ligase (New England BioLabs).

    Positive Control:

    Article Title: Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification
    Article Snippet: SacII digestion analysis of CDR-H3 region mutants The presence of unmutated DNA was also analysed from cm-plate clones by digesting 2 µl of the WO375-B1 amplicons with 1 U SacII (NEB) for 4 h at 37°C in the manufacturer's recommended buffer. .. In addition, a phagemid pEB07-scFv[STOP] containing the SacII site was amplified with the same WO375-B1 primers and used as a positive control to assess the completeness of the SacII-digestions. pEB07-scFv[NONSTOP] lacking the SacII site was used as a negative control.

    Electrophoresis:

    Article Title: Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei
    Article Snippet: Fifteen micrograms of chromosomal DNA was digested with 30 U of SacII (New England BioLabs, Ipswich, MA, USA). .. The resulting DNA fragments were separated by electrophoresis on an 0.8% (wt/vol) agarose gel, denatured in 0.4 M NaOH, and transferred by capillary force onto a Biodyne B 0.45-μm nylon membrane (Pall Corporation, Port Washington, NY, USA) using 10× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate).

    Ex Vivo:

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition
    Article Snippet: Paragraph title: Ex vivo assay: ... 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.

    Incubation:

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition
    Article Snippet: Resulting supernatants were incubated in the presence of ~0.1 mg/mL proteinase K for 2–3 hours at 55°C. .. 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.

    Expressing:

    Article Title: Basement membrane collagen IV: Isolation of functional domains
    Article Snippet: Paragraph title: 5 RECOMBINANT EXPRESSION OF COLLAGEN IV FRAGMENTS ... Phusion DNA polymerase; ApaI, NheI, and SacII restriction enzymes; T4 DNA ligase (New England BioLabs).

    Article Title: Oligodeoxynucleotides Can Transiently Up- and Downregulate CHS Gene Expression in Flax by Changing DNA Methylation in a Sequence-Specific Manner
    Article Snippet: .. DNA accessibility for restriction The DNA accessibility for restriction was established in the control and W.92 transgenic plants with stabilized modulation of CHS gene expression (Lorenc-Kukula et al., ) by using restriction enzymes AatII, PvuI, SacII, and Sau3AI (New England Biolabs). .. The first three enzymes were selected on the basis of the predicted cut sites in the vicinity of the +1,273 -CCGG- motif (NEB cutter V2.0).

    Article Title: Cloning of a functional mannose-6-phosphate reductase (M6PR) gene homolog from Egyptian celery plants (Apium graveolens): overexpression in non-mannitol producing plants resulted in mannitol accumulation in transgenic individuals
    Article Snippet: Paragraph title: Cloning into plant expression vector ... Plasmid DNA isolated from the selected clones was digested with Sac ΙΙ (NEB, Cat #R0157S) enzyme, and Klenow fragment enzyme (NEB, Cat #M0210S) was employed to create blunt ends, then the linear plasmids were digested with Sal Ι (NEB, Cat #R0138S) enzyme to release the gene from the pGEM T-easy vector.

    Article Title: A Metalloprotease Homolog Venom Protein From a Parasitoid Wasp Suppresses the Toll Pathway in Host Hemocytes
    Article Snippet: SacI (NEB) and SacII (NEB) restriction sites were incorporated into the primers for directional cloning of the gene. .. PCR-amplified gene fragments were first cloned into PGEM-Teasy (Promega) and then cloned into the expression vector pIZT/V5-His (Invitrogen).

    Modification:

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition
    Article Snippet: For all experiments, DNA was isolated 48 hours post-transfection using a modified Hirt method, as described ( ). .. 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.

    Transformation Assay:

    Article Title: In vitro functional assessment of natural HIV-1 group M Vpu sequences using a universal priming approach
    Article Snippet: Vpu amplicons were verified by gel electrophoresis, gel extracted (GeneJET; ThermoFisher Scientific), digested with AscI and SacII (New England Biolabs) and ligated at a 3:1 molar ratio with linearized pSel-GFP and pSelRRE -GFP vectors. .. Ligations were transformed into E. cloni ).

    Hybridization:

    Article Title: Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei
    Article Snippet: Fifteen micrograms of chromosomal DNA was digested with 30 U of SacII (New England BioLabs, Ipswich, MA, USA). .. A 1.5-μg quantity of biotinylated DNA probe was used for hybridization at 65°C overnight.

    Transfection:

    Article Title: A Metalloprotease Homolog Venom Protein From a Parasitoid Wasp Suppresses the Toll Pathway in Host Hemocytes
    Article Snippet: Paragraph title: Transfection of IOZCAS-Ha-I and cell extracts ... SacI (NEB) and SacII (NEB) restriction sites were incorporated into the primers for directional cloning of the gene.

    Southern Blot:

    Article Title: Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei
    Article Snippet: Paragraph title: Southern blot analysis. ... Fifteen micrograms of chromosomal DNA was digested with 30 U of SacII (New England BioLabs, Ipswich, MA, USA).

    Ligation:

    Article Title: Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection
    Article Snippet: Plasmid pSFS2 was used as backbone for the first round of ligation, producing plasmid p3ALS7 ( Table S1 ). .. The purified downstream homology region and the pSFS2 plasmid were digested with the combination of SacII/SacI restriction enzymes (New England Biolabs, Ipswich, MA, USA) and then ligated together with T4 DNA ligase (Promega) in order to create plasmid p3ALS7 ( Table S3 ).

    Sequencing:

    Article Title: In vitro functional assessment of natural HIV-1 group M Vpu sequences using a universal priming approach
    Article Snippet: Paragraph title: 2.3 Cloning and Vpu sequence verification ... Vpu amplicons were verified by gel electrophoresis, gel extracted (GeneJET; ThermoFisher Scientific), digested with AscI and SacII (New England Biolabs) and ligated at a 3:1 molar ratio with linearized pSel-GFP and pSelRRE -GFP vectors.

    Article Title: Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection
    Article Snippet: The purified downstream homology region and the pSFS2 plasmid were digested with the combination of SacII/SacI restriction enzymes (New England Biolabs, Ipswich, MA, USA) and then ligated together with T4 DNA ligase (Promega) in order to create plasmid p3ALS7 ( Table S3 ). .. Plasmid p35ALS7 was sequenced by Eurofins MWG Operon, using M13 forward sequencing primer (−20), 17-mer and M13 reverse sequencing primer (−26), 17-mer and primer But237 ( Table S2 ).

    Sonication:

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition
    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. .. After UV crosslinking, pre-hybridization of the membranes was carried out by incubation with a 0.25M sodium phosphate (pH 7.2)/ 7% SDS/ 100 μg/ml sonicated sperm DNA buffer, and incubation at 65˚C, ≥2 hrs.

    Recombinant:

    Article Title: Basement membrane collagen IV: Isolation of functional domains
    Article Snippet: Paragraph title: 5 RECOMBINANT EXPRESSION OF COLLAGEN IV FRAGMENTS ... Phusion DNA polymerase; ApaI, NheI, and SacII restriction enzymes; T4 DNA ligase (New England BioLabs).

    DNA Extraction:

    Article Title: Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection
    Article Snippet: Plasmidic DNA (pDNA) was isolated with small-scale plasmid DNA isolation (Miniprep) assay. .. The purified downstream homology region and the pSFS2 plasmid were digested with the combination of SacII/SacI restriction enzymes (New England Biolabs, Ipswich, MA, USA) and then ligated together with T4 DNA ligase (Promega) in order to create plasmid p3ALS7 ( Table S3 ).

    Nucleic Acid Electrophoresis:

    Article Title: In vitro functional assessment of natural HIV-1 group M Vpu sequences using a universal priming approach
    Article Snippet: .. Vpu amplicons were verified by gel electrophoresis, gel extracted (GeneJET; ThermoFisher Scientific), digested with AscI and SacII (New England Biolabs) and ligated at a 3:1 molar ratio with linearized pSel-GFP and pSelRRE -GFP vectors. .. Ligations were transformed into E. cloni ).

    Mutagenesis:

    Article Title: Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection
    Article Snippet: Paragraph title: Construction of the als7△/als7△ null mutant strain ... The purified downstream homology region and the pSFS2 plasmid were digested with the combination of SacII/SacI restriction enzymes (New England Biolabs, Ipswich, MA, USA) and then ligated together with T4 DNA ligase (Promega) in order to create plasmid p3ALS7 ( Table S3 ).

