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  • 99
    New England Biolabs spei
    Over-expressing L1-ectodomain in MDA-MB-468 cells . (A) Schematic diagram of Lvv 1879 vector containing L1ED. 3350 bp L1 ectodomain fragment was amplified and inserted into Lvv 1879 via <t>SpeI</t> and <t>XhoI</t> restriction enzyme sites. The constructed lentivirus was used to infect MDA-MB-468 cells to establish a new stable cell line. (B) Immunostaining and FACS analysis of L1CAM level in MDA-MB-468-L1ED compared to mock vector infected and plain MDA-MB-468 cells. (C) TCA precipitation and western blotting examining over-expressed L1 ectodomain release in MDA-MB-468-L1ED culture medium by monoclonal antibody 5G3. The amount of cell associated L1 in pellets was probed by polyclonal antibody NCAM-L1 (C-20).
    Spei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1984 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    spei - by Bioz Stars, 2020-04
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    99
    Thermo Fisher bcui spei
    Restriction patterns of plasmid DNA from E. coli transconjugants of E. coli , partial K. pneumoniae and S. marcescens isolates, and partial original S. marcescens isolates. Lane 1, E. coli transconjugant of E. coli E1; lanes 2 to 4, E. coli transconjugants of K. pneumoniae K1, K8, and K10; lanes 5 to 7, E. coli transconjugants of S. marcescens S1, S10, and S20; lanes 8 and 9, original S. marcescens S1 and S10; lane 10, E. coli EC600 as a negative control; M, 1-kb DNA ladder (MBI Fermentas). Plasmid DNA was digested with <t>EcoRI,</t> HindIII, and <t>BcuI</t> endonucleases.
    Bcui Spei, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs spei hf
    Restriction patterns of plasmid DNA from E. coli transconjugants of E. coli , partial K. pneumoniae and S. marcescens isolates, and partial original S. marcescens isolates. Lane 1, E. coli transconjugant of E. coli E1; lanes 2 to 4, E. coli transconjugants of K. pneumoniae K1, K8, and K10; lanes 5 to 7, E. coli transconjugants of S. marcescens S1, S10, and S20; lanes 8 and 9, original S. marcescens S1 and S10; lane 10, E. coli EC600 as a negative control; M, 1-kb DNA ladder (MBI Fermentas). Plasmid DNA was digested with <t>EcoRI,</t> HindIII, and <t>BcuI</t> endonucleases.
    Spei Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega spei promega enzymes
    (a to d) DNA fingerprints obtained by AP-PCR. (a) Primer 1; (b) primer 4; (c) primer 5; (d) primer 6 (data for the OPA primers are not shown). Lanes: 1, clinical isolate from our patient; 2 to 4, isolates from our patient's sponge; 5 to 7, isolates from three different sponges from other households; 8, clinical strain from control patient. In all cases, the first lane on the left corresponds to DNA ladders used as weight markers. (e) PFGE analysis with isolates iñ lanes 1, 2, and 3 from panels a to d. The three lanes on the left correspond to DNA digestion with Spe I, and the three lanes on the right correspond to DNA digestion with <t>Xba</t> I.
    Spei Promega Enzymes, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    GE Healthcare spei
    Molecular comparison of bla OXA-18 -producing P. aeruginosa isolates. (A and B) <t>PFGE</t> with <t>SpeI-restricted</t> DNA (A) and bla OXA-18 hybridization of the SpeI PFGE gel (B). Lane 1, P. aeruginosa MUS; lane 2, P. aeruginosa 1-63; lane 3, P. aeruginosa 1-22; lane
    Spei, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 98/100, based on 127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    spei  (Roche)
    95
    Roche spei
    Representative XbaI and <t>SpeI</t> <t>PFGE</t> profiles obtained for the 91 S. enterica serotype Typhi isolates under study. The PFGE profile numbering is indicated. The dendrograms generated by the BioNumerics software show the results of cluster analysis on the
    Spei, supplied by Roche, used in various techniques. Bioz Stars score: 95/100, based on 229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher spei
    Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows <t>SpeI</t> resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show BsmBI and SpeI resistant PCR fragments amplified from N. benthamiana genomic <t>DNA.</t> d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively
    Spei, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1771 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Bio-Rad spei
    Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows <t>SpeI</t> resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show BsmBI and SpeI resistant PCR fragments amplified from N. benthamiana genomic <t>DNA.</t> d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively
    Spei, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    spei - by Bioz Stars, 2020-04
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    86
    Boehringer Mannheim spei
    Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows <t>SpeI</t> resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show BsmBI and SpeI resistant PCR fragments amplified from N. benthamiana genomic <t>DNA.</t> d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively
    Spei, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore spei
    Minichromosome <t>DNA</t> is cut at only one site by <t>SpeI</t> or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.
    Spei, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega zrai spei
    Minichromosome <t>DNA</t> is cut at only one site by <t>SpeI</t> or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.
    Zrai Spei, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cellfree Sciences spei site
    Minichromosome <t>DNA</t> is cut at only one site by <t>SpeI</t> or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.
    Spei Site, supplied by Cellfree Sciences, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher spei enzyme
    Minichromosome <t>DNA</t> is cut at only one site by <t>SpeI</t> or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.
    Spei Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa spei digestion
    Minichromosome <t>DNA</t> is cut at only one site by <t>SpeI</t> or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.
    Spei Digestion, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Genewiz nsii spei fragment
    Minichromosome <t>DNA</t> is cut at only one site by <t>SpeI</t> or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.
    Nsii Spei Fragment, supplied by Genewiz, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher restriction enzyme spei
    Minichromosome <t>DNA</t> is cut at only one site by <t>SpeI</t> or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.
    Restriction Enzyme Spei, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Stratagene spei digested pbluescript
    Minichromosome <t>DNA</t> is cut at only one site by <t>SpeI</t> or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.
    Spei Digested Pbluescript, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Stratagene spei
    Minichromosome <t>DNA</t> is cut at only one site by <t>SpeI</t> or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.
    Spei, supplied by Stratagene, used in various techniques. Bioz Stars score: 94/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    spei - by Bioz Stars, 2020-04
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    95
    Addgene inc spei digested pcfj151
    Minichromosome <t>DNA</t> is cut at only one site by <t>SpeI</t> or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.
    Spei Digested Pcfj151, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    TaKaRa restriction enzyme spei
    Minichromosome <t>DNA</t> is cut at only one site by <t>SpeI</t> or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.
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    Roche spei restriction enzyme
    Simultaneous cleavage of phage <t>DNA</t> by CRISPR1-Cas and CRISPR3-Cas systems of S. thermophilus BIMs. A . Representation of the phage 2972 genome. Arrows symbolize the open reading frames (ORFs) as identified previously [18] and colors indicate associated transcriptional modules [24] . Three protospacers (PS61, PS78 and PS85) are positioned below the genome and the CRISPR-Cas systems that have acquired the respective spacers are specified (CRISPR1 or CRISPR3). B . The fragment sizes obtained from restriction and/or CRISPR digestion are specified. Probes used in the Southern blot assays are also indicated and details of the primers used are presented in Supplemental Table S1 . C . Comparison by Southern blot assays of CRISPR-Cas cleavage occurring in the two selected BIMs S61/S78, and S61/S85, which share the S61 spacer (CRISPR3), but not a second spacer (CRISPR3 and CRISPR1, respectively). The Southern profiles show that independent cleavage of CRISPR1-Cas (PS85) and CRISPR3-Cas (PS61 and PS78) systems can occur simultaneously, when infected by phage 2972. Corresponding cleavage bands are identified with asterisks, and their sizes are specified. The two BIMs were infected with phage 2972 for 45 minutes before total DNA was extracted. Five micrograms of <t>SpeI-restricted</t> DNA was then loaded in each lane. NI, non-infected strain. C+, 10 ng of 2972 phage DNA digested with SpeI.
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    Thermo Fisher spei restriction sites
    Simultaneous cleavage of phage <t>DNA</t> by CRISPR1-Cas and CRISPR3-Cas systems of S. thermophilus BIMs. A . Representation of the phage 2972 genome. Arrows symbolize the open reading frames (ORFs) as identified previously [18] and colors indicate associated transcriptional modules [24] . Three protospacers (PS61, PS78 and PS85) are positioned below the genome and the CRISPR-Cas systems that have acquired the respective spacers are specified (CRISPR1 or CRISPR3). B . The fragment sizes obtained from restriction and/or CRISPR digestion are specified. Probes used in the Southern blot assays are also indicated and details of the primers used are presented in Supplemental Table S1 . C . Comparison by Southern blot assays of CRISPR-Cas cleavage occurring in the two selected BIMs S61/S78, and S61/S85, which share the S61 spacer (CRISPR3), but not a second spacer (CRISPR3 and CRISPR1, respectively). The Southern profiles show that independent cleavage of CRISPR1-Cas (PS85) and CRISPR3-Cas (PS61 and PS78) systems can occur simultaneously, when infected by phage 2972. Corresponding cleavage bands are identified with asterisks, and their sizes are specified. The two BIMs were infected with phage 2972 for 45 minutes before total DNA was extracted. Five micrograms of <t>SpeI-restricted</t> DNA was then loaded in each lane. NI, non-infected strain. C+, 10 ng of 2972 phage DNA digested with SpeI.
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    SibEnzyme spei
    Simultaneous cleavage of phage <t>DNA</t> by CRISPR1-Cas and CRISPR3-Cas systems of S. thermophilus BIMs. A . Representation of the phage 2972 genome. Arrows symbolize the open reading frames (ORFs) as identified previously [18] and colors indicate associated transcriptional modules [24] . Three protospacers (PS61, PS78 and PS85) are positioned below the genome and the CRISPR-Cas systems that have acquired the respective spacers are specified (CRISPR1 or CRISPR3). B . The fragment sizes obtained from restriction and/or CRISPR digestion are specified. Probes used in the Southern blot assays are also indicated and details of the primers used are presented in Supplemental Table S1 . C . Comparison by Southern blot assays of CRISPR-Cas cleavage occurring in the two selected BIMs S61/S78, and S61/S85, which share the S61 spacer (CRISPR3), but not a second spacer (CRISPR3 and CRISPR1, respectively). The Southern profiles show that independent cleavage of CRISPR1-Cas (PS85) and CRISPR3-Cas (PS61 and PS78) systems can occur simultaneously, when infected by phage 2972. Corresponding cleavage bands are identified with asterisks, and their sizes are specified. The two BIMs were infected with phage 2972 for 45 minutes before total DNA was extracted. Five micrograms of <t>SpeI-restricted</t> DNA was then loaded in each lane. NI, non-infected strain. C+, 10 ng of 2972 phage DNA digested with SpeI.
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    Addgene inc spei
    Simultaneous cleavage of phage <t>DNA</t> by CRISPR1-Cas and CRISPR3-Cas systems of S. thermophilus BIMs. A . Representation of the phage 2972 genome. Arrows symbolize the open reading frames (ORFs) as identified previously [18] and colors indicate associated transcriptional modules [24] . Three protospacers (PS61, PS78 and PS85) are positioned below the genome and the CRISPR-Cas systems that have acquired the respective spacers are specified (CRISPR1 or CRISPR3). B . The fragment sizes obtained from restriction and/or CRISPR digestion are specified. Probes used in the Southern blot assays are also indicated and details of the primers used are presented in Supplemental Table S1 . C . Comparison by Southern blot assays of CRISPR-Cas cleavage occurring in the two selected BIMs S61/S78, and S61/S85, which share the S61 spacer (CRISPR3), but not a second spacer (CRISPR3 and CRISPR1, respectively). The Southern profiles show that independent cleavage of CRISPR1-Cas (PS85) and CRISPR3-Cas (PS61 and PS78) systems can occur simultaneously, when infected by phage 2972. Corresponding cleavage bands are identified with asterisks, and their sizes are specified. The two BIMs were infected with phage 2972 for 45 minutes before total DNA was extracted. Five micrograms of <t>SpeI-restricted</t> DNA was then loaded in each lane. NI, non-infected strain. C+, 10 ng of 2972 phage DNA digested with SpeI.
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    spei  (TaKaRa)
    99
    TaKaRa spei
    (A) PFGE of <t>SpeI-,</t> <t>XbaI-,</t> and HpaI-digested genomic DNA from multidrug-resistant P. aeruginosa IMCJ2.S1. (B) Southern blotting of the same gels with an aac(6′)-Iae probe. Lanes M, HindIII-digested λ phage DNA as a size marker.
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    spei  (Toyobo)
    86
    Toyobo spei
    (A) PFGE of <t>SpeI-,</t> <t>XbaI-,</t> and HpaI-digested genomic DNA from multidrug-resistant P. aeruginosa IMCJ2.S1. (B) Southern blotting of the same gels with an aac(6′)-Iae probe. Lanes M, HindIII-digested λ phage DNA as a size marker.
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    Reiss Manufacturing spey sj
    (A) PFGE of <t>SpeI-,</t> <t>XbaI-,</t> and HpaI-digested genomic DNA from multidrug-resistant P. aeruginosa IMCJ2.S1. (B) Southern blotting of the same gels with an aac(6′)-Iae probe. Lanes M, HindIII-digested λ phage DNA as a size marker.
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    Bio-Rad enzyme spei
    PFGE profiles of genomic <t>DNAs</t> of PER-1-positive P. aeruginosa isolates after digestion with <t>SpeI.</t> The profile of isolate Pa6NI, which was identical to that of Pa34SM, and that of isolate Pa105TR, which was different from that of Pa144TR by three bands,
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    TaKaRa ncoi spei
    PFGE profiles of genomic <t>DNAs</t> of PER-1-positive P. aeruginosa isolates after digestion with <t>SpeI.</t> The profile of isolate Pa6NI, which was identical to that of Pa34SM, and that of isolate Pa105TR, which was different from that of Pa144TR by three bands,
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    Image Search Results


    Over-expressing L1-ectodomain in MDA-MB-468 cells . (A) Schematic diagram of Lvv 1879 vector containing L1ED. 3350 bp L1 ectodomain fragment was amplified and inserted into Lvv 1879 via SpeI and XhoI restriction enzyme sites. The constructed lentivirus was used to infect MDA-MB-468 cells to establish a new stable cell line. (B) Immunostaining and FACS analysis of L1CAM level in MDA-MB-468-L1ED compared to mock vector infected and plain MDA-MB-468 cells. (C) TCA precipitation and western blotting examining over-expressed L1 ectodomain release in MDA-MB-468-L1ED culture medium by monoclonal antibody 5G3. The amount of cell associated L1 in pellets was probed by polyclonal antibody NCAM-L1 (C-20).

    Journal: Cancer Cell International

    Article Title: Soluble L1CAM promotes breast cancer cell adhesion and migration in vitro, but not invasion

    doi: 10.1186/1475-2867-10-34

    Figure Lengend Snippet: Over-expressing L1-ectodomain in MDA-MB-468 cells . (A) Schematic diagram of Lvv 1879 vector containing L1ED. 3350 bp L1 ectodomain fragment was amplified and inserted into Lvv 1879 via SpeI and XhoI restriction enzyme sites. The constructed lentivirus was used to infect MDA-MB-468 cells to establish a new stable cell line. (B) Immunostaining and FACS analysis of L1CAM level in MDA-MB-468-L1ED compared to mock vector infected and plain MDA-MB-468 cells. (C) TCA precipitation and western blotting examining over-expressed L1 ectodomain release in MDA-MB-468-L1ED culture medium by monoclonal antibody 5G3. The amount of cell associated L1 in pellets was probed by polyclonal antibody NCAM-L1 (C-20).

    Article Snippet: The following primers with SpeI or XhoI (NEB, Ipswich, MA) cleavage sites were used to amplify the sequence: sense, 5'-GAAACTAGTCGCCGGGAAAG-3'; antisense, 5'- GCCTCGAGGAGGGAGCC- 3'.

    Techniques: Expressing, Multiple Displacement Amplification, Plasmid Preparation, Amplification, Construct, Stable Transfection, Immunostaining, FACS, Infection, TCA Precipitation, Western Blot

    Plasmid stability. GFP expression was induced with 500 ng/ml of ATc. ON culture of day 0 was supplemented with 15 μg/ml kanamycin, while ON cultures of days 1–4 were supplemented with 50 μg/ml ampicillin. (A) GFP expression in F. novicida U112 pFNMB1 msfgfp ON cultures of day 0 and day 4. (B) GFP expression in F. novicida U112 pFNMB2 msfgfp ON cultures of day 0 and day 4. (A,B) Images are a merge of phase contrast and GFP channel. 26 × 39 μm fields of view are shown. Scale bar represent 5 μm. Representative replicates are shown. Three independent experiments were performed. (C) Survival assay performed with ON cultures of F. novicida U112 pFNMB1 msfgfp and F. novicida U112 pFNMB2 msfgfp plated on ampicillin and ampicillin/kanamycin plates. Three independent experiments were performed. (D) Digestion of pFNMB1 msfgfp with SacI and SpeI restriction enzymes. (E) Digestion of pFNMB2 msfgfp with SacI and SpeI restriction enzymes. (D,E) Plasmids were passaged in F. novicida for 4 days before being transformed into E. coli . Controls were isolated directly from E. coli . Representative replicates are shown. Three independent experiments were performed.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida

    doi: 10.3389/fcimb.2018.00284

    Figure Lengend Snippet: Plasmid stability. GFP expression was induced with 500 ng/ml of ATc. ON culture of day 0 was supplemented with 15 μg/ml kanamycin, while ON cultures of days 1–4 were supplemented with 50 μg/ml ampicillin. (A) GFP expression in F. novicida U112 pFNMB1 msfgfp ON cultures of day 0 and day 4. (B) GFP expression in F. novicida U112 pFNMB2 msfgfp ON cultures of day 0 and day 4. (A,B) Images are a merge of phase contrast and GFP channel. 26 × 39 μm fields of view are shown. Scale bar represent 5 μm. Representative replicates are shown. Three independent experiments were performed. (C) Survival assay performed with ON cultures of F. novicida U112 pFNMB1 msfgfp and F. novicida U112 pFNMB2 msfgfp plated on ampicillin and ampicillin/kanamycin plates. Three independent experiments were performed. (D) Digestion of pFNMB1 msfgfp with SacI and SpeI restriction enzymes. (E) Digestion of pFNMB2 msfgfp with SacI and SpeI restriction enzymes. (D,E) Plasmids were passaged in F. novicida for 4 days before being transformed into E. coli . Controls were isolated directly from E. coli . Representative replicates are shown. Three independent experiments were performed.

    Article Snippet: The next day, colonies were grown in liquid LB supplemented with 50 μg/ml kanamycin, plasmid DNA was isolated and 250 ng of each plasmid was digested with SacI-HF and SpeI restriction enzymes (New England BioLabs) for 1 h. As control, both plasmids were additionally isolated from E. coli directly, without passaging in F. novicida , and digested identically.

    Techniques: Plasmid Preparation, Expressing, Clonogenic Cell Survival Assay, Transformation Assay, Isolation

    PFGE profiles of Spe I-digested genomic DNAs from 21 Pseudomonas isolates carrying IMP- or VIM-type MBL genes. (A) Lanes 1 to 9 and 11, VIM-3-producing P. aeruginosa isolates NTU-23/99, PS-1205/00, PB-59, NTU-34/99, NTU-39/00, PS-64/99, NTU-26/99, NTU-33/99, NTU-43/00, and NTU-79/00, respectively; lane 10, VIM-2-producing P. putida isolate PS-693/00; lane M, a bacteriophage lambda ladder. (B) Lanes 1, 4, 6, 9, and 10, IMP-1 producer isolates PB-207, NTU-92/99, PB-166, PB-101, and PB-24, respectively; lanes 2, 3, 5, 7, and 8, VIM-2 producers isolates PS-1153/00, NTU-91/99, NTU-93/99, PS-579/00, and PS-679/00, respectively; lane M, a bacteriophage lambda ladder.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Metallo-?-Lactamases in Clinical Pseudomonas Isolates in Taiwan and Identification of VIM-3, a Novel Variant of the VIM-2 Enzyme

    doi: 10.1128/AAC.45.8.2224-2228.2001

    Figure Lengend Snippet: PFGE profiles of Spe I-digested genomic DNAs from 21 Pseudomonas isolates carrying IMP- or VIM-type MBL genes. (A) Lanes 1 to 9 and 11, VIM-3-producing P. aeruginosa isolates NTU-23/99, PS-1205/00, PB-59, NTU-34/99, NTU-39/00, PS-64/99, NTU-26/99, NTU-33/99, NTU-43/00, and NTU-79/00, respectively; lane 10, VIM-2-producing P. putida isolate PS-693/00; lane M, a bacteriophage lambda ladder. (B) Lanes 1, 4, 6, 9, and 10, IMP-1 producer isolates PB-207, NTU-92/99, PB-166, PB-101, and PB-24, respectively; lanes 2, 3, 5, 7, and 8, VIM-2 producers isolates PS-1153/00, NTU-91/99, NTU-93/99, PS-579/00, and PS-679/00, respectively; lane M, a bacteriophage lambda ladder.

    Article Snippet: Genomic DNAs prepared by the procedure of Piggot et al. ( ) were digested overnight with 10 U of Spe I (New England Biolabs, Beverly, Mass.) and subjected to pulsed-field gel electrophoresis (PFGE) as described elsewhere ( , ).

    Techniques:

    Restriction patterns of plasmid DNA from E. coli transconjugants of E. coli , partial K. pneumoniae and S. marcescens isolates, and partial original S. marcescens isolates. Lane 1, E. coli transconjugant of E. coli E1; lanes 2 to 4, E. coli transconjugants of K. pneumoniae K1, K8, and K10; lanes 5 to 7, E. coli transconjugants of S. marcescens S1, S10, and S20; lanes 8 and 9, original S. marcescens S1 and S10; lane 10, E. coli EC600 as a negative control; M, 1-kb DNA ladder (MBI Fermentas). Plasmid DNA was digested with EcoRI, HindIII, and BcuI endonucleases.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Emergence of Serratia marcescens, Klebsiella pneumoniae, and Escherichia coli Isolates Possessing the Plasmid-Mediated Carbapenem-Hydrolyzing β-Lactamase KPC-2 in Intensive Care Units of a Chinese Hospital

    doi: 10.1128/AAC.01539-07

    Figure Lengend Snippet: Restriction patterns of plasmid DNA from E. coli transconjugants of E. coli , partial K. pneumoniae and S. marcescens isolates, and partial original S. marcescens isolates. Lane 1, E. coli transconjugant of E. coli E1; lanes 2 to 4, E. coli transconjugants of K. pneumoniae K1, K8, and K10; lanes 5 to 7, E. coli transconjugants of S. marcescens S1, S10, and S20; lanes 8 and 9, original S. marcescens S1 and S10; lane 10, E. coli EC600 as a negative control; M, 1-kb DNA ladder (MBI Fermentas). Plasmid DNA was digested with EcoRI, HindIII, and BcuI endonucleases.

    Article Snippet: Plasmid DNAs were obtained with an Axyprep plasmid miniprep kit (Axygen Scientific) and were digested by various endonucleases, including EcoRI, HindIII, and BcuI (MBI Fermentas, Lithuania).

    Techniques: Plasmid Preparation, Negative Control

    (a to d) DNA fingerprints obtained by AP-PCR. (a) Primer 1; (b) primer 4; (c) primer 5; (d) primer 6 (data for the OPA primers are not shown). Lanes: 1, clinical isolate from our patient; 2 to 4, isolates from our patient's sponge; 5 to 7, isolates from three different sponges from other households; 8, clinical strain from control patient. In all cases, the first lane on the left corresponds to DNA ladders used as weight markers. (e) PFGE analysis with isolates iñ lanes 1, 2, and 3 from panels a to d. The three lanes on the left correspond to DNA digestion with Spe I, and the three lanes on the right correspond to DNA digestion with Xba I.

    Journal: Journal of Clinical Microbiology

    Article Title: Infection of Hickman Catheter by Pseudomonas (formerly Flavimonas) oryzihabitans Traced to a Synthetic Bath Sponge

    doi:

    Figure Lengend Snippet: (a to d) DNA fingerprints obtained by AP-PCR. (a) Primer 1; (b) primer 4; (c) primer 5; (d) primer 6 (data for the OPA primers are not shown). Lanes: 1, clinical isolate from our patient; 2 to 4, isolates from our patient's sponge; 5 to 7, isolates from three different sponges from other households; 8, clinical strain from control patient. In all cases, the first lane on the left corresponds to DNA ladders used as weight markers. (e) PFGE analysis with isolates iñ lanes 1, 2, and 3 from panels a to d. The three lanes on the left correspond to DNA digestion with Spe I, and the three lanes on the right correspond to DNA digestion with Xba I.

    Article Snippet: The low-frequency-of-cleavage restriction endonucleases Xba I and Spe I (Promega) were selected for use for DNA digestion.

    Techniques: Polymerase Chain Reaction

    Molecular comparison of bla OXA-18 -producing P. aeruginosa isolates. (A and B) PFGE with SpeI-restricted DNA (A) and bla OXA-18 hybridization of the SpeI PFGE gel (B). Lane 1, P. aeruginosa MUS; lane 2, P. aeruginosa 1-63; lane 3, P. aeruginosa 1-22; lane

    Journal:

    Article Title: Genetic Structure Associated with blaOXA-18, Encoding a Clavulanic Acid-Inhibited Extended-Spectrum Oxacillinase ▿

    doi: 10.1128/AAC.00403-08

    Figure Lengend Snippet: Molecular comparison of bla OXA-18 -producing P. aeruginosa isolates. (A and B) PFGE with SpeI-restricted DNA (A) and bla OXA-18 hybridization of the SpeI PFGE gel (B). Lane 1, P. aeruginosa MUS; lane 2, P. aeruginosa 1-63; lane 3, P. aeruginosa 1-22; lane

    Article Snippet: PFGE was performed using SpeI (Amersham Biosciences) as previously described ( ).

    Techniques: Hybridization

    Representative XbaI and SpeI PFGE profiles obtained for the 91 S. enterica serotype Typhi isolates under study. The PFGE profile numbering is indicated. The dendrograms generated by the BioNumerics software show the results of cluster analysis on the

    Journal:

    Article Title: Clonal Expansion and Microevolution of Quinolone-Resistant Salmonella enterica Serotype Typhi in Vietnam from 1996 to 2004 ▿

    doi: 10.1128/JCM.00948-07

    Figure Lengend Snippet: Representative XbaI and SpeI PFGE profiles obtained for the 91 S. enterica serotype Typhi isolates under study. The PFGE profile numbering is indicated. The dendrograms generated by the BioNumerics software show the results of cluster analysis on the

    Article Snippet: PFGE of XbaI (Roche)- and SpeI (Roche)-digested genomic DNA was carried out with all 91 Nalr serotype Typhi isolates in this study, as described previously ( ).

    Techniques: Generated, Software

    Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows SpeI resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show BsmBI and SpeI resistant PCR fragments amplified from N. benthamiana genomic DNA. d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively

    Journal: Plant Methods

    Article Title: A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard

    doi: 10.1186/s13007-016-0101-2

    Figure Lengend Snippet: Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows SpeI resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show BsmBI and SpeI resistant PCR fragments amplified from N. benthamiana genomic DNA. d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively

    Article Snippet: The resulting PCR products were purified with the QIAquick PCR purification kit (QIAGEN) following the manufacturer’s protocol and restriction reactions were set up with 500 ng of purified DNA and the corresponding restriction enzyme; BsmBI (Fermentas) for XT1 and SpeI (Fermentas) for XT2.

    Techniques: Mutagenesis, CRISPR, Expressing, Diagnostic Assay, Sequencing, Polymerase Chain Reaction, Amplification, Clone Assay

    Heightened ISR in EIF2B4 A392D cells. (A) Flow cytometry analysis of CHOP :: GFP and XBP1 :: Turquoise dual reporter-containing parental CHO-S21 and EIF2B4 A392D mutant cells. The cells were untreated (UT) or stimulated with 250 nM thapsigargin (Tg) or 0.5 mM histidinol (His) for 24 hours. Note the enhanced response of the CHOP :: GFP ISR reporter. (B) Bar diagram of the median ± S.D. of the reporter gene activity from experiments as shown in “A”. N = 3, *P = 0.0057 for Tg, *P = 0.037 for His, Unpaired t test. (C) Experimental design for tracking EIF2B4 A392D mutations. A fluorescent protein-marked sgRNA/Cas9 plasmid targeting EIF2B4 and a wildtype or EIF2B4 A392D mutant repair template marked by a silent Spe I mutation were co-transfected into CHO-S21 cells. Transfected cells (selected by FACS), were treated with histidinol and divided into four bins (Bin #1 to #4) by level of CHOP :: GFP expression. After recovery, genomic DNA was isolated from cells in each bin and the targeted region of EIF2B4 was amplified by PCR and digested with Spe I to reveal frequency of targeting by either repair template. (D) PCR fragments digested with Spe I from genomic DNA of the indicated bins, visualized on an agarose gel. Shown is an image of a representative experiment reproduced twice. (E) Plot of the distribution of Spe I digested fragments in the four bins of transduced cells from the experiment in “D”. The band intensities of the digested fragments (reporting on recombination of the wildtype or mutant repair template) were normalized to total PCR product intensity and the distribution of the relative frequency of recombination in the different bins was plotted. Note the enrichment for recombination of the EIF2B4 A392D mutant repair template in the ISR High bin.

    Journal: PLoS ONE

    Article Title: Paradoxical Sensitivity to an Integrated Stress Response Blocking Mutation in Vanishing White Matter Cells

    doi: 10.1371/journal.pone.0166278

    Figure Lengend Snippet: Heightened ISR in EIF2B4 A392D cells. (A) Flow cytometry analysis of CHOP :: GFP and XBP1 :: Turquoise dual reporter-containing parental CHO-S21 and EIF2B4 A392D mutant cells. The cells were untreated (UT) or stimulated with 250 nM thapsigargin (Tg) or 0.5 mM histidinol (His) for 24 hours. Note the enhanced response of the CHOP :: GFP ISR reporter. (B) Bar diagram of the median ± S.D. of the reporter gene activity from experiments as shown in “A”. N = 3, *P = 0.0057 for Tg, *P = 0.037 for His, Unpaired t test. (C) Experimental design for tracking EIF2B4 A392D mutations. A fluorescent protein-marked sgRNA/Cas9 plasmid targeting EIF2B4 and a wildtype or EIF2B4 A392D mutant repair template marked by a silent Spe I mutation were co-transfected into CHO-S21 cells. Transfected cells (selected by FACS), were treated with histidinol and divided into four bins (Bin #1 to #4) by level of CHOP :: GFP expression. After recovery, genomic DNA was isolated from cells in each bin and the targeted region of EIF2B4 was amplified by PCR and digested with Spe I to reveal frequency of targeting by either repair template. (D) PCR fragments digested with Spe I from genomic DNA of the indicated bins, visualized on an agarose gel. Shown is an image of a representative experiment reproduced twice. (E) Plot of the distribution of Spe I digested fragments in the four bins of transduced cells from the experiment in “D”. The band intensities of the digested fragments (reporting on recombination of the wildtype or mutant repair template) were normalized to total PCR product intensity and the distribution of the relative frequency of recombination in the different bins was plotted. Note the enrichment for recombination of the EIF2B4 A392D mutant repair template in the ISR High bin.

    Article Snippet: The PCR products were digested by Spe I (Bcu I, ThermoFisher) and run on 2% agarose gel containing SYTO60 red fluorescent nucleic acid stain (ThermoFisher).

    Techniques: Flow Cytometry, Cytometry, Mutagenesis, Activity Assay, Plasmid Preparation, Transfection, FACS, Expressing, Isolation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Minichromosome DNA is cut at only one site by SpeI or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.

    Journal: Nucleic Acids Research

    Article Title: DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA

    doi: 10.1093/nar/gks723

    Figure Lengend Snippet: Minichromosome DNA is cut at only one site by SpeI or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.

    Article Snippet: Restriction fragments of this DNA cut with SpeI or SwaI (100 U/ml) were separated by PFGE in 1% LMP agarose at 190 V/cm for 7 or 20 h and switch time ramped linearly from 0.4 to 6 or 0.3 to 3 s, respectively, excised from gels and purified on Ultrafree columns (Millipore).

    Techniques: Incubation, Produced, Hybridization, Isolation, Fluorescence In Situ Hybridization

    Sites of breakage of minichromosome DNA in γ-irradiated cells. ( A ) SpeI and SwaI EBV DNA fragments used as probes for PFGE gels. ( B ) Hybridization of these probes to gels of DNA from control cells ( C ) or cells irradiated with 100 Gy (Irrad) restricted by the same enzyme; the PFGE conditions differed according to the length of fragments to be detected. Arrows show fragments predicted from the minichromosome DNA sequence; the origin of the weakly-hybridizing fragments is discussed in the text. (C) Fibre-FISH; the hybridization probes and procedure were as described in Figure 2 E. Images show representative linear molecules from irradiated cells (100 Gy); probes are green and DNA is red, and the extremities of the molecule represent the site of breakage. ( D ) Distribution of the breakage site expressed as the % of 55 circular DNA molecules in which the break occurred in one of four quadrants.

    Journal: Nucleic Acids Research

    Article Title: DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA

    doi: 10.1093/nar/gks723

    Figure Lengend Snippet: Sites of breakage of minichromosome DNA in γ-irradiated cells. ( A ) SpeI and SwaI EBV DNA fragments used as probes for PFGE gels. ( B ) Hybridization of these probes to gels of DNA from control cells ( C ) or cells irradiated with 100 Gy (Irrad) restricted by the same enzyme; the PFGE conditions differed according to the length of fragments to be detected. Arrows show fragments predicted from the minichromosome DNA sequence; the origin of the weakly-hybridizing fragments is discussed in the text. (C) Fibre-FISH; the hybridization probes and procedure were as described in Figure 2 E. Images show representative linear molecules from irradiated cells (100 Gy); probes are green and DNA is red, and the extremities of the molecule represent the site of breakage. ( D ) Distribution of the breakage site expressed as the % of 55 circular DNA molecules in which the break occurred in one of four quadrants.

    Article Snippet: Restriction fragments of this DNA cut with SpeI or SwaI (100 U/ml) were separated by PFGE in 1% LMP agarose at 190 V/cm for 7 or 20 h and switch time ramped linearly from 0.4 to 6 or 0.3 to 3 s, respectively, excised from gels and purified on Ultrafree columns (Millipore).

    Techniques: Irradiation, Hybridization, Sequencing, Fluorescence In Situ Hybridization

    Data and flow illustrations representing the coupling of SPE and PCR sample preparation steps on the MGA device using elastomeric valves and flow control preset by channel design. ( a ) Flow control between SPE and PCR was accomplished by using differential

    Journal:

    Article Title: A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability

    doi: 10.1073/pnas.0604663103

    Figure Lengend Snippet: Data and flow illustrations representing the coupling of SPE and PCR sample preparation steps on the MGA device using elastomeric valves and flow control preset by channel design. ( a ) Flow control between SPE and PCR was accomplished by using differential

    Article Snippet: The SPE and PCR domains, along with the syringe used to deliver master mix, were silanized by using Sigmacote (Sigma-Aldrich, St. Louis, MO).

    Techniques: Flow Cytometry, Polymerase Chain Reaction, Sample Prep

    PFGE banding patterns after Spe I digestion. Lanes 1 to 3, VIM-1-producing P. putida isolates from the general ICU (lane 1, isolate VA-304/99; lane 2, isolate VA-523/99; lane 3, isolate VA-420/00); lane 4, multidrug-resistant P. putida isolate from the nephrology department; lanes 5 and 6, two antibiotic-susceptible P. putida isolates obtained from the general ICU at the same hospital in 2000; lane L, λ phage DNA concatemers as a size marker.

    Journal: Journal of Clinical Microbiology

    Article Title: Nosocomial Infections Caused by Multidrug-Resistant Isolates of Pseudomonas putida Producing VIM-1 Metallo-?-Lactamase

    doi: 10.1128/JCM.40.11.4051-4055.2002

    Figure Lengend Snippet: PFGE banding patterns after Spe I digestion. Lanes 1 to 3, VIM-1-producing P. putida isolates from the general ICU (lane 1, isolate VA-304/99; lane 2, isolate VA-523/99; lane 3, isolate VA-420/00); lane 4, multidrug-resistant P. putida isolate from the nephrology department; lanes 5 and 6, two antibiotic-susceptible P. putida isolates obtained from the general ICU at the same hospital in 2000; lane L, λ phage DNA concatemers as a size marker.

    Article Snippet: Restrictions were carried out overnight at 37°C with 10 U of Spe I (Sigma, Milan, Italy).

    Techniques: Marker

    Ability of antibodies against either wild-type SPE A or mutants to neutralize the superantigenicity of SPE A. Dilutions of antibodies were mixed with wild-type SPE A (1 μg/well) 1 h prior to addition of rabbit splenocytes. Cultures were incubated for 3 days at 37°C in a 7% CO 2 incubator, and then 1 μCi of [ 3 H]thymidine was added per well. DNA was harvested after an additional day of incubation. Error bars indicate standard deviations.

    Journal: Infection and Immunity

    Article Title: Toxoids of Streptococcal Pyrogenic Exotoxin A Are Protective in Rabbit Models of Streptococcal Toxic Shock Syndrome

    doi:

    Figure Lengend Snippet: Ability of antibodies against either wild-type SPE A or mutants to neutralize the superantigenicity of SPE A. Dilutions of antibodies were mixed with wild-type SPE A (1 μg/well) 1 h prior to addition of rabbit splenocytes. Cultures were incubated for 3 days at 37°C in a 7% CO 2 incubator, and then 1 μCi of [ 3 H]thymidine was added per well. DNA was harvested after an additional day of incubation. Error bars indicate standard deviations.

    Article Snippet: Bound anti-SPE A was detected by goat anti-rabbit immunoglobulin G conjugated with peroxidase (Sigma).

    Techniques: Incubation

    Simultaneous cleavage of phage DNA by CRISPR1-Cas and CRISPR3-Cas systems of S. thermophilus BIMs. A . Representation of the phage 2972 genome. Arrows symbolize the open reading frames (ORFs) as identified previously [18] and colors indicate associated transcriptional modules [24] . Three protospacers (PS61, PS78 and PS85) are positioned below the genome and the CRISPR-Cas systems that have acquired the respective spacers are specified (CRISPR1 or CRISPR3). B . The fragment sizes obtained from restriction and/or CRISPR digestion are specified. Probes used in the Southern blot assays are also indicated and details of the primers used are presented in Supplemental Table S1 . C . Comparison by Southern blot assays of CRISPR-Cas cleavage occurring in the two selected BIMs S61/S78, and S61/S85, which share the S61 spacer (CRISPR3), but not a second spacer (CRISPR3 and CRISPR1, respectively). The Southern profiles show that independent cleavage of CRISPR1-Cas (PS85) and CRISPR3-Cas (PS61 and PS78) systems can occur simultaneously, when infected by phage 2972. Corresponding cleavage bands are identified with asterisks, and their sizes are specified. The two BIMs were infected with phage 2972 for 45 minutes before total DNA was extracted. Five micrograms of SpeI-restricted DNA was then loaded in each lane. NI, non-infected strain. C+, 10 ng of 2972 phage DNA digested with SpeI.

    Journal: PLoS ONE

    Article Title: Cleavage of Phage DNA by the Streptococcus thermophilus CRISPR3-Cas System

    doi: 10.1371/journal.pone.0040913

    Figure Lengend Snippet: Simultaneous cleavage of phage DNA by CRISPR1-Cas and CRISPR3-Cas systems of S. thermophilus BIMs. A . Representation of the phage 2972 genome. Arrows symbolize the open reading frames (ORFs) as identified previously [18] and colors indicate associated transcriptional modules [24] . Three protospacers (PS61, PS78 and PS85) are positioned below the genome and the CRISPR-Cas systems that have acquired the respective spacers are specified (CRISPR1 or CRISPR3). B . The fragment sizes obtained from restriction and/or CRISPR digestion are specified. Probes used in the Southern blot assays are also indicated and details of the primers used are presented in Supplemental Table S1 . C . Comparison by Southern blot assays of CRISPR-Cas cleavage occurring in the two selected BIMs S61/S78, and S61/S85, which share the S61 spacer (CRISPR3), but not a second spacer (CRISPR3 and CRISPR1, respectively). The Southern profiles show that independent cleavage of CRISPR1-Cas (PS85) and CRISPR3-Cas (PS61 and PS78) systems can occur simultaneously, when infected by phage 2972. Corresponding cleavage bands are identified with asterisks, and their sizes are specified. The two BIMs were infected with phage 2972 for 45 minutes before total DNA was extracted. Five micrograms of SpeI-restricted DNA was then loaded in each lane. NI, non-infected strain. C+, 10 ng of 2972 phage DNA digested with SpeI.

    Article Snippet: Five micrograms of DNA from a phage-infected phage-resistant (BIM) of S. thermophilus and 1 µg of DNA from a phage-infected wild-type strain were digested with SpeI restriction enzyme (Roche) before migration on six replicated 0.8% agarose gel (one per probe).

    Techniques: CRISPR, Southern Blot, Infection

    (A) PFGE of SpeI-, XbaI-, and HpaI-digested genomic DNA from multidrug-resistant P. aeruginosa IMCJ2.S1. (B) Southern blotting of the same gels with an aac(6′)-Iae probe. Lanes M, HindIII-digested λ phage DNA as a size marker.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Multidrug-Resistant Pseudomonas aeruginosa Strain That Caused an Outbreak in a Neurosurgery Ward and Its aac(6′)-Iae Gene Cassette Encoding a Novel Aminoglycoside Acetyltransferase

    doi: 10.1128/AAC.49.9.3734-3742.2005

    Figure Lengend Snippet: (A) PFGE of SpeI-, XbaI-, and HpaI-digested genomic DNA from multidrug-resistant P. aeruginosa IMCJ2.S1. (B) Southern blotting of the same gels with an aac(6′)-Iae probe. Lanes M, HindIII-digested λ phage DNA as a size marker.

    Article Snippet: Genomic DNA from P. aeruginosa was prepared by the procedure of Grundmann et al. ( ) and digested overnight with 10 U of SpeI, XbaI, or HpaI (Takara Bio).

    Techniques: Southern Blot, Marker

    PFGE profiles of genomic DNAs of PER-1-positive P. aeruginosa isolates after digestion with SpeI. The profile of isolate Pa6NI, which was identical to that of Pa34SM, and that of isolate Pa105TR, which was different from that of Pa144TR by three bands,

    Journal:

    Article Title: Multifocal Detection of Multidrug-Resistant Pseudomonas aeruginosa Producing the PER-1 Extended-Spectrum ?-Lactamase in Northern Italy

    doi: 10.1128/JCM.42.6.2523-2529.2004

    Figure Lengend Snippet: PFGE profiles of genomic DNAs of PER-1-positive P. aeruginosa isolates after digestion with SpeI. The profile of isolate Pa6NI, which was identical to that of Pa34SM, and that of isolate Pa105TR, which was different from that of Pa144TR by three bands,

    Article Snippet: Digestion of the genomic DNAs was carried out with the enzyme SpeI, used as recommended by the enzyme manufacturer (Bio-Rad).

    Techniques: