i ceu i  (New England Biolabs)


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    Name:
    I CeuI
    Description:
    I CeuI 2 500 units
    Catalog Number:
    r0699l
    Price:
    302
    Size:
    2 500 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs i ceu i
    I CeuI
    I CeuI 2 500 units
    https://www.bioz.com/result/i ceu i/product/New England Biolabs
    Average 94 stars, based on 32 article reviews
    Price from $9.99 to $1999.99
    i ceu i - by Bioz Stars, 2020-07
    94/100 stars

    Images

    1) Product Images from "Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii"

    Article Title: Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evu119

    Estimation of Xenorhabdus poinarii strains genome size by PFGE of I- Ceu I-hydrolyzed genomic DNA. The separation of I- Ceu I fragments was optimized by using different electrophoresis conditions for fragments of different sizes: ( A ) a pulse ramp from 150 to 400 s for 45 h for I- Ceu I fragments between 500 and 4,000 kb in size; ( B ) a pulse ramp from 5 to 35 s for 24 h for fragments of less than 500 kb in size. Schematic representations of the I- Ceu I PFGE patterns under two sets of migration conditions, making it possible to separate fragments from 500 to 4,000 kb in size ( C ) and fragments from 10 to 500 kb in size ( D ), were also shown. Lane 1: Saccharomyces cerevisiae (strain 972h); lane 2: X. bovienii SS-2004; lane 3: X. poinarii AZ26; lane 4: X. poinarii G6; lane 5: X. poinarii SK72; lane 6: X. poinarii CU01; lane 7: X. poinarii NC33; lane 8: X. doucetiae FRM16; lane 9: Hansenula wingei (strain YB-4662-VIA). Dashed bands around 120 kb in strains Xp_AZ26 (lane 3) and Xp_SK72 (lane 4) correspond to fragments with a lower staining intensity, probably plasmids. *Although these bands are difficult to see on the gel photography, there were directly distinguishable on the gel and their sizes were confirmed by the theorical I- Ceu I pattern of the genome sequences of X. bovienii SS-2004 and X. poinarii G6. Fragment and genome sizes of the four unsequenced X. poinarii strains were evaluated with the X. poinarii G6 , X. bovienii SS-2004, and X. doucetiae FRM16 genomes used as a reference (lanes 2, 4, and 8) and molecular weight ladders (lanes 1 and 9).
    Figure Legend Snippet: Estimation of Xenorhabdus poinarii strains genome size by PFGE of I- Ceu I-hydrolyzed genomic DNA. The separation of I- Ceu I fragments was optimized by using different electrophoresis conditions for fragments of different sizes: ( A ) a pulse ramp from 150 to 400 s for 45 h for I- Ceu I fragments between 500 and 4,000 kb in size; ( B ) a pulse ramp from 5 to 35 s for 24 h for fragments of less than 500 kb in size. Schematic representations of the I- Ceu I PFGE patterns under two sets of migration conditions, making it possible to separate fragments from 500 to 4,000 kb in size ( C ) and fragments from 10 to 500 kb in size ( D ), were also shown. Lane 1: Saccharomyces cerevisiae (strain 972h); lane 2: X. bovienii SS-2004; lane 3: X. poinarii AZ26; lane 4: X. poinarii G6; lane 5: X. poinarii SK72; lane 6: X. poinarii CU01; lane 7: X. poinarii NC33; lane 8: X. doucetiae FRM16; lane 9: Hansenula wingei (strain YB-4662-VIA). Dashed bands around 120 kb in strains Xp_AZ26 (lane 3) and Xp_SK72 (lane 4) correspond to fragments with a lower staining intensity, probably plasmids. *Although these bands are difficult to see on the gel photography, there were directly distinguishable on the gel and their sizes were confirmed by the theorical I- Ceu I pattern of the genome sequences of X. bovienii SS-2004 and X. poinarii G6. Fragment and genome sizes of the four unsequenced X. poinarii strains were evaluated with the X. poinarii G6 , X. bovienii SS-2004, and X. doucetiae FRM16 genomes used as a reference (lanes 2, 4, and 8) and molecular weight ladders (lanes 1 and 9).

    Techniques Used: Electrophoresis, Migration, Staining, Molecular Weight

    2) Product Images from "CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes"

    Article Title: CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17180-w

    Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.
    Figure Legend Snippet: Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.

    Techniques Used: Plasmid Preparation, CRISPR, Multiplexing, Expressing, Amplification, Polymerase Chain Reaction, Clone Assay, Homologous Recombination, RNA Expression

    3) Product Images from "CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes"

    Article Title: CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17180-w

    Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.
    Figure Legend Snippet: Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.

    Techniques Used: Plasmid Preparation, CRISPR, Multiplexing, Expressing, Amplification, Polymerase Chain Reaction, Clone Assay, Homologous Recombination, RNA Expression

    4) Product Images from "Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures"

    Article Title: Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evq056

    ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.
    Figure Legend Snippet: ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.

    Techniques Used:

    Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.
    Figure Legend Snippet: Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.

    Techniques Used: Sequencing, Blocking Assay

    ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.
    Figure Legend Snippet: ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.

    Techniques Used:

    5) Product Images from "VanA-Type Enterococci from Humans, Animals, and Food: Species Distribution, Population Structure, Tn1546 Typing and Location, and Virulence Determinants ▿"

    Article Title: VanA-Type Enterococci from Humans, Animals, and Food: Species Distribution, Population Structure, Tn1546 Typing and Location, and Virulence Determinants ▿

    Journal:

    doi: 10.1128/AEM.02239-06

    PFGE of S1-digested (A) and I-CeuI-digested (C) total DNA and corresponding vanA (B and E) and 16S rRNA gene (D) hybridization. Lane 1, E. faecium HI-MI28; lane 2, E. faecium HI-MI34; lane 3, E. faecium HI-MI60; lane 4, E. faecium HI-MI32; lane 5, E.
    Figure Legend Snippet: PFGE of S1-digested (A) and I-CeuI-digested (C) total DNA and corresponding vanA (B and E) and 16S rRNA gene (D) hybridization. Lane 1, E. faecium HI-MI28; lane 2, E. faecium HI-MI34; lane 3, E. faecium HI-MI60; lane 4, E. faecium HI-MI32; lane 5, E.

    Techniques Used: Hybridization

    6) Product Images from "Chromosomally Encoded blaCMY-2 Located on a Novel SXT/R391-Related Integrating Conjugative Element in a Proteus mirabilis Clinical Isolate ▿"

    Article Title: Chromosomally Encoded blaCMY-2 Located on a Novel SXT/R391-Related Integrating Conjugative Element in a Proteus mirabilis Clinical Isolate ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00111-10

    Localization of bla CMY-2 and int in P. mirabilis TUM4660 and its transconjugant. (A) Whole genomic DNAs of P. mirabilis TUM4660 (lane 1), E. coli ML4909 (lane 2), and E. coli TUM4670 (lane 3) were digested with I-CeuI, and the restricted fragments were subjected to pulsed-field gel electrophoresis. DNA fragments were transferred to a nylon membrane and hybridized with probes specific to the 23S rRNA gene (B), bla CMY-2 (C), and int (D).
    Figure Legend Snippet: Localization of bla CMY-2 and int in P. mirabilis TUM4660 and its transconjugant. (A) Whole genomic DNAs of P. mirabilis TUM4660 (lane 1), E. coli ML4909 (lane 2), and E. coli TUM4670 (lane 3) were digested with I-CeuI, and the restricted fragments were subjected to pulsed-field gel electrophoresis. DNA fragments were transferred to a nylon membrane and hybridized with probes specific to the 23S rRNA gene (B), bla CMY-2 (C), and int (D).

    Techniques Used: Pulsed-Field Gel, Electrophoresis

    7) Product Images from "Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures"

    Article Title: Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evq056

    ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.
    Figure Legend Snippet: ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.

    Techniques Used:

    Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.
    Figure Legend Snippet: Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.

    Techniques Used: Sequencing, Blocking Assay

    ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.
    Figure Legend Snippet: ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.

    Techniques Used:

    8) Product Images from "Transposon-Assisted Cloning and Traceless Mutagenesis of Adenoviruses: Development of a Novel Vector Based on Species D †"

    Article Title: Transposon-Assisted Cloning and Traceless Mutagenesis of Adenoviruses: Development of a Novel Vector Based on Species D †

    Journal: Journal of Virology

    doi: 10.1128/JVI.00687-06

    Generation of the wt Ad19a genome upon Tn removal from B19aT51. (A) Schematic representation of the precise removal of the Tn. The KnR gene (arrow) of the Tn (open double arrows) was removed in vitro by I-SceI/I-CeuI meganuclease double digestion (meganuclease sites are indicated by gray lines) followed by end filling and ligation, generating B19aT51ΔKn. In parallel, a PCR was performed using primers specific to the Tn ends (open arrows) flanked by 40-bp homologies to the target sites in Ad (black and gray boxes). In the forward primer the entire target repeat (12345) was incorporated, whereas only the last 3 bp of the right target repeat were included into the homology region of the reverse primer. Target repeats are indicated on either side of the Tn by black and gray numbers. This Tn-containing PCR fragment was introduced into the B19aT51ΔKn by ET recombination, whereby the orientation of the Tn in the newly generated BAC B19aT51T becomes reversed. (B) Tn removal from B19aT51T. B19aT51T was treated with TnsABC* transposase, which excises the Tn, leaving compatible 3-base-long 5′ overhangs on the BAC ends. Simple ligation reconstitutes the 5-bp wt Ad target sequence, thereby generating a BAC containing the wt Ad19a genome (B19a). (C) Restriction analysis of BAC clones and their derived Ads. The XhoI patterns of B19aT51, B19aT51ΔKn, and B19aT51T are shown in lanes 1 to 3, respectively, with Tn-containing fragments indicated by asterisks. The HindIII pattern of BACs (lanes 5 to 7) and reconstituted viruses (lanes 8 and 9) is also shown. Fragment B-derived bands are indicated by asterisks. A HindIII-PacI double digest of B19a DNA releases the end fragments (C and one of the DD′ fragments) from the vector backbone (black arrowhead), eliminating fragment a (lane 7). BAC-derived Ad19a, Ad19aB; wt Ad19a, Ad19a. M indicates the lane of DNA markers (NEB) with numbers in kilobases.
    Figure Legend Snippet: Generation of the wt Ad19a genome upon Tn removal from B19aT51. (A) Schematic representation of the precise removal of the Tn. The KnR gene (arrow) of the Tn (open double arrows) was removed in vitro by I-SceI/I-CeuI meganuclease double digestion (meganuclease sites are indicated by gray lines) followed by end filling and ligation, generating B19aT51ΔKn. In parallel, a PCR was performed using primers specific to the Tn ends (open arrows) flanked by 40-bp homologies to the target sites in Ad (black and gray boxes). In the forward primer the entire target repeat (12345) was incorporated, whereas only the last 3 bp of the right target repeat were included into the homology region of the reverse primer. Target repeats are indicated on either side of the Tn by black and gray numbers. This Tn-containing PCR fragment was introduced into the B19aT51ΔKn by ET recombination, whereby the orientation of the Tn in the newly generated BAC B19aT51T becomes reversed. (B) Tn removal from B19aT51T. B19aT51T was treated with TnsABC* transposase, which excises the Tn, leaving compatible 3-base-long 5′ overhangs on the BAC ends. Simple ligation reconstitutes the 5-bp wt Ad target sequence, thereby generating a BAC containing the wt Ad19a genome (B19a). (C) Restriction analysis of BAC clones and their derived Ads. The XhoI patterns of B19aT51, B19aT51ΔKn, and B19aT51T are shown in lanes 1 to 3, respectively, with Tn-containing fragments indicated by asterisks. The HindIII pattern of BACs (lanes 5 to 7) and reconstituted viruses (lanes 8 and 9) is also shown. Fragment B-derived bands are indicated by asterisks. A HindIII-PacI double digest of B19a DNA releases the end fragments (C and one of the DD′ fragments) from the vector backbone (black arrowhead), eliminating fragment a (lane 7). BAC-derived Ad19a, Ad19aB; wt Ad19a, Ad19a. M indicates the lane of DNA markers (NEB) with numbers in kilobases.

    Techniques Used: In Vitro, Ligation, Polymerase Chain Reaction, Generated, BAC Assay, Sequencing, Clone Assay, Derivative Assay, Plasmid Preparation

    9) Product Images from "Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii"

    Article Title: Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evu119

    Estimation of Xenorhabdus poinarii strains genome size by PFGE of I- Ceu I-hydrolyzed genomic DNA. The separation of I- Ceu I fragments was optimized by using different electrophoresis conditions for fragments of different sizes: ( A ) a pulse ramp from 150 to 400 s for 45 h for I- Ceu I fragments between 500 and 4,000 kb in size; ( B ) a pulse ramp from 5 to 35 s for 24 h for fragments of less than 500 kb in size. Schematic representations of the I- Ceu I PFGE patterns under two sets of migration conditions, making it possible to separate fragments from 500 to 4,000 kb in size ( C ) and fragments from 10 to 500 kb in size ( D ), were also shown. Lane 1: Saccharomyces cerevisiae (strain 972h); lane 2: X. bovienii SS-2004; lane 3: X. poinarii AZ26; lane 4: X. poinarii G6; lane 5: X. poinarii SK72; lane 6: X. poinarii CU01; lane 7: X. poinarii NC33; lane 8: X. doucetiae FRM16; lane 9: Hansenula wingei (strain YB-4662-VIA). Dashed bands around 120 kb in strains Xp_AZ26 (lane 3) and Xp_SK72 (lane 4) correspond to fragments with a lower staining intensity, probably plasmids. *Although these bands are difficult to see on the gel photography, there were directly distinguishable on the gel and their sizes were confirmed by the theorical I- Ceu I pattern of the genome sequences of X. bovienii SS-2004 and X. poinarii G6. Fragment and genome sizes of the four unsequenced X. poinarii strains were evaluated with the X. poinarii G6 , X. bovienii SS-2004, and X. doucetiae FRM16 genomes used as a reference (lanes 2, 4, and 8) and molecular weight ladders (lanes 1 and 9).
    Figure Legend Snippet: Estimation of Xenorhabdus poinarii strains genome size by PFGE of I- Ceu I-hydrolyzed genomic DNA. The separation of I- Ceu I fragments was optimized by using different electrophoresis conditions for fragments of different sizes: ( A ) a pulse ramp from 150 to 400 s for 45 h for I- Ceu I fragments between 500 and 4,000 kb in size; ( B ) a pulse ramp from 5 to 35 s for 24 h for fragments of less than 500 kb in size. Schematic representations of the I- Ceu I PFGE patterns under two sets of migration conditions, making it possible to separate fragments from 500 to 4,000 kb in size ( C ) and fragments from 10 to 500 kb in size ( D ), were also shown. Lane 1: Saccharomyces cerevisiae (strain 972h); lane 2: X. bovienii SS-2004; lane 3: X. poinarii AZ26; lane 4: X. poinarii G6; lane 5: X. poinarii SK72; lane 6: X. poinarii CU01; lane 7: X. poinarii NC33; lane 8: X. doucetiae FRM16; lane 9: Hansenula wingei (strain YB-4662-VIA). Dashed bands around 120 kb in strains Xp_AZ26 (lane 3) and Xp_SK72 (lane 4) correspond to fragments with a lower staining intensity, probably plasmids. *Although these bands are difficult to see on the gel photography, there were directly distinguishable on the gel and their sizes were confirmed by the theorical I- Ceu I pattern of the genome sequences of X. bovienii SS-2004 and X. poinarii G6. Fragment and genome sizes of the four unsequenced X. poinarii strains were evaluated with the X. poinarii G6 , X. bovienii SS-2004, and X. doucetiae FRM16 genomes used as a reference (lanes 2, 4, and 8) and molecular weight ladders (lanes 1 and 9).

    Techniques Used: Electrophoresis, Migration, Staining, Molecular Weight

    10) Product Images from "A recombineering based approach for high-throughput conditional knockout targeting vector construction"

    Article Title: A recombineering based approach for high-throughput conditional knockout targeting vector construction

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm163

    Construction of conditional knockout targeting vectors using the new recombineering reagents. ( A ) The genomic structure of a locus with exons 3–5 to be deleted in the cko allele. The Bsd cassette flanked by two rare cutter sites, I-SceI and I-CeuI, is targeted to the 5′ side of the intended deletion region. Subsequently, the loxP-F3-PGK-EM7-Neo-F3 ( Neo ) cassette is targeted to the 3′ side of the deletion region. The point mutation present in the Neo coding sequence of PL452 and PL451 plasmids ( 10 , 36 ) was corrected in this Neo cassette which resulted in higher resistance to Kanamycin in E. coli and a 2-fold increase in the number of G418-resistant ES colonies. Coloured lines represent the short homology arms in recombineering. ( B ) The genomic DNA fragment is retrieved from the BAC to PL611, which has the Amp R gene. In a typical cko vector, we choose 4–5-kb genomic DNA as the left homology arm (5′), and 2–3 kb as the right homology arm (3′). The genomic DNA region to be deleted is generally between 1 and 7 kb. ( C ) The Bsd cassette can conveniently be replaced by a reporter, i.e. lacZ , in a simple ligation reaction. The final targeting vector has the reporter flanked by two FRT sites followed by a lox P site at the 5′ side of the intended deletion region, and a F3 flanked Neo cassette providing positive selection in ES cells. The negative selection marker TK is added to the vector backbone by recombineering. The vector is linearized with the rare-cutter I-PpoI.
    Figure Legend Snippet: Construction of conditional knockout targeting vectors using the new recombineering reagents. ( A ) The genomic structure of a locus with exons 3–5 to be deleted in the cko allele. The Bsd cassette flanked by two rare cutter sites, I-SceI and I-CeuI, is targeted to the 5′ side of the intended deletion region. Subsequently, the loxP-F3-PGK-EM7-Neo-F3 ( Neo ) cassette is targeted to the 3′ side of the deletion region. The point mutation present in the Neo coding sequence of PL452 and PL451 plasmids ( 10 , 36 ) was corrected in this Neo cassette which resulted in higher resistance to Kanamycin in E. coli and a 2-fold increase in the number of G418-resistant ES colonies. Coloured lines represent the short homology arms in recombineering. ( B ) The genomic DNA fragment is retrieved from the BAC to PL611, which has the Amp R gene. In a typical cko vector, we choose 4–5-kb genomic DNA as the left homology arm (5′), and 2–3 kb as the right homology arm (3′). The genomic DNA region to be deleted is generally between 1 and 7 kb. ( C ) The Bsd cassette can conveniently be replaced by a reporter, i.e. lacZ , in a simple ligation reaction. The final targeting vector has the reporter flanked by two FRT sites followed by a lox P site at the 5′ side of the intended deletion region, and a F3 flanked Neo cassette providing positive selection in ES cells. The negative selection marker TK is added to the vector backbone by recombineering. The vector is linearized with the rare-cutter I-PpoI.

    Techniques Used: Knock-Out, Mutagenesis, Sequencing, BAC Assay, Plasmid Preparation, Ligation, Selection, Marker

    11) Product Images from "Characterization of Genes Encoding for Acquired Bacitracin Resistance in Clostridium perfringens"

    Article Title: Characterization of Genes Encoding for Acquired Bacitracin Resistance in Clostridium perfringens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044449

    PFGE and hybridization analysis of I-CeuI and MluI double-digested DNA of the bacitracin resistant C. perfringens strain c1261_A. PFGE analysis of C. perfringens strain c1261_A total DNA (A). Southern blot of C. perfringens isolate c1261_A total DNA probed with rrn (B) and with bcrB (C). Sizes (in kilobases) are indicated on the left.
    Figure Legend Snippet: PFGE and hybridization analysis of I-CeuI and MluI double-digested DNA of the bacitracin resistant C. perfringens strain c1261_A. PFGE analysis of C. perfringens strain c1261_A total DNA (A). Southern blot of C. perfringens isolate c1261_A total DNA probed with rrn (B) and with bcrB (C). Sizes (in kilobases) are indicated on the left.

    Techniques Used: Hybridization, Southern Blot

    12) Product Images from "Genetic Characterization of Atypical Citrobacter freundii"

    Article Title: Genetic Characterization of Atypical Citrobacter freundii

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074120

    Pulsed-field gel electrophoresis profiles of l- Ceu I restriction fragments from C. freundii isolates. a) Pulsed-field gel shows Ceu I profiles: line 1, C. freundii E9750 NCTC; lane 2, FMU108327/P; lane 3, FMU108327/A 10 and lane 4; S. Typhimurium LT2 ATCC 700720. The results of Southern blotting analysis appear with capital letters. These indicate the Ceús individual fragments of C. freundii corresponding to those of S . Typhimurium LT2; b) chromosome sizes and molecular weights of individual Ceu I fragments. It also shows the location of every marker gene according to its correspondence in each Ceu I fragment. The Ceu I-C and Ceu I-F fragments of C. freundii were determined by probing the isolated DNA from S. Typhimurium LT2 Ceu I-C and Ceu I-F fragments; c) the gel from panel a were blotted to N+nylon membranes and probed as follows: lanes 1–4 met H ( Table S1 ); lanes 5–8 DNA from C. freundii fragment Ceu I-H and lanes 9–12, met A. Ceu I fragments identified by the probes are indicated inside the images with capital letters.
    Figure Legend Snippet: Pulsed-field gel electrophoresis profiles of l- Ceu I restriction fragments from C. freundii isolates. a) Pulsed-field gel shows Ceu I profiles: line 1, C. freundii E9750 NCTC; lane 2, FMU108327/P; lane 3, FMU108327/A 10 and lane 4; S. Typhimurium LT2 ATCC 700720. The results of Southern blotting analysis appear with capital letters. These indicate the Ceús individual fragments of C. freundii corresponding to those of S . Typhimurium LT2; b) chromosome sizes and molecular weights of individual Ceu I fragments. It also shows the location of every marker gene according to its correspondence in each Ceu I fragment. The Ceu I-C and Ceu I-F fragments of C. freundii were determined by probing the isolated DNA from S. Typhimurium LT2 Ceu I-C and Ceu I-F fragments; c) the gel from panel a were blotted to N+nylon membranes and probed as follows: lanes 1–4 met H ( Table S1 ); lanes 5–8 DNA from C. freundii fragment Ceu I-H and lanes 9–12, met A. Ceu I fragments identified by the probes are indicated inside the images with capital letters.

    Techniques Used: Pulsed-Field Gel, Electrophoresis, Southern Blot, Marker, Isolation

    13) Product Images from "Molecular Characterization of a Carbapenem-Hydrolyzing Class A ?-Lactamase, SFC-1, from Serratia fonticola UTAD54"

    Article Title: Molecular Characterization of a Carbapenem-Hydrolyzing Class A ?-Lactamase, SFC-1, from Serratia fonticola UTAD54

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.48.6.2321-2324.2004

    Hybridizations to I-CeuI fragments generated from the genome of S. fonticola UTAD54 and separated by pulsed-field gel electrophoresis. Lane 1, hybridization with SFC-1 probe; lane 2, hybridization with probe for naturally occurring class A β-lactamases of S. fonticola ; lane 3, hybridization using a probe for rRNA genes; lane 4, concatemers of phage lambda DNA.
    Figure Legend Snippet: Hybridizations to I-CeuI fragments generated from the genome of S. fonticola UTAD54 and separated by pulsed-field gel electrophoresis. Lane 1, hybridization with SFC-1 probe; lane 2, hybridization with probe for naturally occurring class A β-lactamases of S. fonticola ; lane 3, hybridization using a probe for rRNA genes; lane 4, concatemers of phage lambda DNA.

    Techniques Used: Generated, Pulsed-Field Gel, Electrophoresis, Hybridization, Lambda DNA Preparation

    14) Product Images from "Genomic Diversity of Erwinia carotovora subsp. carotovora and Its Correlation with Virulence"

    Article Title: Genomic Diversity of Erwinia carotovora subsp. carotovora and Its Correlation with Virulence

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.70.5.3013-3023.2004

    Genomic fingerprints of E. carotovora subsp. carotovora as determined by PFGE. (A) I-CeuI genomic cleavage patterns of representative E. carotovora subsp. carotovora strains. Strains WPP17 and WPP19 had unique chromosomal structures compared to the chromosomal structures of other E. carotovora subsp. carotovora strains. (B) Detection of large plasmids in E. carotovora subsp. carotovora 71 (serogroup 3) and E. carotovora subsp. carotovora 63 (serogroup 9). Undigested genomic DNA was compared to I-CeuI-digested DNA of the same strains. In the left panel, the arrows indicate the positions of the putative plasmids. Electrophoresis was performed by using pulse times of 25 to 45 s at 200 V for 20 h at 12°C. Lanes M contained markers. Ecc, E. carotovora subsp. carotovora ; SG, serogroup.
    Figure Legend Snippet: Genomic fingerprints of E. carotovora subsp. carotovora as determined by PFGE. (A) I-CeuI genomic cleavage patterns of representative E. carotovora subsp. carotovora strains. Strains WPP17 and WPP19 had unique chromosomal structures compared to the chromosomal structures of other E. carotovora subsp. carotovora strains. (B) Detection of large plasmids in E. carotovora subsp. carotovora 71 (serogroup 3) and E. carotovora subsp. carotovora 63 (serogroup 9). Undigested genomic DNA was compared to I-CeuI-digested DNA of the same strains. In the left panel, the arrows indicate the positions of the putative plasmids. Electrophoresis was performed by using pulse times of 25 to 45 s at 200 V for 20 h at 12°C. Lanes M contained markers. Ecc, E. carotovora subsp. carotovora ; SG, serogroup.

    Techniques Used: Electrophoresis

    Construction of an I-CeuI physical map. (A) I-CeuI cleavage patterns of genomic DNA of WPP14 and I-CeuI-restricted derivatives. (B) Circular genome map based on restriction data in panel A. The mapped I-CeuI sites localized seven rrn operons, which were designated rrnA through rrnG , and the flanking junction I-CeuI fragments were numbered 1 through 7 in ascending order (the smallest fragment was fragment 1).
    Figure Legend Snippet: Construction of an I-CeuI physical map. (A) I-CeuI cleavage patterns of genomic DNA of WPP14 and I-CeuI-restricted derivatives. (B) Circular genome map based on restriction data in panel A. The mapped I-CeuI sites localized seven rrn operons, which were designated rrnA through rrnG , and the flanking junction I-CeuI fragments were numbered 1 through 7 in ascending order (the smallest fragment was fragment 1).

    Techniques Used:

    15) Product Images from "Detection of SGI1/PGI1 Elements and Resistance to Extended-Spectrum Cephalosporins in Proteae of Animal Origin in France"

    Article Title: Detection of SGI1/PGI1 Elements and Resistance to Extended-Spectrum Cephalosporins in Proteae of Animal Origin in France

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.00032

    Chromosomal localization of bla CMY -2 in the relevant P. mirabilis isolates. (A) Whole genomic DNAs of isolates 34381 (lane1), 37665 (lane 2), 38327 (lane 3), 38368 (lane 4), 39165 (lane 5), 39175 (lane 6), 39193 (lane 7), and 39214 (lane 8) were digested with I-Ceu I, and the restricted fragments subjected to PFGE. DNA fragments were transferred to a nylon membrane and hybridized with probes specific to bla CMY -2 (B) , and the 23S rRNA gene (C) . The arrows indicate the bands of interest.
    Figure Legend Snippet: Chromosomal localization of bla CMY -2 in the relevant P. mirabilis isolates. (A) Whole genomic DNAs of isolates 34381 (lane1), 37665 (lane 2), 38327 (lane 3), 38368 (lane 4), 39165 (lane 5), 39175 (lane 6), 39193 (lane 7), and 39214 (lane 8) were digested with I-Ceu I, and the restricted fragments subjected to PFGE. DNA fragments were transferred to a nylon membrane and hybridized with probes specific to bla CMY -2 (B) , and the 23S rRNA gene (C) . The arrows indicate the bands of interest.

    Techniques Used:

    16) Product Images from "Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures"

    Article Title: Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evq056

    ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.
    Figure Legend Snippet: ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.

    Techniques Used:

    Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.
    Figure Legend Snippet: Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.

    Techniques Used: Sequencing, Blocking Assay

    ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.
    Figure Legend Snippet: ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.

    Techniques Used:

    17) Product Images from "Efficient assembly of de novo human artificial chromosomes from large genomic loci"

    Article Title: Efficient assembly of de novo human artificial chromosomes from large genomic loci

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-5-21

    Strategy for construction of a bimolecular, prefabricated, linear HAC vector. Digestion of BAC-CEN and BAC-GEN vectors with the ultra-rare homing endonucleases I-CeuI and PI-SceI permits directional ligation of both
    Figure Legend Snippet: Strategy for construction of a bimolecular, prefabricated, linear HAC vector. Digestion of BAC-CEN and BAC-GEN vectors with the ultra-rare homing endonucleases I-CeuI and PI-SceI permits directional ligation of both "arms" to form a linear HAC vector.

    Techniques Used: HAC Assay, Plasmid Preparation, BAC Assay, Ligation

    18) Product Images from "Genetic and Biochemical Characterization of an Acquired Subgroup B3 Metallo-?-Lactamase Gene, blaAIM-1, and Its Unique Genetic Context in Pseudomonas aeruginosa from Australia"

    Article Title: Genetic and Biochemical Characterization of an Acquired Subgroup B3 Metallo-?-Lactamase Gene, blaAIM-1, and Its Unique Genetic Context in Pseudomonas aeruginosa from Australia

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.05654-11

    Evidence of movement of IS CR15 and bla AIM-1 , and relatedness of AIM-1-positive strains. All panels a marker (lane M), WCH2677 (lane 1), WCH2813 (lane 2), and WCH2837 (lane 3). (A) SpeI digestion. (B) I-CeuI digestion. (C) S1 digestion and probing with
    Figure Legend Snippet: Evidence of movement of IS CR15 and bla AIM-1 , and relatedness of AIM-1-positive strains. All panels a marker (lane M), WCH2677 (lane 1), WCH2813 (lane 2), and WCH2837 (lane 3). (A) SpeI digestion. (B) I-CeuI digestion. (C) S1 digestion and probing with

    Techniques Used: Marker

    Related Articles

    Purification:

    Article Title: CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes
    Article Snippet: .. I-Ceu I and PI-Sce I-digested pAdFTC was dephosphorylated by adding 1 µl calf intestinal alkaline phosphatase (New England Biolabs) and incubated for 1 h at 37 °C and subsequently purified by Ph-Cl extraction followed by EtOH precipitation. .. Finally CBh-Cas9-gRNA or the Tre-Cas9-TetOn3G-gRNA expression cassette were ligated into linearized and dephosphorylated pAdFTC using 400U of T4 DNA ligase (New England Biolabs) and at a molar ratio of vector to insert of 1:3, respectively.

    Incubation:

    Article Title: CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes
    Article Snippet: .. I-Ceu I and PI-Sce I-digested pAdFTC was dephosphorylated by adding 1 µl calf intestinal alkaline phosphatase (New England Biolabs) and incubated for 1 h at 37 °C and subsequently purified by Ph-Cl extraction followed by EtOH precipitation. .. Finally CBh-Cas9-gRNA or the Tre-Cas9-TetOn3G-gRNA expression cassette were ligated into linearized and dephosphorylated pAdFTC using 400U of T4 DNA ligase (New England Biolabs) and at a molar ratio of vector to insert of 1:3, respectively.

    other:

    Article Title: Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures
    Article Snippet: Pulse times used were 1–30 s for Apa I or Sma I and 5–60 s for I-Ceu I.

    Derivative Assay:

    Article Title: Transposon-Assisted Cloning and Traceless Mutagenesis of Adenoviruses: Development of a Novel Vector Based on Species D †
    Article Snippet: .. Transprimer-1 is a Tn 7 -derived mobile DNA element that carries the Kn resistance gene flanked by I-CeuI and I-SceI meganuclease recognition sites and is contained in the Tn donor plasmid pGPS1.1 (NEB). .. Originally, the Transprimer-1 system was developed for sequencing larger segments of DNA , but it can also be successfully utilized to randomly label linear genomic DNA ( ).

    Expressing:

    Article Title: CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes
    Article Snippet: .. The single gRNA shuttle plasmid pShV-CBh-Cas9-gRNAHPV18E6 was digested with I-Ceu I and PI-Sce I (New England Biolabs) to release the CRISPR/Cas9 expression cassette HOM-Cas9-gRNAHPV18E6-HOM. .. Meanwhile RecA expression from the pSC101 plasmid in E. coli GBO5 containing the pHCAdV-eGFP-HOM-CCDB-AMP-HOM was induced by addition of arabinose and bacteria were grown for 40 min before they were rendered electrocompetent by consecutive washing with ice cold ddH2 O for three times .

    CRISPR:

    Article Title: CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes
    Article Snippet: .. The single gRNA shuttle plasmid pShV-CBh-Cas9-gRNAHPV18E6 was digested with I-Ceu I and PI-Sce I (New England Biolabs) to release the CRISPR/Cas9 expression cassette HOM-Cas9-gRNAHPV18E6-HOM. .. Meanwhile RecA expression from the pSC101 plasmid in E. coli GBO5 containing the pHCAdV-eGFP-HOM-CCDB-AMP-HOM was induced by addition of arabinose and bacteria were grown for 40 min before they were rendered electrocompetent by consecutive washing with ice cold ddH2 O for three times .

    Marker:

    Article Title: Chromosomally Encoded blaCMY-2 Located on a Novel SXT/R391-Related Integrating Conjugative Element in a Proteus mirabilis Clinical Isolate ▿
    Article Snippet: .. After digestion of whole-cell DNA with I-CeuI (New England Biolabs, Hertfordshire, United Kingdom), the resultant fragments were separated with a CHEF-Mapper apparatus (Bio-Rad, Hercules, CA) at 14°C, 6 V/cm, and a 120° switch angle, with a nonlinear switch time ramp of 5.3 to 49.9 s for 19.7 h. The sizes of the fragments were determined by comparison with a yeast chromosome pulsed-field gel electrophoresis marker (New England Biolabs). .. After Southern transfer to a Hybond-N+ membrane, the fragments were hybridized with three different PCR-generated probes: the probe for ampC , described above; a probe for the 23S rRNA gene, generated with primers 23S-For and 23S-Rev (Table ); and a probe for int , generated with primers IntFor1 and IntRev1.

    Plasmid Preparation:

    Article Title: CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes
    Article Snippet: .. The single gRNA shuttle plasmid pShV-CBh-Cas9-gRNAHPV18E6 was digested with I-Ceu I and PI-Sce I (New England Biolabs) to release the CRISPR/Cas9 expression cassette HOM-Cas9-gRNAHPV18E6-HOM. .. Meanwhile RecA expression from the pSC101 plasmid in E. coli GBO5 containing the pHCAdV-eGFP-HOM-CCDB-AMP-HOM was induced by addition of arabinose and bacteria were grown for 40 min before they were rendered electrocompetent by consecutive washing with ice cold ddH2 O for three times .

    Article Title: Transposon-Assisted Cloning and Traceless Mutagenesis of Adenoviruses: Development of a Novel Vector Based on Species D †
    Article Snippet: .. Transprimer-1 is a Tn 7 -derived mobile DNA element that carries the Kn resistance gene flanked by I-CeuI and I-SceI meganuclease recognition sites and is contained in the Tn donor plasmid pGPS1.1 (NEB). .. Originally, the Transprimer-1 system was developed for sequencing larger segments of DNA , but it can also be successfully utilized to randomly label linear genomic DNA ( ).

    Pulsed-Field Gel:

    Article Title: Chromosomally Encoded blaCMY-2 Located on a Novel SXT/R391-Related Integrating Conjugative Element in a Proteus mirabilis Clinical Isolate ▿
    Article Snippet: .. After digestion of whole-cell DNA with I-CeuI (New England Biolabs, Hertfordshire, United Kingdom), the resultant fragments were separated with a CHEF-Mapper apparatus (Bio-Rad, Hercules, CA) at 14°C, 6 V/cm, and a 120° switch angle, with a nonlinear switch time ramp of 5.3 to 49.9 s for 19.7 h. The sizes of the fragments were determined by comparison with a yeast chromosome pulsed-field gel electrophoresis marker (New England Biolabs). .. After Southern transfer to a Hybond-N+ membrane, the fragments were hybridized with three different PCR-generated probes: the probe for ampC , described above; a probe for the 23S rRNA gene, generated with primers 23S-For and 23S-Rev (Table ); and a probe for int , generated with primers IntFor1 and IntRev1.

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    New England Biolabs i ceu i
    Estimation of Xenorhabdus poinarii strains genome size by PFGE of I- <t>Ceu</t> I-hydrolyzed genomic DNA. The separation of I- <t>Ceu</t> I fragments was optimized by using different electrophoresis conditions for fragments of different sizes: ( A ) a pulse ramp from 150 to 400 s for 45 h for I- Ceu I fragments between 500 and 4,000 kb in size; ( B ) a pulse ramp from 5 to 35 s for 24 h for fragments of less than 500 kb in size. Schematic representations of the I- Ceu I PFGE patterns under two sets of migration conditions, making it possible to separate fragments from 500 to 4,000 kb in size ( C ) and fragments from 10 to 500 kb in size ( D ), were also shown. Lane 1: Saccharomyces cerevisiae (strain 972h); lane 2: X. bovienii SS-2004; lane 3: X. poinarii AZ26; lane 4: X. poinarii G6; lane 5: X. poinarii SK72; lane 6: X. poinarii CU01; lane 7: X. poinarii NC33; lane 8: X. doucetiae FRM16; lane 9: Hansenula wingei (strain YB-4662-VIA). Dashed bands around 120 kb in strains Xp_AZ26 (lane 3) and Xp_SK72 (lane 4) correspond to fragments with a lower staining intensity, probably plasmids. *Although these bands are difficult to see on the gel photography, there were directly distinguishable on the gel and their sizes were confirmed by the theorical I- Ceu I pattern of the genome sequences of X. bovienii SS-2004 and X. poinarii G6. Fragment and genome sizes of the four unsequenced X. poinarii strains were evaluated with the X. poinarii G6 , X. bovienii SS-2004, and X. doucetiae FRM16 genomes used as a reference (lanes 2, 4, and 8) and molecular weight ladders (lanes 1 and 9).
    I Ceu I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Estimation of Xenorhabdus poinarii strains genome size by PFGE of I- Ceu I-hydrolyzed genomic DNA. The separation of I- Ceu I fragments was optimized by using different electrophoresis conditions for fragments of different sizes: ( A ) a pulse ramp from 150 to 400 s for 45 h for I- Ceu I fragments between 500 and 4,000 kb in size; ( B ) a pulse ramp from 5 to 35 s for 24 h for fragments of less than 500 kb in size. Schematic representations of the I- Ceu I PFGE patterns under two sets of migration conditions, making it possible to separate fragments from 500 to 4,000 kb in size ( C ) and fragments from 10 to 500 kb in size ( D ), were also shown. Lane 1: Saccharomyces cerevisiae (strain 972h); lane 2: X. bovienii SS-2004; lane 3: X. poinarii AZ26; lane 4: X. poinarii G6; lane 5: X. poinarii SK72; lane 6: X. poinarii CU01; lane 7: X. poinarii NC33; lane 8: X. doucetiae FRM16; lane 9: Hansenula wingei (strain YB-4662-VIA). Dashed bands around 120 kb in strains Xp_AZ26 (lane 3) and Xp_SK72 (lane 4) correspond to fragments with a lower staining intensity, probably plasmids. *Although these bands are difficult to see on the gel photography, there were directly distinguishable on the gel and their sizes were confirmed by the theorical I- Ceu I pattern of the genome sequences of X. bovienii SS-2004 and X. poinarii G6. Fragment and genome sizes of the four unsequenced X. poinarii strains were evaluated with the X. poinarii G6 , X. bovienii SS-2004, and X. doucetiae FRM16 genomes used as a reference (lanes 2, 4, and 8) and molecular weight ladders (lanes 1 and 9).

    Journal: Genome Biology and Evolution

    Article Title: Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii

    doi: 10.1093/gbe/evu119

    Figure Lengend Snippet: Estimation of Xenorhabdus poinarii strains genome size by PFGE of I- Ceu I-hydrolyzed genomic DNA. The separation of I- Ceu I fragments was optimized by using different electrophoresis conditions for fragments of different sizes: ( A ) a pulse ramp from 150 to 400 s for 45 h for I- Ceu I fragments between 500 and 4,000 kb in size; ( B ) a pulse ramp from 5 to 35 s for 24 h for fragments of less than 500 kb in size. Schematic representations of the I- Ceu I PFGE patterns under two sets of migration conditions, making it possible to separate fragments from 500 to 4,000 kb in size ( C ) and fragments from 10 to 500 kb in size ( D ), were also shown. Lane 1: Saccharomyces cerevisiae (strain 972h); lane 2: X. bovienii SS-2004; lane 3: X. poinarii AZ26; lane 4: X. poinarii G6; lane 5: X. poinarii SK72; lane 6: X. poinarii CU01; lane 7: X. poinarii NC33; lane 8: X. doucetiae FRM16; lane 9: Hansenula wingei (strain YB-4662-VIA). Dashed bands around 120 kb in strains Xp_AZ26 (lane 3) and Xp_SK72 (lane 4) correspond to fragments with a lower staining intensity, probably plasmids. *Although these bands are difficult to see on the gel photography, there were directly distinguishable on the gel and their sizes were confirmed by the theorical I- Ceu I pattern of the genome sequences of X. bovienii SS-2004 and X. poinarii G6. Fragment and genome sizes of the four unsequenced X. poinarii strains were evaluated with the X. poinarii G6 , X. bovienii SS-2004, and X. doucetiae FRM16 genomes used as a reference (lanes 2, 4, and 8) and molecular weight ladders (lanes 1 and 9).

    Article Snippet: I-Ceu I (New England Biolabs) hydrolysis was performed as previously described ( ).

    Techniques: Electrophoresis, Migration, Staining, Molecular Weight

    Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.

    Journal: Scientific Reports

    Article Title: CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes

    doi: 10.1038/s41598-017-17180-w

    Figure Lengend Snippet: Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.

    Article Snippet: The single gRNA shuttle plasmid pShV-CBh-Cas9-gRNAHPV18E6 was digested with I-Ceu I and PI-Sce I (New England Biolabs) to release the CRISPR/Cas9 expression cassette HOM-Cas9-gRNAHPV18E6-HOM.

    Techniques: Plasmid Preparation, CRISPR, Multiplexing, Expressing, Amplification, Polymerase Chain Reaction, Clone Assay, Homologous Recombination, RNA Expression

    ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.

    Journal: Genome Biology and Evolution

    Article Title: Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

    doi: 10.1093/gbe/evq056

    Figure Lengend Snippet: ( A ) Locations of I- Ceu I recognition sites on the Lactococcus lactis IL1403 chromosome. I- Ceu I cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. ( B ) PFGE patterns of genomic DNA from L. lactis strains.

    Article Snippet: Pulse times used were 1–30 s for Apa I or Sma I and 5–60 s for I-Ceu I.

    Techniques:

    Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.

    Journal: Genome Biology and Evolution

    Article Title: Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

    doi: 10.1093/gbe/evq056

    Figure Lengend Snippet: Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I- Ceu I cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.

    Article Snippet: Pulse times used were 1–30 s for Apa I or Sma I and 5–60 s for I-Ceu I.

    Techniques: Sequencing, Blocking Assay

    ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.

    Journal: Genome Biology and Evolution

    Article Title: Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

    doi: 10.1093/gbe/evq056

    Figure Lengend Snippet: ( A ) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis . ( B ) Relationship between variances of different I- Ceu I fragments standardized by their average size and the average size of the corresponding fragments.

    Article Snippet: Pulse times used were 1–30 s for Apa I or Sma I and 5–60 s for I-Ceu I.

    Techniques: