sfii  (New England Biolabs)


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    Name:
    SfiI
    Description:
    SfiI 15 000 units
    Catalog Number:
    R0123L
    Price:
    290
    Category:
    Restriction Enzymes
    Size:
    15 000 units
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    New England Biolabs sfii
    SfiI
    SfiI 15 000 units
    https://www.bioz.com/result/sfii/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sfii - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "Genotypic Characterization of Vibrio vulnificus Clinical Isolates in Korea"

    Article Title: Genotypic Characterization of Vibrio vulnificus Clinical Isolates in Korea

    Journal: Osong Public Health and Research Perspectives

    doi: 10.1016/j.phrp.2011.04.008

    Yearly distributions of PFGE patterns analyzed by NotI (A) and SfiI (B) for V. vulnificus strains in Korea from 2000 to 2004.
    Figure Legend Snippet: Yearly distributions of PFGE patterns analyzed by NotI (A) and SfiI (B) for V. vulnificus strains in Korea from 2000 to 2004.

    Techniques Used:

    (A) Map of southwestern and southeastern Korea showing sampling locations for clinical isolates of V. vulnificus . Regional distribution of PFGE patterns by NotI (B) and SfiI (C) of V. vulnificus from 2000 to 2004. BS = Busan; GN = Gyeongsangnam-do; JB = Jeollabuk-do; JN = Jeollanam-do; US = Ulsan.
    Figure Legend Snippet: (A) Map of southwestern and southeastern Korea showing sampling locations for clinical isolates of V. vulnificus . Regional distribution of PFGE patterns by NotI (B) and SfiI (C) of V. vulnificus from 2000 to 2004. BS = Busan; GN = Gyeongsangnam-do; JB = Jeollabuk-do; JN = Jeollanam-do; US = Ulsan.

    Techniques Used: Sampling

    2) Product Images from "COLORFUL-Circuit: A Platform for Rapid Multigene Assembly, Delivery, and Expression in Plants"

    Article Title: COLORFUL-Circuit: A Platform for Rapid Multigene Assembly, Delivery, and Expression in Plants

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2016.00246

    Construction and transient expression of fluorescent organelle markers . (A) Schematic view of the modular pC1 (6178 bp), pC2 (6210 bp), pC3 (7163 bp), and pC4 (6351 bp) binary vectors expressing fluorescent organelle markers. The vector backbone (4282 bp) is represented outside of the SfII recongintion sequences ( SfiI A, SfiI B, SfiI C, SfiI D, SfiI E, SfiI F, SfiI G, and SfiI H) that flank the gene cassettes encoding the fluorescent organelle markers C1 (1896 bp), C2 (1928 bp), C3 (2881 bp), and C4 (2069 bp). The vector backbone was amplified from the plasmid pXNS2pat-YFP. UBQ10, polyubiquitin 10 promoter; 35S, cauliflower mosaic virus CaMV 35S promoter; T35, terminator of the cauliflower mosaic virus 35S; Tocs, terminator of octopine synthase; mKate2, TagRFP-T, Venus and mTurquoise2, fluorescent protein encoding genes; LTI6b, low temperature induced protein as a membrane marker; SKL, peroxisomal targeting sequence; MAP4, microtubule binding domain of the mouse microtubule-associated protein 4; N7, nuclear localization signal; Basta R , selectable marker conferring Basta resistance in plants; Amp R , selectable marker conferring ampicillin resistance in E. coli and A. tumefaciens ; LB/RB, left/right borders of T-DNA. The figure is not drawn to scale. (B–E) Agrobacterium -mediated transient expression of the fluorescent organelle markers indicated in (A) . (B) Membrane localization of mKate2-LTI6b produced from pC1. (C) Peroxisomal localization of TagRFP-T-SKL produced from pC2. (D) Microtubule localization of Venus-MAP4 produced from pC3. (E) Nuclear localization of mTurquoise2-N7 produced from pC4. Top, middle and bottom panels in (B–E) represent fluorescence, bright field and overlay of confocal images. The images in (C–E) represent maximum projections of z-stacks obtained by confocal laser scanning microscopy. Scale bar = 50 μm.
    Figure Legend Snippet: Construction and transient expression of fluorescent organelle markers . (A) Schematic view of the modular pC1 (6178 bp), pC2 (6210 bp), pC3 (7163 bp), and pC4 (6351 bp) binary vectors expressing fluorescent organelle markers. The vector backbone (4282 bp) is represented outside of the SfII recongintion sequences ( SfiI A, SfiI B, SfiI C, SfiI D, SfiI E, SfiI F, SfiI G, and SfiI H) that flank the gene cassettes encoding the fluorescent organelle markers C1 (1896 bp), C2 (1928 bp), C3 (2881 bp), and C4 (2069 bp). The vector backbone was amplified from the plasmid pXNS2pat-YFP. UBQ10, polyubiquitin 10 promoter; 35S, cauliflower mosaic virus CaMV 35S promoter; T35, terminator of the cauliflower mosaic virus 35S; Tocs, terminator of octopine synthase; mKate2, TagRFP-T, Venus and mTurquoise2, fluorescent protein encoding genes; LTI6b, low temperature induced protein as a membrane marker; SKL, peroxisomal targeting sequence; MAP4, microtubule binding domain of the mouse microtubule-associated protein 4; N7, nuclear localization signal; Basta R , selectable marker conferring Basta resistance in plants; Amp R , selectable marker conferring ampicillin resistance in E. coli and A. tumefaciens ; LB/RB, left/right borders of T-DNA. The figure is not drawn to scale. (B–E) Agrobacterium -mediated transient expression of the fluorescent organelle markers indicated in (A) . (B) Membrane localization of mKate2-LTI6b produced from pC1. (C) Peroxisomal localization of TagRFP-T-SKL produced from pC2. (D) Microtubule localization of Venus-MAP4 produced from pC3. (E) Nuclear localization of mTurquoise2-N7 produced from pC4. Top, middle and bottom panels in (B–E) represent fluorescence, bright field and overlay of confocal images. The images in (C–E) represent maximum projections of z-stacks obtained by confocal laser scanning microscopy. Scale bar = 50 μm.

    Techniques Used: Expressing, Plasmid Preparation, Amplification, Marker, Sequencing, Binding Assay, Produced, Fluorescence, Confocal Laser Scanning Microscopy

    Generation of COLORFUL-Circuit multigene assemblies . (A) Ligation of the SfiI -cleaved gene cassettes C1, C2, C3, and C4 followed by agarose gel electrophoresis verified the production of higher molecular weight DNA fragments corresponding to the assembled molecules. C1, 1896 bp, and C2, 1928 bp (ligated C1 + C2 = 3824 bp); C2 and C3, 2881 bp (ligated C2 + C3 = 4809 bp); and C3 and C4, 2069 bp (ligated C3 + C4 = 4950 bp). M: GeneRuler™ 1-Kb DNA ladder. (B) DNA assembly of the four individual gene cassettes (C1, C2, C3, and C4) upon ligation. All possible unidirectionally ligated gene cassettes are produced; (C1 + C2 = 3824 bp; C2 + C3 = 4809 bp; C3 + C4 = 4950 bp), (C1 + C2 + C3 = 6705 bp; C2 + C3 + C4 = 6878 bp), and (C1+ C2 + C3 + C4 = 8774 bp). M: GeneRuler™ 1-Kb DNA ladder. (C–E) Modules of the COLORFUL-Circuit multigene binary vectors that allow double-gene cassette assembly (pC2–C3), Triple-gene cassette assembly (pC1–C3) and quadruple-gene cassette assembly [pC1–C4 (version I)], respectively. C1, C2, C3, and C4: individual gene cassettes. The vector backbone was amplified from the plasmid pGreenII. UBQ: polyubiquitin 10 promoter; 35S: cauliflower mosaic virus CaMV 35S promoter; T: terminator of the cauliflower mosaic virus 35S (gray) and terminator of octopine synthase (purple); red, yellow, green and turquoise arrows: fluorescent protein encoding genes of mKate2, TagRFP-T, Venus and mTurquoise2, respectively; LTI6b, low temperature induced protein as a membrane marker; SKL, peroxisomes targeting sequence; MAP4, microtubule binding domain of the mouse microtubule-associated protein 4; N7, nuclear localization signal; Basta R , selectable marker confers Basta resistance in plants; Kan R , selectable marker confers kanamycin resistance in E. coli and A. tumefaciens ; LB/RB, left/right borders of T-DNA. The figures are not drawn to scale.
    Figure Legend Snippet: Generation of COLORFUL-Circuit multigene assemblies . (A) Ligation of the SfiI -cleaved gene cassettes C1, C2, C3, and C4 followed by agarose gel electrophoresis verified the production of higher molecular weight DNA fragments corresponding to the assembled molecules. C1, 1896 bp, and C2, 1928 bp (ligated C1 + C2 = 3824 bp); C2 and C3, 2881 bp (ligated C2 + C3 = 4809 bp); and C3 and C4, 2069 bp (ligated C3 + C4 = 4950 bp). M: GeneRuler™ 1-Kb DNA ladder. (B) DNA assembly of the four individual gene cassettes (C1, C2, C3, and C4) upon ligation. All possible unidirectionally ligated gene cassettes are produced; (C1 + C2 = 3824 bp; C2 + C3 = 4809 bp; C3 + C4 = 4950 bp), (C1 + C2 + C3 = 6705 bp; C2 + C3 + C4 = 6878 bp), and (C1+ C2 + C3 + C4 = 8774 bp). M: GeneRuler™ 1-Kb DNA ladder. (C–E) Modules of the COLORFUL-Circuit multigene binary vectors that allow double-gene cassette assembly (pC2–C3), Triple-gene cassette assembly (pC1–C3) and quadruple-gene cassette assembly [pC1–C4 (version I)], respectively. C1, C2, C3, and C4: individual gene cassettes. The vector backbone was amplified from the plasmid pGreenII. UBQ: polyubiquitin 10 promoter; 35S: cauliflower mosaic virus CaMV 35S promoter; T: terminator of the cauliflower mosaic virus 35S (gray) and terminator of octopine synthase (purple); red, yellow, green and turquoise arrows: fluorescent protein encoding genes of mKate2, TagRFP-T, Venus and mTurquoise2, respectively; LTI6b, low temperature induced protein as a membrane marker; SKL, peroxisomes targeting sequence; MAP4, microtubule binding domain of the mouse microtubule-associated protein 4; N7, nuclear localization signal; Basta R , selectable marker confers Basta resistance in plants; Kan R , selectable marker confers kanamycin resistance in E. coli and A. tumefaciens ; LB/RB, left/right borders of T-DNA. The figures are not drawn to scale.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Molecular Weight, Produced, Plasmid Preparation, Amplification, Marker, Sequencing, Binding Assay

    Structural design of the COLORFUL-Circuit vectors and assembly . (A) Schematic view showing the modular design of the C1 gene expression cassette (1896 bp). The gene cassettes C1, C1.1, C1.2, and C1.3 only differ in the color coded unique SfiI overhangs ( SfiI -A, SfiI -B, SfiI -C, SfiI -D, SfiI -E, SfiI -F, SfiI -G, and SfiI -H). UBQ10, polyubiquitin 10 promoter; mKate2, far-red fluorescent protein encoding gene; LTI6b, low temperature induced protein as a membrane marker; T35S, terminator of the cauliflower mosaic virus 35S; SfiI, RsrII, EcoRI, XbaI/SpeI , recognition sites of the corresponding restriction enzymes. (B) Diagram depicting the concept of COLORFUL-Circuit assembly of four tandemly arranged gene cassettes (C1–C1.1–C1.2–C1.3). The SfiI unique overhangs flanking each gene cassette allow combinatorial assembly, after SfiI cleavage and subsequent DNA ligation. The gray arrows indicate transcriptional orientations of the gene cassettes. SfiI overhangs with the same color flank one gene cassette. Note, that figure elements are not drawn to scale.
    Figure Legend Snippet: Structural design of the COLORFUL-Circuit vectors and assembly . (A) Schematic view showing the modular design of the C1 gene expression cassette (1896 bp). The gene cassettes C1, C1.1, C1.2, and C1.3 only differ in the color coded unique SfiI overhangs ( SfiI -A, SfiI -B, SfiI -C, SfiI -D, SfiI -E, SfiI -F, SfiI -G, and SfiI -H). UBQ10, polyubiquitin 10 promoter; mKate2, far-red fluorescent protein encoding gene; LTI6b, low temperature induced protein as a membrane marker; T35S, terminator of the cauliflower mosaic virus 35S; SfiI, RsrII, EcoRI, XbaI/SpeI , recognition sites of the corresponding restriction enzymes. (B) Diagram depicting the concept of COLORFUL-Circuit assembly of four tandemly arranged gene cassettes (C1–C1.1–C1.2–C1.3). The SfiI unique overhangs flanking each gene cassette allow combinatorial assembly, after SfiI cleavage and subsequent DNA ligation. The gray arrows indicate transcriptional orientations of the gene cassettes. SfiI overhangs with the same color flank one gene cassette. Note, that figure elements are not drawn to scale.

    Techniques Used: Expressing, Marker, DNA Ligation

    3) Product Images from "A Novel Quantitative Kinase Assay Using Bacterial Surface Display and Flow Cytometry"

    Article Title: A Novel Quantitative Kinase Assay Using Bacterial Surface Display and Flow Cytometry

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080474

    BacKin assay procedure. (A) Map of plasmid pBAD33-eCPX-abltide (left) and schematic representation of abltide fused to eCPX expressed on the cell surface of an E. coli cell transformed with pBAD33-eCPX-abltide (right). The plasmid carries the arabinose promoter pBAD and resistance to chloramphenicol (CM r ). The recognition sites for the restriction enzyme SfiI and annealing region are show. (B) Schematic representation of the BacKin assay: 1. E. coli cells transformed with eCPX-substrate plasmid are incubated at 37°C and shaken to grow until mid log phase; 2. Substrate (e.g. abltide) expression and display on the bacteria surface is induced with arabinose; 3. Substrate phosphorylation, cells are incubated with kinase (e.g. Abl kinase) and an excess of ATP; 4. Phosphorylated substrate is labeled by incubation of cells with biotinylated-anti-phosphotyrosine antibody (biotin-anti-PY) followed by incubation with streptavidin-phycoerythrin (SAPE); 5. Mean fluorescence of kinase-treated cells is quantified by flow cytometry and compared with mean fluorescence of kinase untreated cells.
    Figure Legend Snippet: BacKin assay procedure. (A) Map of plasmid pBAD33-eCPX-abltide (left) and schematic representation of abltide fused to eCPX expressed on the cell surface of an E. coli cell transformed with pBAD33-eCPX-abltide (right). The plasmid carries the arabinose promoter pBAD and resistance to chloramphenicol (CM r ). The recognition sites for the restriction enzyme SfiI and annealing region are show. (B) Schematic representation of the BacKin assay: 1. E. coli cells transformed with eCPX-substrate plasmid are incubated at 37°C and shaken to grow until mid log phase; 2. Substrate (e.g. abltide) expression and display on the bacteria surface is induced with arabinose; 3. Substrate phosphorylation, cells are incubated with kinase (e.g. Abl kinase) and an excess of ATP; 4. Phosphorylated substrate is labeled by incubation of cells with biotinylated-anti-phosphotyrosine antibody (biotin-anti-PY) followed by incubation with streptavidin-phycoerythrin (SAPE); 5. Mean fluorescence of kinase-treated cells is quantified by flow cytometry and compared with mean fluorescence of kinase untreated cells.

    Techniques Used: Plasmid Preparation, Transformation Assay, Incubation, Expressing, Labeling, Fluorescence, Flow Cytometry, Cytometry

    4) Product Images from "Herpesvirus telomeric repeats facilitate genomic integration into host telomeres and mobilization of viral DNA during reactivation"

    Article Title: Herpesvirus telomeric repeats facilitate genomic integration into host telomeres and mobilization of viral DNA during reactivation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20101402

    Integration does not occur in host telomeres in the absence of viral TMRs. (A) Schematic representation and corresponding PFGE and Southern blot analysis of LCL DNA digested with SfiI. Fragment sizes generated by SfiI digestion of integrated and nonintegrated MDV genomes are depicted and sizes are given. The size of the linear MDV genome observed during lytic replication is indicated by an arrow. Results are representative of three independent Southern blot analyses. (B) Southern blotting of DNA of LCL derived from animals infected with vTE1 and digested with BclI. Potential intragenomic and telomeric integration sites are indicated. Results are representative of three independent Southern blot analyses. (C) Quantification of MDV copies in tumor cells. Results are shown as mean herpesvirus genome copies detected by the TR L probe relative to B2M in three independent experiments. The data are shown relative to LCL CU482 derived from a vRB-1B–infected chicken with standard deviations (error bars).
    Figure Legend Snippet: Integration does not occur in host telomeres in the absence of viral TMRs. (A) Schematic representation and corresponding PFGE and Southern blot analysis of LCL DNA digested with SfiI. Fragment sizes generated by SfiI digestion of integrated and nonintegrated MDV genomes are depicted and sizes are given. The size of the linear MDV genome observed during lytic replication is indicated by an arrow. Results are representative of three independent Southern blot analyses. (B) Southern blotting of DNA of LCL derived from animals infected with vTE1 and digested with BclI. Potential intragenomic and telomeric integration sites are indicated. Results are representative of three independent Southern blot analyses. (C) Quantification of MDV copies in tumor cells. Results are shown as mean herpesvirus genome copies detected by the TR L probe relative to B2M in three independent experiments. The data are shown relative to LCL CU482 derived from a vRB-1B–infected chicken with standard deviations (error bars).

    Techniques Used: Southern Blot, Generated, Derivative Assay, Infection

    5) Product Images from "Evolution and Spread of a Multidrug-Resistant Proteus mirabilis Clone with Chromosomal AmpC-Type Cephalosporinases in Europe ▿"

    Article Title: Evolution and Spread of a Multidrug-Resistant Proteus mirabilis Clone with Chromosomal AmpC-Type Cephalosporinases in Europe ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01736-10

    Dendrogram based on NotI+SfiI PFGE patterns, indicating clusters of the resulting types, as well as ribotypes. The bands arising after NotI+SfiI PFGE for each isolate are shown. The number next to the isolate code denotes the CMY variant. PFGE type clusters
    Figure Legend Snippet: Dendrogram based on NotI+SfiI PFGE patterns, indicating clusters of the resulting types, as well as ribotypes. The bands arising after NotI+SfiI PFGE for each isolate are shown. The number next to the isolate code denotes the CMY variant. PFGE type clusters

    Techniques Used: Variant Assay

    6) Product Images from "DNA looping by two-site restriction endonucleases: heterogeneous probability distributions for loop size and unbinding force"

    Article Title: DNA looping by two-site restriction endonucleases: heterogeneous probability distributions for loop size and unbinding force

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl382

    Typical DNA force-extension plots. ( A ) The two-site enzyme BsgI tested on the LBAC-B template, which has four recognition sites. The one detected loop had a measured length of 946 bp, in excellent agreement with the separation between the binding sites at positions 8342 and 9287 bp (a distance of 945 bp). ( B ) Sau3AI on the LBAC-A template, which has 55 recognition sites. ( C ) Control experiments: (i) the two-site enzyme Sau3AI was tested on bacteriophage phiX174 DNA, which contains no copies of its recognition sequence; (ii) the one-site enzyme HaeIII was tested on LBAC-B DNA containing 36 copies of its recognition sequence; (iii) the two-site enzyme SfiI was tested on LBAC-B DNA containing one copy of its recognition sequence and (iv) LBAC-B DNA, containing 26 FokI binding sites, incubated in the FokI reaction buffer, but no FokI added.
    Figure Legend Snippet: Typical DNA force-extension plots. ( A ) The two-site enzyme BsgI tested on the LBAC-B template, which has four recognition sites. The one detected loop had a measured length of 946 bp, in excellent agreement with the separation between the binding sites at positions 8342 and 9287 bp (a distance of 945 bp). ( B ) Sau3AI on the LBAC-A template, which has 55 recognition sites. ( C ) Control experiments: (i) the two-site enzyme Sau3AI was tested on bacteriophage phiX174 DNA, which contains no copies of its recognition sequence; (ii) the one-site enzyme HaeIII was tested on LBAC-B DNA containing 36 copies of its recognition sequence; (iii) the two-site enzyme SfiI was tested on LBAC-B DNA containing one copy of its recognition sequence and (iv) LBAC-B DNA, containing 26 FokI binding sites, incubated in the FokI reaction buffer, but no FokI added.

    Techniques Used: Binding Assay, Sequencing, Incubation

    7) Product Images from "Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation"

    Article Title: Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201404157

    Premutation hESCs, which do not expand to full mutation, have a different replication program than FXS hESCs and a similar replication program to nonaffected hESCs. (A) The stability of the CGG repeats in premutation hESCs was analyzed by EcoRI and EagI Southern blotting. (B) Schematic of the steps of SMARD: First, cells are pulsed with IdU and CldU. Cells are then embedded in agarose and lysed. After digestion with a restriction enzyme, DNA molecules are separated by PFGE. Target segments were identified by Southern blotting and stretched on silanized glass slides. The replicated DNA molecules are detected by immunostaining and FISH. (C and D) The replication program was analyzed by SMARD in premutation and FXS hESCs as previously described ( Gerhardt et al., 2014 ). (top) Map of the 102-kb PmeI segment containing the SNP variant T/C and the CGG repeats. The positions of the FISH probes are marked in blue and through the DNA molecules by yellow lines. (bottom) Photomicrographs of labeled DNA molecules from WCMC37 FXS hESCs (C) and WCMC5 premutation hESCs (D) are ordered according to replication fork (yellow arrowheads) progression; replication initiation, 5′→3′ and 3′→5′ directions, and termination are depicted. In contrast to FXS hESCs, the premutation hESCs WCMC5 contains a replication origin 53 kb upstream of the repeats. (E) The replication program was analyzed by SMARD at the FMR1 locus containing the CGG repeats in premutation hESCs (120-kb SfiI segment). The CGG repeats are indicated by a red vertical line.
    Figure Legend Snippet: Premutation hESCs, which do not expand to full mutation, have a different replication program than FXS hESCs and a similar replication program to nonaffected hESCs. (A) The stability of the CGG repeats in premutation hESCs was analyzed by EcoRI and EagI Southern blotting. (B) Schematic of the steps of SMARD: First, cells are pulsed with IdU and CldU. Cells are then embedded in agarose and lysed. After digestion with a restriction enzyme, DNA molecules are separated by PFGE. Target segments were identified by Southern blotting and stretched on silanized glass slides. The replicated DNA molecules are detected by immunostaining and FISH. (C and D) The replication program was analyzed by SMARD in premutation and FXS hESCs as previously described ( Gerhardt et al., 2014 ). (top) Map of the 102-kb PmeI segment containing the SNP variant T/C and the CGG repeats. The positions of the FISH probes are marked in blue and through the DNA molecules by yellow lines. (bottom) Photomicrographs of labeled DNA molecules from WCMC37 FXS hESCs (C) and WCMC5 premutation hESCs (D) are ordered according to replication fork (yellow arrowheads) progression; replication initiation, 5′→3′ and 3′→5′ directions, and termination are depicted. In contrast to FXS hESCs, the premutation hESCs WCMC5 contains a replication origin 53 kb upstream of the repeats. (E) The replication program was analyzed by SMARD at the FMR1 locus containing the CGG repeats in premutation hESCs (120-kb SfiI segment). The CGG repeats are indicated by a red vertical line.

    Techniques Used: Mutagenesis, Southern Blot, Immunostaining, Fluorescence In Situ Hybridization, Variant Assay, Labeling

    A replication initiation site upstream of the repeats overlaps with an SNP in nonaffected cells and is absent in FXS hESCs. (A and C) The previously identified replication initiation sites ∼50 kb upstream of the repeats ( Gerhardt et al., 2014 ) were mapped more in detail by nascent strand abundance. (top) Map of the FMR1 locus upstream of the repeats. The SfiI restriction enzyme site and position of primers used in the nascent strand abundance assay are indicated. SNP primer pair binds at the SNP T/C (ss71651738 or WEX70) on the X chromosome (Chr. X; Fig. S1, A and B ), which cosegregates with a chromosome haplotype at the highest risk for repeat expansion ( Table S1 ; Ennis et al., 2007 ). Primer pairs A, B1, B2, B3, and C amplify surrounding genomic regions. (A, bottom) Nascent strand DNA was isolated from nonaffected hESCs (H14) and fetal fibroblast (GM00011). Nascent strand enrichment at the SNP was determined by real-time PCR using specific primer pairs. (B) As a control, the nascent strand enrichment was determined at the well-characterized Or6 replication origin ( Gerhardt et al., 2006 ) on chromosome 12 (with Or6 primer pair) and at the adjacent control region (N primer pair). (C and D) Nascent strand DNA was isolated from FXS hESCs SI-214 and WCMC37 upstream of the CGG repeats (C) and at a control region (D). Enrichment of the nascent strand at the SNP and neighboring genomic regions was determined by real-time PCR with primer pairs shown above the map. No nascent strand enrichment was obtained for primer B. There was no significant enrichment of nascent strand DNA in FXS hESCs at the SNP in comparisons to control region C in contrast to nonaffected cells in three separate experiments (SI-214: **, P = 0.075; WCMC37: **, P = 0.81; H14: *, P
    Figure Legend Snippet: A replication initiation site upstream of the repeats overlaps with an SNP in nonaffected cells and is absent in FXS hESCs. (A and C) The previously identified replication initiation sites ∼50 kb upstream of the repeats ( Gerhardt et al., 2014 ) were mapped more in detail by nascent strand abundance. (top) Map of the FMR1 locus upstream of the repeats. The SfiI restriction enzyme site and position of primers used in the nascent strand abundance assay are indicated. SNP primer pair binds at the SNP T/C (ss71651738 or WEX70) on the X chromosome (Chr. X; Fig. S1, A and B ), which cosegregates with a chromosome haplotype at the highest risk for repeat expansion ( Table S1 ; Ennis et al., 2007 ). Primer pairs A, B1, B2, B3, and C amplify surrounding genomic regions. (A, bottom) Nascent strand DNA was isolated from nonaffected hESCs (H14) and fetal fibroblast (GM00011). Nascent strand enrichment at the SNP was determined by real-time PCR using specific primer pairs. (B) As a control, the nascent strand enrichment was determined at the well-characterized Or6 replication origin ( Gerhardt et al., 2006 ) on chromosome 12 (with Or6 primer pair) and at the adjacent control region (N primer pair). (C and D) Nascent strand DNA was isolated from FXS hESCs SI-214 and WCMC37 upstream of the CGG repeats (C) and at a control region (D). Enrichment of the nascent strand at the SNP and neighboring genomic regions was determined by real-time PCR with primer pairs shown above the map. No nascent strand enrichment was obtained for primer B. There was no significant enrichment of nascent strand DNA in FXS hESCs at the SNP in comparisons to control region C in contrast to nonaffected cells in three separate experiments (SI-214: **, P = 0.075; WCMC37: **, P = 0.81; H14: *, P

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction

    8) Product Images from "Coincident Resection at Both Ends of Random, ?-Induced Double-Strand Breaks Requires MRX (MRN), Sae2 (Ctp1), and Mre11-Nuclease"

    Article Title: Coincident Resection at Both Ends of Random, ?-Induced Double-Strand Breaks Requires MRX (MRN), Sae2 (Ctp1), and Mre11-Nuclease

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003420

    Detection of 1-end and 2-end resections at a DSB based on rare restriction cutter analysis of 1D PFGE-shifted molecules and subsequent 2D-PFGE. A. Diagram of approach to determine whether the m* and m** PFGE-shift bands of the broken Chr III are due to 1-end or 2-end resections. Cells are induced for I- Sce I, creating a single DSB in Chr III. The m band shown at the left of the gel-slice corresponds to a linear molecule with unresected DSB ends, and the m** band is proposed to be due to 2-end resections. The diagrammed molecules on the right of the gel slice represent two scenarios of resection that could account for the partial shift m* band. In scenario A molecules have extensive resection at only one or the other end while in scenario B both ends of the broken molecules have undergone only a small amount of resection. Diagrammed in the lower part of (A) is the subsequent treatment of the gel slice with the rare cutter SfiI (“S”), followed by PFGE in a second dimension and probing for only the right fragment, identified by a horizontal short red line. The m and m** bands containing molecules with 0- or 2-ends resected, respectively, would give rise to single spots. Scenario A predicts 2 spots for the m*, corresponding to molecules with no “right end” resection and those with a resected “right end.” Scenario B would only yield molecules with the right end resected. B. Stained PFGE gel (SYBR Gold, Invitrogen) of DNA from Δrad50 cells that are induced for I- Sce I for 6 hr. The region of the lane containing the m, m* and m** molecules is indicated. C. Second dimension PFGE analysis of Δrad50 using SYBR Gold stain and probe to “right” fragment. An unstained gel slice (“1 st dimension”) was equilibrated in TE (10 mM Tris, pH 8, 1 mM EDTA), treated with SfiI (New England Biolabs; 400 units in 4 ml total reaction volume including gel slice) for 5 hr at 50°C, followed by overnight treatment in 1% sarkosyl and 1 mg/ml proteinase K at 37°C and subsequent equilibration with running buffer and run in the second dimension. The left image corresponds to the SYBR Gold stained gel. The 3 forms (m**, m* and m) of I- Sce I linearized circle Chr III are identified above and the positions of other chromosomes are identified under the image. In the right image, a Southern transfer of the same gel is probed with DNA that is specific to the right end (also see Figure S2A ) along with a small amount of labeled genomic probe to identify chromosome fragment positions on the gel. Note that in the SYBR Gold stained gel, the SfiI fragments of the m* band (in the blue box) are well-separated, indicating two distinct populations of the probed “right fragments.” D. Second dimension PFGE analysis of Δrad50 and Δrad51 using pure “right” fragment probe. The probe pattern for Δrad50 corresponds to 1-end resections, as described in scenario A of (A), while the Δrad51 pattern reveals resection at both ends of a DSB but no 1-end resections. E. Resection of the “left” side of the in vivo I-SceI cut chromosome III. The scheme described in (A) was followed except that the probe was specific for the left end of the I-SceI break. For Δrad50 the m* position yields both unshifted and shifted molecules after SfiI digestion, consistent with 1-end resection as described for the right end in (D). There is a bias in 1-end resection among the m* molecules in that the “right” sides of the DSBs are more frequently resected than the “left” sides (also see Figure 3 and Figure S3 ). Note that for Δrad51 there are no molecules at the m* position, which corresponds to 1-end resection. Similar results were found in another experiment that compared Δrad50 and Δrad52 (see Figure S3 ).
    Figure Legend Snippet: Detection of 1-end and 2-end resections at a DSB based on rare restriction cutter analysis of 1D PFGE-shifted molecules and subsequent 2D-PFGE. A. Diagram of approach to determine whether the m* and m** PFGE-shift bands of the broken Chr III are due to 1-end or 2-end resections. Cells are induced for I- Sce I, creating a single DSB in Chr III. The m band shown at the left of the gel-slice corresponds to a linear molecule with unresected DSB ends, and the m** band is proposed to be due to 2-end resections. The diagrammed molecules on the right of the gel slice represent two scenarios of resection that could account for the partial shift m* band. In scenario A molecules have extensive resection at only one or the other end while in scenario B both ends of the broken molecules have undergone only a small amount of resection. Diagrammed in the lower part of (A) is the subsequent treatment of the gel slice with the rare cutter SfiI (“S”), followed by PFGE in a second dimension and probing for only the right fragment, identified by a horizontal short red line. The m and m** bands containing molecules with 0- or 2-ends resected, respectively, would give rise to single spots. Scenario A predicts 2 spots for the m*, corresponding to molecules with no “right end” resection and those with a resected “right end.” Scenario B would only yield molecules with the right end resected. B. Stained PFGE gel (SYBR Gold, Invitrogen) of DNA from Δrad50 cells that are induced for I- Sce I for 6 hr. The region of the lane containing the m, m* and m** molecules is indicated. C. Second dimension PFGE analysis of Δrad50 using SYBR Gold stain and probe to “right” fragment. An unstained gel slice (“1 st dimension”) was equilibrated in TE (10 mM Tris, pH 8, 1 mM EDTA), treated with SfiI (New England Biolabs; 400 units in 4 ml total reaction volume including gel slice) for 5 hr at 50°C, followed by overnight treatment in 1% sarkosyl and 1 mg/ml proteinase K at 37°C and subsequent equilibration with running buffer and run in the second dimension. The left image corresponds to the SYBR Gold stained gel. The 3 forms (m**, m* and m) of I- Sce I linearized circle Chr III are identified above and the positions of other chromosomes are identified under the image. In the right image, a Southern transfer of the same gel is probed with DNA that is specific to the right end (also see Figure S2A ) along with a small amount of labeled genomic probe to identify chromosome fragment positions on the gel. Note that in the SYBR Gold stained gel, the SfiI fragments of the m* band (in the blue box) are well-separated, indicating two distinct populations of the probed “right fragments.” D. Second dimension PFGE analysis of Δrad50 and Δrad51 using pure “right” fragment probe. The probe pattern for Δrad50 corresponds to 1-end resections, as described in scenario A of (A), while the Δrad51 pattern reveals resection at both ends of a DSB but no 1-end resections. E. Resection of the “left” side of the in vivo I-SceI cut chromosome III. The scheme described in (A) was followed except that the probe was specific for the left end of the I-SceI break. For Δrad50 the m* position yields both unshifted and shifted molecules after SfiI digestion, consistent with 1-end resection as described for the right end in (D). There is a bias in 1-end resection among the m* molecules in that the “right” sides of the DSBs are more frequently resected than the “left” sides (also see Figure 3 and Figure S3 ). Note that for Δrad51 there are no molecules at the m* position, which corresponds to 1-end resection. Similar results were found in another experiment that compared Δrad50 and Δrad52 (see Figure S3 ).

    Techniques Used: Staining, Labeling, In Vivo

    Related Articles

    Lysis:

    Article Title: Herpesvirus telomeric repeats facilitate genomic integration into host telomeres and mobilization of viral DNA during reactivation
    Article Snippet: .. 1 × 107 /ml LCLs were embedded in 1% agarose and digested at 50°C for 48 h hours in lysis buffer (0.5 M EDTA and 1% wt/vol N -laurylsarcosine) containing 1 mg/ml proteinase K. Proteinase K was inactivated with 0.01 mM phenylmethanesulfonyl fluoride, and agarose plugs were digested with either SfiI or BclI (New England Biolabs) overnight according to the manufacturer’s instructions. .. DNA fragments were resolved via PFGE using the CHEF-DR III system (Bio-Rad Laboratories).

    Incubation:

    Article Title: Diphtheria in the Republic of Georgia: Use of Molecular Typing Techniques for Characterization of Corynebacterium diphtheriae Strains
    Article Snippet: .. The plugs were incubated (30 min, room temperature) with restriction enzyme buffer, the DNA in the plugs was digested by incubation (50°C, 5 h) of the plugs with Sfi I (New England Biolabs, Beverly, Mass.), and electrophoresis was performed with 1% SeaKem Gold agarose in 0.5× Tris-borate-EDTA buffer. .. For block 1, the voltage was 183 V, the initial switch time was 8 s, the final switch time was 20 s, and the duration was 20 h; for block 2, the voltage was 183 V, the initial switch time was 5 s, the final switch time was 8 s, and the duration was 20 h. The PFGE patterns were normalized with lambda DNA and low-range PFGE molecular weight markers (Bio-Rad).

    Electrophoresis:

    Article Title: Diphtheria in the Republic of Georgia: Use of Molecular Typing Techniques for Characterization of Corynebacterium diphtheriae Strains
    Article Snippet: .. The plugs were incubated (30 min, room temperature) with restriction enzyme buffer, the DNA in the plugs was digested by incubation (50°C, 5 h) of the plugs with Sfi I (New England Biolabs, Beverly, Mass.), and electrophoresis was performed with 1% SeaKem Gold agarose in 0.5× Tris-borate-EDTA buffer. .. For block 1, the voltage was 183 V, the initial switch time was 8 s, the final switch time was 20 s, and the duration was 20 h; for block 2, the voltage was 183 V, the initial switch time was 5 s, the final switch time was 8 s, and the duration was 20 h. The PFGE patterns were normalized with lambda DNA and low-range PFGE molecular weight markers (Bio-Rad).

    Pulsed-Field Gel:

    Article Title: Chromosomal Integration of a Cephalosporinase Gene from Acinetobacter baumannii into Oligella urethralis as a Source of Acquired Resistance to ?-Lactams
    Article Snippet: The restriction fragments were separated by conventional electrophoresis ( ). .. Macrorestriction analysis of whole-cell DNA of O. urethralis COH-1 was done by the pulsed-field gel electrophoresis (PFGE) technique with Xba I, Spe I, Dra I, Apa I, and Sfi I (New England Biolabs), as reported previously ( , ). .. To search for the chromosomal integration of the β-lactamase genes, whole-cell DNA of O. urethralis COH-1 was restricted with Ceu I fragment (New England Biolabs), which recognizes a 26-bp sequence in rrn genes coding for the 23S large-subunit rRNA ( ).

    Polymerase Chain Reaction:

    Article Title: A Novel Quantitative Kinase Assay Using Bacterial Surface Display and Flow Cytometry
    Article Snippet: .. The product of PCR reaction and the vector pBAD33-eCPX were digested with SFiI (New England Biolabs) and ligated. .. The ligation product (pBAD33-eCPX-abltide) was desalted and electroporated into electrocompetent E. coli MC1061 with 2 mm electroporation cuvette and pulse at 2.5 kV, 50 µF, and 100 Ω.

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens
    Article Snippet: .. Sfi I digestion was used to define clones containing inserts; 4 μL of the above PCR reactions were digested with SfiI according to the manufacturer's protocol (NEB). .. Products were analyzed on 1% agarose gel, and plasmids of validated clones prepared using Plasmid Mini Kit (Qiagen) and sequenced with 5′U6 or 3′H1 primer.

    Plasmid Preparation:

    Article Title: A Novel Quantitative Kinase Assay Using Bacterial Surface Display and Flow Cytometry
    Article Snippet: .. The product of PCR reaction and the vector pBAD33-eCPX were digested with SFiI (New England Biolabs) and ligated. .. The ligation product (pBAD33-eCPX-abltide) was desalted and electroporated into electrocompetent E. coli MC1061 with 2 mm electroporation cuvette and pulse at 2.5 kV, 50 µF, and 100 Ω.

    Article Title: COLORFUL-Circuit: A Platform for Rapid Multigene Assembly, Delivery, and Expression in Plants
    Article Snippet: Meanwhile, four binary vector backbones were PCR-amplified from the plasmid pXNS2pat-YFP (accession number KF499077; Dahncke and Witte, ) using four pairs of primers that add distinct SfiI cleavage sites in the flanks of the backbones to specifically allow ligation with the overhangs of either C1, C1.1, C1.2, or C1.3 gene cassette as indicated in Supplementary Figure . .. The gene cassettes C1, C1.1, C1.2, or C1.3 and their corresponding vector backbones were cleaved with SfiI (NEB, Germany), separated on agarose gel electrophoresis, and then cleaned up from the gel using innuPREP DOUBLEpure Kit (Analytik Jena, Germany). .. For the SfiI cleavage, 0.5–1 μg DNA, 2 μl CutSmart® buffer and 0.2–0.5 μl SfiI (20 unit/μl) were added in 20 μl total volume, and then incubated for 1–16 h at 50°C in a thermocycler (MyCycler™, Bio-Rad, USA).

    Agarose Gel Electrophoresis:

    Article Title: COLORFUL-Circuit: A Platform for Rapid Multigene Assembly, Delivery, and Expression in Plants
    Article Snippet: Meanwhile, four binary vector backbones were PCR-amplified from the plasmid pXNS2pat-YFP (accession number KF499077; Dahncke and Witte, ) using four pairs of primers that add distinct SfiI cleavage sites in the flanks of the backbones to specifically allow ligation with the overhangs of either C1, C1.1, C1.2, or C1.3 gene cassette as indicated in Supplementary Figure . .. The gene cassettes C1, C1.1, C1.2, or C1.3 and their corresponding vector backbones were cleaved with SfiI (NEB, Germany), separated on agarose gel electrophoresis, and then cleaned up from the gel using innuPREP DOUBLEpure Kit (Analytik Jena, Germany). .. For the SfiI cleavage, 0.5–1 μg DNA, 2 μl CutSmart® buffer and 0.2–0.5 μl SfiI (20 unit/μl) were added in 20 μl total volume, and then incubated for 1–16 h at 50°C in a thermocycler (MyCycler™, Bio-Rad, USA).

    Clone Assay:

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens
    Article Snippet: .. Sfi I digestion was used to define clones containing inserts; 4 μL of the above PCR reactions were digested with SfiI according to the manufacturer's protocol (NEB). .. Products were analyzed on 1% agarose gel, and plasmids of validated clones prepared using Plasmid Mini Kit (Qiagen) and sequenced with 5′U6 or 3′H1 primer.

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    New England Biolabs sfi i digestion
    Schematic diagram of the RNA interference expression vector pRNAi-U6H1/Neo. The vector contains a multiple cloning sites with two BsmB I restriction sites to permit cloning of Fok I-digested sRNA fragments and an <t>Sfi</t> I site to assist in screening for recombinants.
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    Schematic diagram of the RNA interference expression vector pRNAi-U6H1/Neo. The vector contains a multiple cloning sites with two BsmB I restriction sites to permit cloning of Fok I-digested sRNA fragments and an Sfi I site to assist in screening for recombinants.

    Journal: Nucleic Acid Therapeutics

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens

    doi: 10.1089/nat.2014.0514

    Figure Lengend Snippet: Schematic diagram of the RNA interference expression vector pRNAi-U6H1/Neo. The vector contains a multiple cloning sites with two BsmB I restriction sites to permit cloning of Fok I-digested sRNA fragments and an Sfi I site to assist in screening for recombinants.

    Article Snippet: Sfi I digestion was used to define clones containing inserts; 4 μL of the above PCR reactions were digested with SfiI according to the manufacturer's protocol (NEB).

    Techniques: Expressing, Plasmid Preparation, Clone Assay

    Yearly distributions of PFGE patterns analyzed by NotI (A) and SfiI (B) for V. vulnificus strains in Korea from 2000 to 2004.

    Journal: Osong Public Health and Research Perspectives

    Article Title: Genotypic Characterization of Vibrio vulnificus Clinical Isolates in Korea

    doi: 10.1016/j.phrp.2011.04.008

    Figure Lengend Snippet: Yearly distributions of PFGE patterns analyzed by NotI (A) and SfiI (B) for V. vulnificus strains in Korea from 2000 to 2004.

    Article Snippet: Lysed plugs were digested with NotI and SfiI (New England Biolabs, Boston, MA, USA), and PFGE was performed in 1% agarose gels in 0.5 × Tris–borate–EDTA buffer at 14°C using a CHEF mapper apparatus (Bio-Rad, Richmond, CA, USA) at 6 V/cm.

    Techniques:

    (A) Map of southwestern and southeastern Korea showing sampling locations for clinical isolates of V. vulnificus . Regional distribution of PFGE patterns by NotI (B) and SfiI (C) of V. vulnificus from 2000 to 2004. BS = Busan; GN = Gyeongsangnam-do; JB = Jeollabuk-do; JN = Jeollanam-do; US = Ulsan.

    Journal: Osong Public Health and Research Perspectives

    Article Title: Genotypic Characterization of Vibrio vulnificus Clinical Isolates in Korea

    doi: 10.1016/j.phrp.2011.04.008

    Figure Lengend Snippet: (A) Map of southwestern and southeastern Korea showing sampling locations for clinical isolates of V. vulnificus . Regional distribution of PFGE patterns by NotI (B) and SfiI (C) of V. vulnificus from 2000 to 2004. BS = Busan; GN = Gyeongsangnam-do; JB = Jeollabuk-do; JN = Jeollanam-do; US = Ulsan.

    Article Snippet: Lysed plugs were digested with NotI and SfiI (New England Biolabs, Boston, MA, USA), and PFGE was performed in 1% agarose gels in 0.5 × Tris–borate–EDTA buffer at 14°C using a CHEF mapper apparatus (Bio-Rad, Richmond, CA, USA) at 6 V/cm.

    Techniques: Sampling

    Dendrogram generated from the Jaccard similarity coefficient computed for 14 serogroup 12 and 33 serogroup 1 (monoclonal subtype Bellingham) strains after pulsed-field gel electrophoresis of Sfi I-restricted chromosomal DNAs. Strains from our patient are in boldface and are underlined. For strains that were isolated from the same source and were indistinguishable, one representative strain is shown.

    Journal: Journal of Clinical Microbiology

    Article Title: Nosocomial Pneumonia Caused by Three Genetically Different Strains of Legionella pneumophila and Detection of These Strains in the Hospital Water Supply

    doi:

    Figure Lengend Snippet: Dendrogram generated from the Jaccard similarity coefficient computed for 14 serogroup 12 and 33 serogroup 1 (monoclonal subtype Bellingham) strains after pulsed-field gel electrophoresis of Sfi I-restricted chromosomal DNAs. Strains from our patient are in boldface and are underlined. For strains that were isolated from the same source and were indistinguishable, one representative strain is shown.

    Article Snippet: For macrorestriction analysis (MRA), chromosomal DNAs were digested overnight with Sfi I, Asc I, and Not I (New England Biolabs, Schwahlbach, Germany) and separated with the CHEF III System (BioRad Laboratories, Munich, Germany) ( ).

    Techniques: Generated, Pulsed-Field Gel, Electrophoresis, Isolation

    MRA of Sfi I-cleaved DNAs of L. pneumophila strains isolated from the patient and from the hospital water supply. DNA sizes are indicated on the left. Lanes: Y, yeast chromosomal DNA standard; L, lambda concatemers; 1, Köln P2 (sg12); 2, Köln U1 (sg12); 3, Köln U2 (sg12); 4, Köln P4 (sg12); 5, Köln P1 (sg12); 6, Köln U6 (sg12); 7, Köln U8 (sg12); 8, Köln P3 (sg1); 9, Köln P6 (sg1); 10, Köln U4 (sg1); 11, Köln U7 (sg1); 12, Köln U3 (sg12); and 13, Köln U5 (sg12).

    Journal: Journal of Clinical Microbiology

    Article Title: Nosocomial Pneumonia Caused by Three Genetically Different Strains of Legionella pneumophila and Detection of These Strains in the Hospital Water Supply

    doi:

    Figure Lengend Snippet: MRA of Sfi I-cleaved DNAs of L. pneumophila strains isolated from the patient and from the hospital water supply. DNA sizes are indicated on the left. Lanes: Y, yeast chromosomal DNA standard; L, lambda concatemers; 1, Köln P2 (sg12); 2, Köln U1 (sg12); 3, Köln U2 (sg12); 4, Köln P4 (sg12); 5, Köln P1 (sg12); 6, Köln U6 (sg12); 7, Köln U8 (sg12); 8, Köln P3 (sg1); 9, Köln P6 (sg1); 10, Köln U4 (sg1); 11, Köln U7 (sg1); 12, Köln U3 (sg12); and 13, Köln U5 (sg12).

    Article Snippet: For macrorestriction analysis (MRA), chromosomal DNAs were digested overnight with Sfi I, Asc I, and Not I (New England Biolabs, Schwahlbach, Germany) and separated with the CHEF III System (BioRad Laboratories, Munich, Germany) ( ).

    Techniques: Isolation

    PFGE profiles of Sfi I-, Apa I-, Dra I-, Spe I-, and Xba I-digested whole-cell DNA of O. urethralis COH-1 (A; lanes 1 to 5) and their Southern transfer, followed by hybridization with internal probes specific for bla ABA-1 (B) and bla CARB-8 (C). Lanes M, molecular size markers

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Chromosomal Integration of a Cephalosporinase Gene from Acinetobacter baumannii into Oligella urethralis as a Source of Acquired Resistance to ?-Lactams

    doi: 10.1128/AAC.47.5.1536-1542.2003

    Figure Lengend Snippet: PFGE profiles of Sfi I-, Apa I-, Dra I-, Spe I-, and Xba I-digested whole-cell DNA of O. urethralis COH-1 (A; lanes 1 to 5) and their Southern transfer, followed by hybridization with internal probes specific for bla ABA-1 (B) and bla CARB-8 (C). Lanes M, molecular size markers

    Article Snippet: Macrorestriction analysis of whole-cell DNA of O. urethralis COH-1 was done by the pulsed-field gel electrophoresis (PFGE) technique with Xba I, Spe I, Dra I, Apa I, and Sfi I (New England Biolabs), as reported previously ( , ).

    Techniques: Hybridization