    Isolation:

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition
    Article Snippet: For all experiments, DNA was isolated 48 hours post-transfection using a modified Hirt method, as described ( ). .. 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.

    Article Title: Oligodeoxynucleotides Can Transiently Up- and Downregulate CHS Gene Expression in Flax by Changing DNA Methylation in a Sequence-Specific Manner
    Article Snippet: DNA accessibility for restriction The DNA accessibility for restriction was established in the control and W.92 transgenic plants with stabilized modulation of CHS gene expression (Lorenc-Kukula et al., ) by using restriction enzymes AatII, PvuI, SacII, and Sau3AI (New England Biolabs). .. The genomic DNA was isolated using the DNeasy Plant Mini Kit (Qiagen) according to the manufacturer's protocol.

    Article Title: Cloning of a functional mannose-6-phosphate reductase (M6PR) gene homolog from Egyptian celery plants (Apium graveolens): overexpression in non-mannitol producing plants resulted in mannitol accumulation in transgenic individuals
    Article Snippet: .. Plasmid DNA isolated from the selected clones was digested with Sac ΙΙ (NEB, Cat #R0157S) enzyme, and Klenow fragment enzyme (NEB, Cat #M0210S) was employed to create blunt ends, then the linear plasmids were digested with Sal Ι (NEB, Cat #R0138S) enzyme to release the gene from the pGEM T-easy vector. .. The product was run on 1% agarose gel and the full-length gene band was purified.

    Article Title: Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection
    Article Snippet: Plasmidic DNA (pDNA) was isolated with small-scale plasmid DNA isolation (Miniprep) assay. .. The purified downstream homology region and the pSFS2 plasmid were digested with the combination of SacII/SacI restriction enzymes (New England Biolabs, Ipswich, MA, USA) and then ligated together with T4 DNA ligase (Promega) in order to create plasmid p3ALS7 ( Table S3 ).

    Labeling:

    Article Title: Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei
    Article Snippet: Fifteen micrograms of chromosomal DNA was digested with 30 U of SacII (New England BioLabs, Ipswich, MA, USA). .. Labeling of the probe was performed by using the Klenow fragment (exo− ) (Thermo Fisher Scientific), random hexamer primers, and biotin-11-dUTP (Jena Bioscience, Jena, Germany).

    Purification:

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition
    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. .. 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: Basement membrane collagen IV: Isolation of functional domains
    Article Snippet: Recombinant proteins fused with an amino terminal FLAG tag have been purified from the conditioned medium using affinity chromatography on anti-FLAG agarose and elution with the FLAG peptide. .. Phusion DNA polymerase; ApaI, NheI, and SacII restriction enzymes; T4 DNA ligase (New England BioLabs).

    Article Title: Cloning of a functional mannose-6-phosphate reductase (M6PR) gene homolog from Egyptian celery plants (Apium graveolens): overexpression in non-mannitol producing plants resulted in mannitol accumulation in transgenic individuals
    Article Snippet: Plasmid DNA isolated from the selected clones was digested with Sac ΙΙ (NEB, Cat #R0157S) enzyme, and Klenow fragment enzyme (NEB, Cat #M0210S) was employed to create blunt ends, then the linear plasmids were digested with Sal Ι (NEB, Cat #R0138S) enzyme to release the gene from the pGEM T-easy vector. .. The product was run on 1% agarose gel and the full-length gene band was purified.

    Article Title: Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection
    Article Snippet: .. The purified downstream homology region and the pSFS2 plasmid were digested with the combination of SacII/SacI restriction enzymes (New England Biolabs, Ipswich, MA, USA) and then ligated together with T4 DNA ligase (Promega) in order to create plasmid p3ALS7 ( Table S3 ). .. This plasmid was propagated in E. coli DH10β cells made competent by calcium chloride method. p3ALS7 and the purified upstream homology region of CpALS7 were digested with ApaI and XhoI restriction enzymes (New England Biolabs).

    Article Title: Characterization of SSR42, a Novel Virulence Factor Regulatory RNA That Contributes to the Pathogenesis of a Staphylococcus aureus USA300 Representative
    Article Snippet: .. Following PCR amplification, the products were gel purified and digested with the SacII restriction enzyme (New England BioLabs, Ipswich, MA) and then ligated using T4 DNA ligase (Invitrogen). .. The ligated products containing a deletion of SSR42 were then inserted into the pKOR1 vector using in vitro recombination via BP Clonase (Invitrogen), propagated in Escherichia coli strain DH5α, and purified using Plasmid Miniprep kits (Qiagen, Valencia, CA).

