sacii  (New England Biolabs)


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    Name:
    SacII
    Description:
    SacII 10 000 units
    Catalog Number:
    R0157L
    Price:
    261
    Category:
    Restriction Enzymes
    Size:
    10 000 units
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    New England Biolabs sacii
    SacII
    SacII 10 000 units
    https://www.bioz.com/result/sacii/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sacii - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "Age-dependent ribosomal DNA variations and their effect on cellular function in mammalian cells"

    Article Title: Age-dependent ribosomal DNA variations and their effect on cellular function in mammalian cells

    Journal: bioRxiv

    doi: 10.1101/2020.07.10.196840

    rDNA methylation in old and young bone marrow cells rDNA methylation was detected by digestion with a methylation sensitive enzyme SacII. ( A ) Position of the probe for Southern blot analysis in ( BC ) and recognition sites for BamHI and SacII. ( BC ) Ratio of methylated rDNA copies in young and old mice. (Top panel) Southern analysis to detect the ratio of undigested band by SacII. The positions of undigested bands are indicated by arrowheads on the left-hand side of the panels. (Middle panel) Detection of the SWI5 gene as a loading control. A single copy gene SWI5 was detected on the same filter used in the upper panel. (Bottom panel) Analysis of rDNA that failed to digest with SacII. The signal intensities of the undigested rDNA (SacII+) and non-digested (SacII-) bands were measured and the ratios plotted. The black dots show the results for the lower bands (black arrowhead, in the Top panel) and the blue dots are for the upper bands (blue arrowhead in the Top panel). ID# is the identification number of individual mice that were used to isolate the bone marrow cells (same as Figure 2 ). p values are calculated from the average of young and old mice.
    Figure Legend Snippet: rDNA methylation in old and young bone marrow cells rDNA methylation was detected by digestion with a methylation sensitive enzyme SacII. ( A ) Position of the probe for Southern blot analysis in ( BC ) and recognition sites for BamHI and SacII. ( BC ) Ratio of methylated rDNA copies in young and old mice. (Top panel) Southern analysis to detect the ratio of undigested band by SacII. The positions of undigested bands are indicated by arrowheads on the left-hand side of the panels. (Middle panel) Detection of the SWI5 gene as a loading control. A single copy gene SWI5 was detected on the same filter used in the upper panel. (Bottom panel) Analysis of rDNA that failed to digest with SacII. The signal intensities of the undigested rDNA (SacII+) and non-digested (SacII-) bands were measured and the ratios plotted. The black dots show the results for the lower bands (black arrowhead, in the Top panel) and the blue dots are for the upper bands (blue arrowhead in the Top panel). ID# is the identification number of individual mice that were used to isolate the bone marrow cells (same as Figure 2 ). p values are calculated from the average of young and old mice.

    Techniques Used: Methylation, Southern Blot, Mouse 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 "Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11"

    Article Title: Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11

    Journal: bioRxiv

    doi: 10.1101/2020.05.29.123570

    Map of pPv121-MCS vector. The multiple cloning site of the vector comprises BamHI, HindIII, XhoI and SacII sites.
    Figure Legend Snippet: Map of pPv121-MCS vector. The multiple cloning site of the vector comprises BamHI, HindIII, XhoI and SacII sites.

    Techniques Used: Plasmid Preparation, Clone Assay

    4) 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

    5) 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

    6) 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

    7) Product Images from "Rapid method for generating designer algal mitochondrial genomes"

    Article Title: Rapid method for generating designer algal mitochondrial genomes

    Journal: bioRxiv

    doi: 10.1101/2019.12.19.882662

    Design, amplification, and analyses of cloned P. tricornutum mitochondrial genomes. A) Plasmid maps of P. tricornutum ’s mitochondrial genomes cloned using PCR (top) or TAR (bottom) methods. For the PCR-based cloning method, the relative sizes and positions of the eight mitochondrial fragments (blue), and the two plasmid backbone fragments (orange) are shown. For the TAR-based cloning method, the relative sizes and positions of the linearized genome (blue) and the two plasmid backbone fragments (orange) are shown. In addition, the six MPX PCR amplicons used for diagnostic screening are indicated (green). These images were generated using Geneious version 2020.0, created by Biomatters. B) Agarose gel electrophoresis of the 10 PCR amplified fragments of the P. tricornutum mitochondrial genome for PCR-based cloning including the pAGE3.0 backbone (Fragments 6 and 7). The resulting amplicon sizes for Fragments 1 to 10 are 5217, 5190, 5197, 5175, 2765, 9124, 8571, 5605, 5299, and 9264 bp, respectively. C) MPX PCR screen of six cloned algal mitochondrial genomes isolated from E. coli . The expected size for each MPX amplicon is indicated by its name (in bp). D) Diagnostic restriction digest of six cloned algal mitochondrial genomes using SrfI and SacII. For the pPT-PCR clones, the expected band sizes are 39925, 8758, 7177, 3276, and 707 bp; and for the pPT-TAR clones, the expected band sizes are 70647, 11506, 8758, 3276, and 707 bp. Notes: 1) We did not see a size difference between the 70.6-kbp and 39.9-kbp fragments, which is most likely due to the electrophoresis conditions (1% agarose gel, 100 V for 90 minutes); 2) The 707-bp band is present but very faint. For all gels, we used NEB 2-log ladder.
    Figure Legend Snippet: Design, amplification, and analyses of cloned P. tricornutum mitochondrial genomes. A) Plasmid maps of P. tricornutum ’s mitochondrial genomes cloned using PCR (top) or TAR (bottom) methods. For the PCR-based cloning method, the relative sizes and positions of the eight mitochondrial fragments (blue), and the two plasmid backbone fragments (orange) are shown. For the TAR-based cloning method, the relative sizes and positions of the linearized genome (blue) and the two plasmid backbone fragments (orange) are shown. In addition, the six MPX PCR amplicons used for diagnostic screening are indicated (green). These images were generated using Geneious version 2020.0, created by Biomatters. B) Agarose gel electrophoresis of the 10 PCR amplified fragments of the P. tricornutum mitochondrial genome for PCR-based cloning including the pAGE3.0 backbone (Fragments 6 and 7). The resulting amplicon sizes for Fragments 1 to 10 are 5217, 5190, 5197, 5175, 2765, 9124, 8571, 5605, 5299, and 9264 bp, respectively. C) MPX PCR screen of six cloned algal mitochondrial genomes isolated from E. coli . The expected size for each MPX amplicon is indicated by its name (in bp). D) Diagnostic restriction digest of six cloned algal mitochondrial genomes using SrfI and SacII. For the pPT-PCR clones, the expected band sizes are 39925, 8758, 7177, 3276, and 707 bp; and for the pPT-TAR clones, the expected band sizes are 70647, 11506, 8758, 3276, and 707 bp. Notes: 1) We did not see a size difference between the 70.6-kbp and 39.9-kbp fragments, which is most likely due to the electrophoresis conditions (1% agarose gel, 100 V for 90 minutes); 2) The 707-bp band is present but very faint. For all gels, we used NEB 2-log ladder.

    Techniques Used: Amplification, Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Diagnostic Assay, Generated, Agarose Gel Electrophoresis, Isolation, Electrophoresis

    8) Product Images from "Age-Dependent Ribosomal DNA Variations in Mice"

    Article Title: Age-Dependent Ribosomal DNA Variations in Mice

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00368-20

    rDNA methylation in old and young bone marrow cells. rDNA methylation was detected by digestion with the methylation-sensitive enzyme SacII. (A) Position of the probe for Southern blot analysis in panels B and C and recognition sites for BamHI and SacII. (B and C) Ratio of copy numbers of methylated rDNA in young and old mice. Southern blot analysis was used to detect the ratio in undigested bands by SacII (top). The positions of undigested bands are indicated by arrowheads on the left-hand side of the panels. The SWI5 gene served as a loading control (middle). A single-copy gene, SWI5, was detected on the same filter used for the experiment shown in the upper panel. Analysis of rDNA that failed to digest with SacII was performed (bottom). The signal intensities of the undigested rDNA (SacII + ) and nondigested (SacII − ) bands were measured, and the ratios were plotted. The black and blue dots show the results corresponding to the bands indicated by the black and blue arrowheads, respectively, in the top panel. Identification (ID) numbers of individual mice that were used to isolate the bone marrow cells are given ( Fig. 3 ). P values were calculated from the average of the values for young and old mice.
    Figure Legend Snippet: rDNA methylation in old and young bone marrow cells. rDNA methylation was detected by digestion with the methylation-sensitive enzyme SacII. (A) Position of the probe for Southern blot analysis in panels B and C and recognition sites for BamHI and SacII. (B and C) Ratio of copy numbers of methylated rDNA in young and old mice. Southern blot analysis was used to detect the ratio in undigested bands by SacII (top). The positions of undigested bands are indicated by arrowheads on the left-hand side of the panels. The SWI5 gene served as a loading control (middle). A single-copy gene, SWI5, was detected on the same filter used for the experiment shown in the upper panel. Analysis of rDNA that failed to digest with SacII was performed (bottom). The signal intensities of the undigested rDNA (SacII + ) and nondigested (SacII − ) bands were measured, and the ratios were plotted. The black and blue dots show the results corresponding to the bands indicated by the black and blue arrowheads, respectively, in the top panel. Identification (ID) numbers of individual mice that were used to isolate the bone marrow cells are given ( Fig. 3 ). P values were calculated from the average of the values for young and old mice.

    Techniques Used: Methylation, Southern Blot, Mouse Assay

    9) Product Images from "Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11"

    Article Title: Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11

    Journal: bioRxiv

    doi: 10.1101/2020.05.29.123570

    Map of pPv121-MCS vector. The multiple cloning site of the vector comprises BamHI, HindIII, XhoI and SacII sites.
    Figure Legend Snippet: Map of pPv121-MCS vector. The multiple cloning site of the vector comprises BamHI, HindIII, XhoI and SacII sites.

    Techniques Used: Plasmid Preparation, Clone Assay

    Related Articles

    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: 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 ). .. 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).

    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) ( ).

    Southern Blot:

    Article Title: Age-dependent ribosomal DNA variations and their effect on cellular function in mammalian cells
    Article Snippet: .. Southern blot analysis to detect rDNA For Southern blot analysis 150 ng of mouse DNA was digested with 10 units of BamHI-HF (NEB, and ), NdeI (NEB, and ) and SacII (NEB, ) overnight at 37°C. ..

    Clone Assay:

    Article Title: Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification
    Article Snippet: The re-streaked cm & amp-mutants were PCR-amplified with the A1-pAK400rev pair from the secondary cm-plates with both 26 and 31 cycles. .. 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.

    Plasmid Preparation:

    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 ). .. 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).

    Amplification:

    Article Title: Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11
    Article Snippet: A DNA fragment for the AMV RTα gene was synthesized by Eurofins Genomics K.K, Tokyo, Japan. .. The AMV RTα gene was then amplified by PCR with specific primers ( ) and inserted into pPv121-MCS digested with BamHI and SacII using NEBuilder HiFi DNA Assembly Mater Mix. .. For the pTetO-202bp-AMV RTα expression vector, the AMV RTα sequence was amplified and inserted into pTetO-202bp-Nluc digested with SalI and BglII using NEBuilder HiFi DNA Assembly Master Mix.

    Polymerase Chain Reaction:

    Article Title: Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11
    Article Snippet: A DNA fragment for the AMV RTα gene was synthesized by Eurofins Genomics K.K, Tokyo, Japan. .. The AMV RTα gene was then amplified by PCR with specific primers ( ) and inserted into pPv121-MCS digested with BamHI and SacII using NEBuilder HiFi DNA Assembly Mater Mix. .. For the pTetO-202bp-AMV RTα expression vector, the AMV RTα sequence was amplified and inserted into pTetO-202bp-Nluc digested with SalI and BglII using NEBuilder HiFi DNA Assembly Master Mix.

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    New England Biolabs sacii restriction enzymes
    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, <t>ApaI/SacII</t> 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.
    Sacii Restriction Enzymes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    rDNA methylation in old and young bone marrow cells rDNA methylation was detected by digestion with a methylation sensitive enzyme SacII. ( A ) Position of the probe for Southern blot analysis in ( BC ) and recognition sites for BamHI and SacII. ( BC ) Ratio of methylated rDNA copies in young and old mice. (Top panel) Southern analysis to detect the ratio of undigested band by SacII. The positions of undigested bands are indicated by arrowheads on the left-hand side of the panels. (Middle panel) Detection of the SWI5 gene as a loading control. A single copy gene SWI5 was detected on the same filter used in the upper panel. (Bottom panel) Analysis of rDNA that failed to digest with SacII. The signal intensities of the undigested rDNA (SacII+) and non-digested (SacII-) bands were measured and the ratios plotted. The black dots show the results for the lower bands (black arrowhead, in the Top panel) and the blue dots are for the upper bands (blue arrowhead in the Top panel). ID# is the identification number of individual mice that were used to isolate the bone marrow cells (same as Figure 2 ). p values are calculated from the average of young and old mice.

    Journal: bioRxiv

    Article Title: Age-dependent ribosomal DNA variations and their effect on cellular function in mammalian cells

    doi: 10.1101/2020.07.10.196840

    Figure Lengend Snippet: rDNA methylation in old and young bone marrow cells rDNA methylation was detected by digestion with a methylation sensitive enzyme SacII. ( A ) Position of the probe for Southern blot analysis in ( BC ) and recognition sites for BamHI and SacII. ( BC ) Ratio of methylated rDNA copies in young and old mice. (Top panel) Southern analysis to detect the ratio of undigested band by SacII. The positions of undigested bands are indicated by arrowheads on the left-hand side of the panels. (Middle panel) Detection of the SWI5 gene as a loading control. A single copy gene SWI5 was detected on the same filter used in the upper panel. (Bottom panel) Analysis of rDNA that failed to digest with SacII. The signal intensities of the undigested rDNA (SacII+) and non-digested (SacII-) bands were measured and the ratios plotted. The black dots show the results for the lower bands (black arrowhead, in the Top panel) and the blue dots are for the upper bands (blue arrowhead in the Top panel). ID# is the identification number of individual mice that were used to isolate the bone marrow cells (same as Figure 2 ). p values are calculated from the average of young and old mice.

    Article Snippet: Southern blot analysis to detect rDNA For Southern blot analysis 150 ng of mouse DNA was digested with 10 units of BamHI-HF (NEB, and ), NdeI (NEB, and ) and SacII (NEB, ) overnight at 37°C.

    Techniques: Methylation, Southern Blot, Mouse Assay

    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:

    Map of pPv121-MCS vector. The multiple cloning site of the vector comprises BamHI, HindIII, XhoI and SacII sites.

    Journal: bioRxiv

    Article Title: Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11

    doi: 10.1101/2020.05.29.123570

    Figure Lengend Snippet: Map of pPv121-MCS vector. The multiple cloning site of the vector comprises BamHI, HindIII, XhoI and SacII sites.

    Article Snippet: The AMV RTα gene was then amplified by PCR with specific primers ( ) and inserted into pPv121-MCS digested with BamHI and SacII using NEBuilder HiFi DNA Assembly Mater Mix.

    Techniques: Plasmid Preparation, Clone Assay