    Polymerase Chain Reaction:

    Article Title: Basement membrane collagen IV: Isolation of functional domains
    Article Snippet: PCR thermocycler (Mastercycler, Eppendorf). .. Phusion DNA polymerase; ApaI, NheI, and SacII restriction enzymes; T4 DNA ligase (New England BioLabs).

    Article Title: A Metalloprotease Homolog Venom Protein From a Parasitoid Wasp Suppresses the Toll Pathway in Host Hemocytes
    Article Snippet: Transfection of IOZCAS-Ha-I and cell extracts The VRF1151−483 was PCR amplified with venom apparatus cDNA as the template and the primers listed in Supplementary Table . .. SacI (NEB) and SacII (NEB) restriction sites were incorporated into the primers for directional cloning of the gene.

    Article Title: Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection
    Article Snippet: The amplified fragments were purified from PCR mixtures with the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA), following the manufacturer's instructions. .. The purified downstream homology region and the pSFS2 plasmid were digested with the combination of SacII/SacI restriction enzymes (New England Biolabs, Ipswich, MA, USA) and then ligated together with T4 DNA ligase (Promega) in order to create plasmid p3ALS7 ( Table S3 ).

    Article Title: Characterization of SSR42, a Novel Virulence Factor Regulatory RNA That Contributes to the Pathogenesis of a Staphylococcus aureus USA300 Representative
    Article Snippet: .. Following PCR amplification, the products were gel purified and digested with the SacII restriction enzyme (New England BioLabs, Ipswich, MA) and then ligated using T4 DNA ligase (Invitrogen). .. The ligated products containing a deletion of SSR42 were then inserted into the pKOR1 vector using in vitro recombination via BP Clonase (Invitrogen), propagated in Escherichia coli strain DH5α, and purified using Plasmid Miniprep kits (Qiagen, Valencia, CA).

    Lysis:

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition
    Article Snippet: Briefly, adherent cells were washed with 1M Tris-buffered saline (50 mM Tris-HCl, pH 7.0 and 150 mM NaCl) followed by lysis in 50 mM Tris–HCl pH 7.0, 20 mM EDTA, 10 mM NaCl, 10% SDS, 0.2 mg/ml proteinase K (Sigma-Aldrich). .. 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.

    Plasmid Preparation:

    Article Title: Basement membrane collagen IV: Isolation of functional domains
    Article Snippet: Human kidney cDNA (Clontech). pRc-X expression vector ( ). .. Phusion DNA polymerase; ApaI, NheI, and SacII restriction enzymes; T4 DNA ligase (New England BioLabs).

    Article Title: Cloning of a functional mannose-6-phosphate reductase (M6PR) gene homolog from Egyptian celery plants (Apium graveolens): overexpression in non-mannitol producing plants resulted in mannitol accumulation in transgenic individuals
    Article Snippet: .. Plasmid DNA isolated from the selected clones was digested with Sac ΙΙ (NEB, Cat #R0157S) enzyme, and Klenow fragment enzyme (NEB, Cat #M0210S) was employed to create blunt ends, then the linear plasmids were digested with Sal Ι (NEB, Cat #R0138S) enzyme to release the gene from the pGEM T-easy vector. .. The product was run on 1% agarose gel and the full-length gene band was purified.

    Article Title: A Metalloprotease Homolog Venom Protein From a Parasitoid Wasp Suppresses the Toll Pathway in Host Hemocytes
    Article Snippet: SacI (NEB) and SacII (NEB) restriction sites were incorporated into the primers for directional cloning of the gene. .. PCR-amplified gene fragments were first cloned into PGEM-Teasy (Promega) and then cloned into the expression vector pIZT/V5-His (Invitrogen).

    Article Title: Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection
    Article Snippet: .. The purified downstream homology region and the pSFS2 plasmid were digested with the combination of SacII/SacI restriction enzymes (New England Biolabs, Ipswich, MA, USA) and then ligated together with T4 DNA ligase (Promega) in order to create plasmid p3ALS7 ( Table S3 ). .. This plasmid was propagated in E. coli DH10β cells made competent by calcium chloride method. p3ALS7 and the purified upstream homology region of CpALS7 were digested with ApaI and XhoI restriction enzymes (New England Biolabs).

    Article Title: Characterization of SSR42, a Novel Virulence Factor Regulatory RNA That Contributes to the Pathogenesis of a Staphylococcus aureus USA300 Representative
    Article Snippet: SSR42 was deleted from the S. aureus UAMS-1 and LAC chromosomes by using the pKOR1 allelic replacement vector as previously described ( ). .. Following PCR amplification, the products were gel purified and digested with the SacII restriction enzyme (New England BioLabs, Ipswich, MA) and then ligated using T4 DNA ligase (Invitrogen).

    Negative Control:

    Article Title: Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification
    Article Snippet: SacII digestion analysis of CDR-H3 region mutants The presence of unmutated DNA was also analysed from cm-plate clones by digesting 2 µl of the WO375-B1 amplicons with 1 U SacII (NEB) for 4 h at 37°C in the manufacturer's recommended buffer. .. In addition, a phagemid pEB07-scFv[STOP] containing the SacII site was amplified with the same WO375-B1 primers and used as a positive control to assess the completeness of the SacII-digestions. pEB07-scFv[NONSTOP] lacking the SacII site was used as a negative control.

    Affinity Chromatography:

    Article Title: Basement membrane collagen IV: Isolation of functional domains
    Article Snippet: Recombinant proteins fused with an amino terminal FLAG tag have been purified from the conditioned medium using affinity chromatography on anti-FLAG agarose and elution with the FLAG peptide. .. Phusion DNA polymerase; ApaI, NheI, and SacII restriction enzymes; T4 DNA ligase (New England BioLabs).

    Agarose Gel Electrophoresis:

    Article Title: Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei
    Article Snippet: Fifteen micrograms of chromosomal DNA was digested with 30 U of SacII (New England BioLabs, Ipswich, MA, USA). .. The resulting DNA fragments were separated by electrophoresis on an 0.8% (wt/vol) agarose gel, denatured in 0.4 M NaOH, and transferred by capillary force onto a Biodyne B 0.45-μm nylon membrane (Pall Corporation, Port Washington, NY, USA) using 10× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate).

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition
    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. .. 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: Cloning of a functional mannose-6-phosphate reductase (M6PR) gene homolog from Egyptian celery plants (Apium graveolens): overexpression in non-mannitol producing plants resulted in mannitol accumulation in transgenic individuals
    Article Snippet: Plasmid DNA isolated from the selected clones was digested with Sac ΙΙ (NEB, Cat #R0157S) enzyme, and Klenow fragment enzyme (NEB, Cat #M0210S) was employed to create blunt ends, then the linear plasmids were digested with Sal Ι (NEB, Cat #R0138S) enzyme to release the gene from the pGEM T-easy vector. .. The product was run on 1% agarose gel and the full-length gene band was purified.

    Article Title: Diversity of Proteolytic Clostridium botulinum Strains, Determined by a Pulsed-Field Gel Electrophoresis Approach
    Article Snippet: Nine rare-cutting restriction enzymes, ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI (New England Biolabs), were chosen for testing the cleavage of DNA of proteolytic C. botulinum . .. Samples were electrophoresed at 8°C through a 1% (wt/vol) agarose gel (Seakem Gold agarose; BMA, Rockland, Maine) in 0.5× Tris-borate-EDTA buffer (Amresco, Solon, Ohio) at 200 V for 20 to 22 h with a Gene Navigator system (Pharmacia, Uppsala, Sweden) with a hexagonal electrode.

    In Vitro:

    Article Title: Characterization of SSR42, a Novel Virulence Factor Regulatory RNA That Contributes to the Pathogenesis of a Staphylococcus aureus USA300 Representative
    Article Snippet: Following PCR amplification, the products were gel purified and digested with the SacII restriction enzyme (New England BioLabs, Ipswich, MA) and then ligated using T4 DNA ligase (Invitrogen). .. The ligated products containing a deletion of SSR42 were then inserted into the pKOR1 vector using in vitro recombination via BP Clonase (Invitrogen), propagated in Escherichia coli strain DH5α, and purified using Plasmid Miniprep kits (Qiagen, Valencia, CA).

    Transgenic Assay:

    Article Title: Oligodeoxynucleotides Can Transiently Up- and Downregulate CHS Gene Expression in Flax by Changing DNA Methylation in a Sequence-Specific Manner
    Article Snippet: .. DNA accessibility for restriction The DNA accessibility for restriction was established in the control and W.92 transgenic plants with stabilized modulation of CHS gene expression (Lorenc-Kukula et al., ) by using restriction enzymes AatII, PvuI, SacII, and Sau3AI (New England Biolabs). .. The first three enzymes were selected on the basis of the predicted cut sites in the vicinity of the +1,273 -CCGG- motif (NEB cutter V2.0).

    Ethanol Precipitation:

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

    Random Hexamer Labeling:

    Article Title: Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei
    Article Snippet: Fifteen micrograms of chromosomal DNA was digested with 30 U of SacII (New England BioLabs, Ipswich, MA, USA). .. Labeling of the probe was performed by using the Klenow fragment (exo− ) (Thermo Fisher Scientific), random hexamer primers, and biotin-11-dUTP (Jena Bioscience, Jena, Germany).

    Knock-Out:

    Article Title: Characterization of SSR42, a Novel Virulence Factor Regulatory RNA That Contributes to the Pathogenesis of a Staphylococcus aureus USA300 Representative
    Article Snippet: Following PCR amplification, the products were gel purified and digested with the SacII restriction enzyme (New England BioLabs, Ipswich, MA) and then ligated using T4 DNA ligase (Invitrogen). .. The resultant pKOR1::SSR42 knockout vector was then electroporated into competent, restriction-deficient S. aureus RN4220 (1.8 kV, 500 Ω, 10 μF), and transformants were selected for on TSB agar plates in the presence of chloramphenicol (10 μg ml−1 ) at 30°C.

    Produced:

    Article Title: Cloning of a functional mannose-6-phosphate reductase (M6PR) gene homolog from Egyptian celery plants (Apium graveolens): overexpression in non-mannitol producing plants resulted in mannitol accumulation in transgenic individuals
    Article Snippet: Plasmid DNA isolated from the selected clones was digested with Sac ΙΙ (NEB, Cat #R0157S) enzyme, and Klenow fragment enzyme (NEB, Cat #M0210S) was employed to create blunt ends, then the linear plasmids were digested with Sal Ι (NEB, Cat #R0138S) enzyme to release the gene from the pGEM T-easy vector. .. Blunt ends were produced by Klenow enzyme.

    FLAG-tag:

    Article Title: Basement membrane collagen IV: Isolation of functional domains
    Article Snippet: Recombinant proteins fused with an amino terminal FLAG tag have been purified from the conditioned medium using affinity chromatography on anti-FLAG agarose and elution with the FLAG peptide. .. Phusion DNA polymerase; ApaI, NheI, and SacII restriction enzymes; T4 DNA ligase (New England BioLabs).

    Gel Extraction:

    Article Title: Basement membrane collagen IV: Isolation of functional domains
    Article Snippet: Phusion DNA polymerase; ApaI, NheI, and SacII restriction enzymes; T4 DNA ligase (New England BioLabs). .. NucleoSpin gel extraction and NucleoSpin plasmid purification kits (Macherey-Nagel).

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    New England Biolabs sacii digestion analysis
    Colony PCR analysis of the progeny of two primary clones (A.1 and B.1) originating from the Kunkel mutagenesis of <t>CDR-H3</t> loop. (I) PCR with wild-type-template- specific primer A1 and pAK400rev. 900 bp product is formed, if template is present. (II) <t>SacII</t> digestion analysis of PCR products (940 bp) primed with WO375 and B1 that hybridize outside the CDR-H3 loop. Both wild-type and mutated DNA is amplified, but only the wild-type DNA is cut to 814 bp and 126 bp fragments. (A.2a–d) Daughter colonies of A.1; (B.2a–d) Daughter colonies of B.1; (−) SacII resistant control, (+) SacII sensitive control, (L) 1 kb Fermentas DNA Ladder bands 1500–500 bp.
    Sacii Digestion Analysis, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Colony PCR analysis of the progeny of two primary clones (A.1 and B.1) originating from the Kunkel mutagenesis of CDR-H3 loop. (I) PCR with wild-type-template- specific primer A1 and pAK400rev. 900 bp product is formed, if template is present. (II) SacII digestion analysis of PCR products (940 bp) primed with WO375 and B1 that hybridize outside the CDR-H3 loop. Both wild-type and mutated DNA is amplified, but only the wild-type DNA is cut to 814 bp and 126 bp fragments. (A.2a–d) Daughter colonies of A.1; (B.2a–d) Daughter colonies of B.1; (−) SacII resistant control, (+) SacII sensitive control, (L) 1 kb Fermentas DNA Ladder bands 1500–500 bp.

    Journal: PLoS ONE

    Article Title: Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

    doi: 10.1371/journal.pone.0031817

    Figure Lengend Snippet: Colony PCR analysis of the progeny of two primary clones (A.1 and B.1) originating from the Kunkel mutagenesis of CDR-H3 loop. (I) PCR with wild-type-template- specific primer A1 and pAK400rev. 900 bp product is formed, if template is present. (II) SacII digestion analysis of PCR products (940 bp) primed with WO375 and B1 that hybridize outside the CDR-H3 loop. Both wild-type and mutated DNA is amplified, but only the wild-type DNA is cut to 814 bp and 126 bp fragments. (A.2a–d) Daughter colonies of A.1; (B.2a–d) Daughter colonies of B.1; (−) SacII resistant control, (+) SacII sensitive control, (L) 1 kb Fermentas DNA Ladder bands 1500–500 bp.

    Article Snippet: SacII digestion analysis of CDR-H3 region mutants The presence of unmutated DNA was also analysed from cm-plate clones by digesting 2 µl of the WO375-B1 amplicons with 1 U SacII (NEB) for 4 h at 37°C in the manufacturer's recommended buffer.

    Techniques: Polymerase Chain Reaction, Clone Assay, Mutagenesis, Amplification

    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

    Expression of recombinant α1–α6 NC1 domains. (A) Map of the pRc-X expression vector showing positions of BM40 signal peptide, FLAG tag and NheI, ApaI/SacII sites used for cloning human NC1 domains. (B) SDS-PAGE of purified α1–α6 NC1 proteins (2 µg/lane). Small variations of MW are due to the presence of short additional Gly-X-Y repeats from collagenous domain in the α1, α3, α4, and α5 NC1s.

    Journal: Methods in cell biology

    Article Title: Basement membrane collagen IV: Isolation of functional domains

    doi: 10.1016/bs.mcb.2017.08.010

    Figure Lengend Snippet: Expression of recombinant α1–α6 NC1 domains. (A) Map of the pRc-X expression vector showing positions of BM40 signal peptide, FLAG tag and NheI, ApaI/SacII sites used for cloning human NC1 domains. (B) SDS-PAGE of purified α1–α6 NC1 proteins (2 µg/lane). Small variations of MW are due to the presence of short additional Gly-X-Y repeats from collagenous domain in the α1, α3, α4, and α5 NC1s.

    Article Snippet: Phusion DNA polymerase; ApaI, NheI, and SacII restriction enzymes; T4 DNA ligase (New England BioLabs).

    Techniques: Expressing, Recombinant, Plasmid Preparation, FLAG-tag, Clone Assay, SDS Page, Purification

    Digestion patterns of five proteolytic C. botulinum strains, two of type A (ATCC 3502 and ATCC 19397), two of type B (FT 243 and 4B), and one of type F (ATCC 35415), using the rare-cutting restriction enzymes SacII and SmaI. The pulse time ramp was 1

    Journal:

    Article Title: Diversity of Proteolytic Clostridium botulinum Strains, Determined by a Pulsed-Field Gel Electrophoresis Approach

    doi: 10.1128/AEM.71.3.1311-1317.2005

    Figure Lengend Snippet: Digestion patterns of five proteolytic C. botulinum strains, two of type A (ATCC 3502 and ATCC 19397), two of type B (FT 243 and 4B), and one of type F (ATCC 35415), using the rare-cutting restriction enzymes SacII and SmaI. The pulse time ramp was 1

    Article Snippet: Nine rare-cutting restriction enzymes, ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI (New England Biolabs), were chosen for testing the cleavage of DNA of proteolytic C. botulinum .

    Techniques: