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  • 99
    New England Biolabs smai
    <t>PFGE-based</t> clustering of C. coli strains investigated in this study. All 68 strains were analyzed by PFGE with <t>SmaI</t> and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Two strains (1004 A
    Smai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1698 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 dna ligase
    <t>PFGE-based</t> clustering of C. coli strains investigated in this study. All 68 strains were analyzed by PFGE with <t>SmaI</t> and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Two strains (1004 A
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25057 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 dna ligase
    XLF L115A does not interact with XRCC4; thus, it does not bridge DNA in vitro but is fully sufficient to stimulate XRCC4/Lig4. (A, left) Schematic of the DNA bridging assay. (Right) Agarose gel showing recovery of DNA fragments bound to streptavidin beads by ethidium bromide staining. Molecular size markers are indicated (kilobases). (B to D) Ethidium bromide staining of agarose gels showing ligation products obtained from in vitro ligation reactions as described in Materials and Methods. Molecular size markers are indicated (kilobases). (B) <t>T4</t> DNA ligase is utilized. (C) XRCC4/Lig4 complexes (0.4 μM) are utilized. Four different concentrations of XLF were utilized: 0.25 μM, 0.5 μM, 1 μM, and 2 μM. (D) XRCC4/Lig4 complexes (0.2 μM) are utilized, with wild-type or mutant XRCC4 as indicated and with wild-type or mutant XLF (0.5 μM). (E, top) Immunoblot analyses of lysates from 293 cells transiently transfected with His-tagged wt and mutant forms of XLF probed with antibodies to XRCC4, XLF, or Lig4. (Bottom) Immunoblot analyses of pulldown fractions recovered from Ni-NTA–agarose beads after 3 h of incubation of cell lysates with beads and subsequent washing. The immunoblot was probed with antibodies to XRCC4, XLF, or Lig4.
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 49958 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs xhoi
    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or <t>PstI</t> ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with <t>XhoI,</t> PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.
    Xhoi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9905 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs bamhi
    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or <t>PstI</t> ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with <t>XhoI,</t> PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.
    Bamhi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11236 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ndei
    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or <t>PstI</t> ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with <t>XhoI,</t> PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.
    Ndei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ecori
    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or <t>PstI</t> ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with <t>XhoI,</t> PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.
    Ecori, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 10938 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dna polymerase i
    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or <t>PstI</t> ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with <t>XhoI,</t> PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.
    Dna Polymerase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 polynucleotide kinase
    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or <t>PstI</t> ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with <t>XhoI,</t> PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 29387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs antarctic phosphatase
    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or <t>PstI</t> ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with <t>XhoI,</t> PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.
    Antarctic Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6925 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs hindiii
    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or <t>PstI</t> ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with <t>XhoI,</t> PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.
    Hindiii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs xbai
    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or <t>PstI</t> ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with <t>XhoI,</t> PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.
    Xbai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ncoi
    PFGE and Southern blot analyses of R. monacensis pMOD658 transformants. (A) Ethidium bromide-stained PFGE gel with DNA from untransformed R. amblyommii isolate WB-8-2 (lane 1), untransformed R. monacensis ). (C) Ethidium bromide-stained PFGE gel with DNA from untransformed R. monacensis (lane 1), Rmona658 (lane 2), Rmona658B (lane 3) digested with <t>NcoI</t> (lane 4) or <t>SmaI</t> (lane 5), and Rmona658B QIAGEN plasmid prep DNA (lane 6) digested with NcoI (lane 7) or SmaI (lane 8). Lane 9 contains 1- and 5-kbp DNA marker ladders with sizes indicated at right. (D) Southern blot of gel shown in panel C hybridized with the GFPuv gene probe. Arrowheads indicate positions of the rickettsial plasmid containing the pMOD658 transposon (DNA bands migrating in a range between 23 and 100 kbp).
    Ncoi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2986 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 dna polymerase
    PFGE and Southern blot analyses of R. monacensis pMOD658 transformants. (A) Ethidium bromide-stained PFGE gel with DNA from untransformed R. amblyommii isolate WB-8-2 (lane 1), untransformed R. monacensis ). (C) Ethidium bromide-stained PFGE gel with DNA from untransformed R. monacensis (lane 1), Rmona658 (lane 2), Rmona658B (lane 3) digested with <t>NcoI</t> (lane 4) or <t>SmaI</t> (lane 5), and Rmona658B QIAGEN plasmid prep DNA (lane 6) digested with NcoI (lane 7) or SmaI (lane 8). Lane 9 contains 1- and 5-kbp DNA marker ladders with sizes indicated at right. (D) Southern blot of gel shown in panel C hybridized with the GFPuv gene probe. Arrowheads indicate positions of the rickettsial plasmid containing the pMOD658 transposon (DNA bands migrating in a range between 23 and 100 kbp).
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9070 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs phusion dna polymerase
    PFGE and Southern blot analyses of R. monacensis pMOD658 transformants. (A) Ethidium bromide-stained PFGE gel with DNA from untransformed R. amblyommii isolate WB-8-2 (lane 1), untransformed R. monacensis ). (C) Ethidium bromide-stained PFGE gel with DNA from untransformed R. monacensis (lane 1), Rmona658 (lane 2), Rmona658B (lane 3) digested with <t>NcoI</t> (lane 4) or <t>SmaI</t> (lane 5), and Rmona658B QIAGEN plasmid prep DNA (lane 6) digested with NcoI (lane 7) or SmaI (lane 8). Lane 9 contains 1- and 5-kbp DNA marker ladders with sizes indicated at right. (D) Southern blot of gel shown in panel C hybridized with the GFPuv gene probe. Arrowheads indicate positions of the rickettsial plasmid containing the pMOD658 transposon (DNA bands migrating in a range between 23 and 100 kbp).
    Phusion Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11721 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs xmai
    PFGE and Southern blot analyses of R. monacensis pMOD658 transformants. (A) Ethidium bromide-stained PFGE gel with DNA from untransformed R. amblyommii isolate WB-8-2 (lane 1), untransformed R. monacensis ). (C) Ethidium bromide-stained PFGE gel with DNA from untransformed R. monacensis (lane 1), Rmona658 (lane 2), Rmona658B (lane 3) digested with <t>NcoI</t> (lane 4) or <t>SmaI</t> (lane 5), and Rmona658B QIAGEN plasmid prep DNA (lane 6) digested with NcoI (lane 7) or SmaI (lane 8). Lane 9 contains 1- and 5-kbp DNA marker ladders with sizes indicated at right. (D) Southern blot of gel shown in panel C hybridized with the GFPuv gene probe. Arrowheads indicate positions of the rickettsial plasmid containing the pMOD658 transposon (DNA bands migrating in a range between 23 and 100 kbp).
    Xmai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1079 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs apai
    <t>ApaI</t> fingerprints of multiple isolates from two patients with closely related <t>SmaI</t> profiles reveal that the strains are genetically different enough to rule out patient-to-patient transmission. The multiple isolates were recovered at different times (shown
    Apai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs cre recombinase
    Design and validation of three alternative approaches for sequence truncation prior to library sequencing. (a) In library 1 we utilized a sticky-end restriction enzyme (SalI) digestion and T4 ligation to remove the static sequence separating the variable genomic fragment of interest with the degenerate DNA barcode (BC) sequence. (b) When the library plasmid was truncated, the barcode could be sequenced together with the variable genetic fragment to generate a look-up table (LUT) using the Ion Torrent sequencing platform. However, the sequencing results from library 1 displayed extensive recombination between barcode and fragment (left in B). This was confirmed to not have been originating from the cloning process through the use of PCR free sequencing using the PacBio sequencer on the non-digested plasmid (centre in B). Using the <t>Cre-recombinase</t> based approach in C, this recombination could be significantly reduced (right in B). (c) In library 2 we replaced the restriction enzyme approach with a Cre-recombinase approach where the same intervening static sequence is removed through the recombination between two loxP sites. (d) In the Cre-based designs we utilize a combination of two mutant loxP sites; loxP-JT15 and loxP-JTZ17 which promote superior Cre-induced recombination compared to wild-type loxP sites as the resulting double-mutant loxp-JT15/JTZ17 has lost the binding capacity of the Cre-recombinase making the recombination a unidirectional event. (e) The loxP-JT15/JTZ17 combination resulted in 79%, 81% and 89% recombined product with 30 minute, 60 minute and overnight Cre-recombination respectively. With restriction enzyme digestion (MluI, which cuts inside the 3′ domain) of the remaining un-recombined product, the remaining fraction of un-truncated plasmid could be removed (last three columns in E). The expected bands are 1007 bp and 464 bp respectively. (f) In the third and final design, we generated two libraries (3 4) where the design is reversed to that the fragment together with the barcode is excised into a mini-plasmid after Cre-recombinase exposure with the fragment and barcode in close proximity.
    Cre Recombinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 724 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs vent polymerase
    Design and validation of three alternative approaches for sequence truncation prior to library sequencing. (a) In library 1 we utilized a sticky-end restriction enzyme (SalI) digestion and T4 ligation to remove the static sequence separating the variable genomic fragment of interest with the degenerate DNA barcode (BC) sequence. (b) When the library plasmid was truncated, the barcode could be sequenced together with the variable genetic fragment to generate a look-up table (LUT) using the Ion Torrent sequencing platform. However, the sequencing results from library 1 displayed extensive recombination between barcode and fragment (left in B). This was confirmed to not have been originating from the cloning process through the use of PCR free sequencing using the PacBio sequencer on the non-digested plasmid (centre in B). Using the <t>Cre-recombinase</t> based approach in C, this recombination could be significantly reduced (right in B). (c) In library 2 we replaced the restriction enzyme approach with a Cre-recombinase approach where the same intervening static sequence is removed through the recombination between two loxP sites. (d) In the Cre-based designs we utilize a combination of two mutant loxP sites; loxP-JT15 and loxP-JTZ17 which promote superior Cre-induced recombination compared to wild-type loxP sites as the resulting double-mutant loxp-JT15/JTZ17 has lost the binding capacity of the Cre-recombinase making the recombination a unidirectional event. (e) The loxP-JT15/JTZ17 combination resulted in 79%, 81% and 89% recombined product with 30 minute, 60 minute and overnight Cre-recombination respectively. With restriction enzyme digestion (MluI, which cuts inside the 3′ domain) of the remaining un-recombined product, the remaining fraction of un-truncated plasmid could be removed (last three columns in E). The expected bands are 1007 bp and 464 bp respectively. (f) In the third and final design, we generated two libraries (3 4) where the design is reversed to that the fragment together with the barcode is excised into a mini-plasmid after Cre-recombinase exposure with the fragment and barcode in close proximity.
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    Bio-Rad chef dr iii system
    Design and validation of three alternative approaches for sequence truncation prior to library sequencing. (a) In library 1 we utilized a sticky-end restriction enzyme (SalI) digestion and T4 ligation to remove the static sequence separating the variable genomic fragment of interest with the degenerate DNA barcode (BC) sequence. (b) When the library plasmid was truncated, the barcode could be sequenced together with the variable genetic fragment to generate a look-up table (LUT) using the Ion Torrent sequencing platform. However, the sequencing results from library 1 displayed extensive recombination between barcode and fragment (left in B). This was confirmed to not have been originating from the cloning process through the use of PCR free sequencing using the PacBio sequencer on the non-digested plasmid (centre in B). Using the <t>Cre-recombinase</t> based approach in C, this recombination could be significantly reduced (right in B). (c) In library 2 we replaced the restriction enzyme approach with a Cre-recombinase approach where the same intervening static sequence is removed through the recombination between two loxP sites. (d) In the Cre-based designs we utilize a combination of two mutant loxP sites; loxP-JT15 and loxP-JTZ17 which promote superior Cre-induced recombination compared to wild-type loxP sites as the resulting double-mutant loxp-JT15/JTZ17 has lost the binding capacity of the Cre-recombinase making the recombination a unidirectional event. (e) The loxP-JT15/JTZ17 combination resulted in 79%, 81% and 89% recombined product with 30 minute, 60 minute and overnight Cre-recombination respectively. With restriction enzyme digestion (MluI, which cuts inside the 3′ domain) of the remaining un-recombined product, the remaining fraction of un-truncated plasmid could be removed (last three columns in E). The expected bands are 1007 bp and 464 bp respectively. (f) In the third and final design, we generated two libraries (3 4) where the design is reversed to that the fragment together with the barcode is excised into a mini-plasmid after Cre-recombinase exposure with the fragment and barcode in close proximity.
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    New England Biolabs kpni
    <t>PFGE-based</t> clustering of C. coli strains investigated in this study. All 68 strains were analyzed by PFGE with SmaI and <t>KpnI,</t> and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Two strains (1004 A
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    New England Biolabs bglii
    A comparison of bla KPC-2 -carrying plasmids originated from two K. pneumoniae clones and two E. coli clones isolated in the same time period. Plasmid restriction analysis of <t>transformants</t> carrying these plasmids showed identity between the K. pneumoniae plasmids (K) and the E. coli plasmids (E). Plasmids from both organisms were digested with <t>BglII</t> (lanes 2 to 5), EcoRV (lanes 6 to 9), and SmaI (lanes 10 to 13) prior to electrophoresis. GeneRuler 1-kb DNA ladder (Fermentas Life Sciences), lane 1 (M); E. coli 386, lanes 2, 6, and 10; K. pneumoniae 523 PFGE type R, lanes 3, 7, and 11; E. coli 547, lanes 4, 8, and 12); K. pneumoniae 531, PFGE type, lanes 5, 9, and 13.
    Bglii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2543 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    PFGE-based clustering of C. coli strains investigated in this study. All 68 strains were analyzed by PFGE with SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Two strains (1004 A

    Journal: Applied and Environmental Microbiology

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys ▿

    doi: 10.1128/AEM.02346-06

    Figure Lengend Snippet: PFGE-based clustering of C. coli strains investigated in this study. All 68 strains were analyzed by PFGE with SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Two strains (1004 A

    Article Snippet: PFGE was performed using SmaI and KpnI (New England Biolabs) by following the protocol described by Ribot et al. ( ) with a few minor modifications; specifically, the prerestriction step was eliminated and the gels were electrophoresed for 22 h. TIFF images of banding patterns resulting from PFGE and fla typing were analyzed by BioNumerics (version 4.6; Applied Maths).

    Techniques:

    PFGE profiles of group I strains. Nineteen MDR strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. The line labeled “a” indicates

    Journal: Applied and Environmental Microbiology

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys ▿

    doi: 10.1128/AEM.02346-06

    Figure Lengend Snippet: PFGE profiles of group I strains. Nineteen MDR strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. The line labeled “a” indicates

    Article Snippet: PFGE was performed using SmaI and KpnI (New England Biolabs) by following the protocol described by Ribot et al. ( ) with a few minor modifications; specifically, the prerestriction step was eliminated and the gels were electrophoresed for 22 h. TIFF images of banding patterns resulting from PFGE and fla typing were analyzed by BioNumerics (version 4.6; Applied Maths).

    Techniques: Labeling

    A conserved SmaI PFGE pattern (type a ) was disseminated among MDR strains of different STs.

    Journal: Applied and Environmental Microbiology

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys ▿

    doi: 10.1128/AEM.02346-06

    Figure Lengend Snippet: A conserved SmaI PFGE pattern (type a ) was disseminated among MDR strains of different STs.

    Article Snippet: PFGE was performed using SmaI and KpnI (New England Biolabs) by following the protocol described by Ribot et al. ( ) with a few minor modifications; specifically, the prerestriction step was eliminated and the gels were electrophoresed for 22 h. TIFF images of banding patterns resulting from PFGE and fla typing were analyzed by BioNumerics (version 4.6; Applied Maths).

    Techniques:

    PFGE profiles of group II strains. Forty-two strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Strains which were not resistant to all six antibiotics

    Journal: Applied and Environmental Microbiology

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys ▿

    doi: 10.1128/AEM.02346-06

    Figure Lengend Snippet: PFGE profiles of group II strains. Forty-two strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Strains which were not resistant to all six antibiotics

    Article Snippet: PFGE was performed using SmaI and KpnI (New England Biolabs) by following the protocol described by Ribot et al. ( ) with a few minor modifications; specifically, the prerestriction step was eliminated and the gels were electrophoresed for 22 h. TIFF images of banding patterns resulting from PFGE and fla typing were analyzed by BioNumerics (version 4.6; Applied Maths).

    Techniques:

    PFGE profiles of 13 strains with various STs but a conserved PFGE profile (type a ). Cluster analysis of the SmaI and KpnI patterns was performed by BioNumerics as described in Materials and Methods.

    Journal: Applied and Environmental Microbiology

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys ▿

    doi: 10.1128/AEM.02346-06

    Figure Lengend Snippet: PFGE profiles of 13 strains with various STs but a conserved PFGE profile (type a ). Cluster analysis of the SmaI and KpnI patterns was performed by BioNumerics as described in Materials and Methods.

    Article Snippet: PFGE was performed using SmaI and KpnI (New England Biolabs) by following the protocol described by Ribot et al. ( ) with a few minor modifications; specifically, the prerestriction step was eliminated and the gels were electrophoresed for 22 h. TIFF images of banding patterns resulting from PFGE and fla typing were analyzed by BioNumerics (version 4.6; Applied Maths).

    Techniques:

    XLF L115A does not interact with XRCC4; thus, it does not bridge DNA in vitro but is fully sufficient to stimulate XRCC4/Lig4. (A, left) Schematic of the DNA bridging assay. (Right) Agarose gel showing recovery of DNA fragments bound to streptavidin beads by ethidium bromide staining. Molecular size markers are indicated (kilobases). (B to D) Ethidium bromide staining of agarose gels showing ligation products obtained from in vitro ligation reactions as described in Materials and Methods. Molecular size markers are indicated (kilobases). (B) T4 DNA ligase is utilized. (C) XRCC4/Lig4 complexes (0.4 μM) are utilized. Four different concentrations of XLF were utilized: 0.25 μM, 0.5 μM, 1 μM, and 2 μM. (D) XRCC4/Lig4 complexes (0.2 μM) are utilized, with wild-type or mutant XRCC4 as indicated and with wild-type or mutant XLF (0.5 μM). (E, top) Immunoblot analyses of lysates from 293 cells transiently transfected with His-tagged wt and mutant forms of XLF probed with antibodies to XRCC4, XLF, or Lig4. (Bottom) Immunoblot analyses of pulldown fractions recovered from Ni-NTA–agarose beads after 3 h of incubation of cell lysates with beads and subsequent washing. The immunoblot was probed with antibodies to XRCC4, XLF, or Lig4.

    Journal: Molecular and Cellular Biology

    Article Title: XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation

    doi: 10.1128/MCB.01503-14

    Figure Lengend Snippet: XLF L115A does not interact with XRCC4; thus, it does not bridge DNA in vitro but is fully sufficient to stimulate XRCC4/Lig4. (A, left) Schematic of the DNA bridging assay. (Right) Agarose gel showing recovery of DNA fragments bound to streptavidin beads by ethidium bromide staining. Molecular size markers are indicated (kilobases). (B to D) Ethidium bromide staining of agarose gels showing ligation products obtained from in vitro ligation reactions as described in Materials and Methods. Molecular size markers are indicated (kilobases). (B) T4 DNA ligase is utilized. (C) XRCC4/Lig4 complexes (0.4 μM) are utilized. Four different concentrations of XLF were utilized: 0.25 μM, 0.5 μM, 1 μM, and 2 μM. (D) XRCC4/Lig4 complexes (0.2 μM) are utilized, with wild-type or mutant XRCC4 as indicated and with wild-type or mutant XLF (0.5 μM). (E, top) Immunoblot analyses of lysates from 293 cells transiently transfected with His-tagged wt and mutant forms of XLF probed with antibodies to XRCC4, XLF, or Lig4. (Bottom) Immunoblot analyses of pulldown fractions recovered from Ni-NTA–agarose beads after 3 h of incubation of cell lysates with beads and subsequent washing. The immunoblot was probed with antibodies to XRCC4, XLF, or Lig4.

    Article Snippet: T4 DNA ligase (New England BioLabs) was used at a final concentration of 8 U/μl.

    Techniques: In Vitro, Agarose Gel Electrophoresis, Staining, Ligation, Mutagenesis, Transfection, Incubation

    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or PstI ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with XhoI, PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.

    Journal: Nucleic Acids Research

    Article Title: RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation

    doi: 10.1093/nar/gkp780

    Figure Lengend Snippet: Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or PstI ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with XhoI, PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.

    Article Snippet: Isolated nuclei from differentiating 3T3-L1 cells were digested with 100 U of PstI, XhoI, SmaI, SpeI and ApaLI (New England Biolabs) for 30 min.

    Techniques: Southern Blot, Staining, Produced, Generated

    PFGE and Southern blot analyses of R. monacensis pMOD658 transformants. (A) Ethidium bromide-stained PFGE gel with DNA from untransformed R. amblyommii isolate WB-8-2 (lane 1), untransformed R. monacensis ). (C) Ethidium bromide-stained PFGE gel with DNA from untransformed R. monacensis (lane 1), Rmona658 (lane 2), Rmona658B (lane 3) digested with NcoI (lane 4) or SmaI (lane 5), and Rmona658B QIAGEN plasmid prep DNA (lane 6) digested with NcoI (lane 7) or SmaI (lane 8). Lane 9 contains 1- and 5-kbp DNA marker ladders with sizes indicated at right. (D) Southern blot of gel shown in panel C hybridized with the GFPuv gene probe. Arrowheads indicate positions of the rickettsial plasmid containing the pMOD658 transposon (DNA bands migrating in a range between 23 and 100 kbp).

    Journal: Applied and Environmental Microbiology

    Article Title: Transposon Insertion Reveals pRM, a Plasmid of Rickettsia monacensis ▿

    doi: 10.1128/AEM.00988-07

    Figure Lengend Snippet: PFGE and Southern blot analyses of R. monacensis pMOD658 transformants. (A) Ethidium bromide-stained PFGE gel with DNA from untransformed R. amblyommii isolate WB-8-2 (lane 1), untransformed R. monacensis ). (C) Ethidium bromide-stained PFGE gel with DNA from untransformed R. monacensis (lane 1), Rmona658 (lane 2), Rmona658B (lane 3) digested with NcoI (lane 4) or SmaI (lane 5), and Rmona658B QIAGEN plasmid prep DNA (lane 6) digested with NcoI (lane 7) or SmaI (lane 8). Lane 9 contains 1- and 5-kbp DNA marker ladders with sizes indicated at right. (D) Southern blot of gel shown in panel C hybridized with the GFPuv gene probe. Arrowheads indicate positions of the rickettsial plasmid containing the pMOD658 transposon (DNA bands migrating in a range between 23 and 100 kbp).

    Article Snippet: Agarose blocks subjected to restriction enzyme digestion with HindIII, PvuI (Promega, Madison, WI), NcoI (New England Biolabs, Beverly, MA), or SmaI (Life Technologies, Rockville, MD) were equilibrated in TE as described above and immersed in 200 μl of the manufacturer's 1× buffer with 10 to 50 units of enzyme and incubated for 4 h at 37°C (25°C for SmaI).

    Techniques: Southern Blot, Staining, Western Blot, Plasmid Preparation, Marker

    ApaI fingerprints of multiple isolates from two patients with closely related SmaI profiles reveal that the strains are genetically different enough to rule out patient-to-patient transmission. The multiple isolates were recovered at different times (shown

    Journal: Journal of Clinical Microbiology

    Article Title: The Streptococcus milleri Population of a Cystic Fibrosis Clinic Reveals Patient Specificity and Intraspecies Diversity ▿

    doi: 10.1128/JCM.00414-10

    Figure Lengend Snippet: ApaI fingerprints of multiple isolates from two patients with closely related SmaI profiles reveal that the strains are genetically different enough to rule out patient-to-patient transmission. The multiple isolates were recovered at different times (shown

    Article Snippet: For restriction digestion, the plugs were preincubated in 300 μl of 1× reaction buffer (Invitrogen, Carlsbad, CA) at room temperature for 15 min and then replaced with fresh 1× reaction buffer supplemented with 90 U of SmaI or ApaI (New England BioLabs, Beverly, MA).

    Techniques: Transmission Assay

    Design and validation of three alternative approaches for sequence truncation prior to library sequencing. (a) In library 1 we utilized a sticky-end restriction enzyme (SalI) digestion and T4 ligation to remove the static sequence separating the variable genomic fragment of interest with the degenerate DNA barcode (BC) sequence. (b) When the library plasmid was truncated, the barcode could be sequenced together with the variable genetic fragment to generate a look-up table (LUT) using the Ion Torrent sequencing platform. However, the sequencing results from library 1 displayed extensive recombination between barcode and fragment (left in B). This was confirmed to not have been originating from the cloning process through the use of PCR free sequencing using the PacBio sequencer on the non-digested plasmid (centre in B). Using the Cre-recombinase based approach in C, this recombination could be significantly reduced (right in B). (c) In library 2 we replaced the restriction enzyme approach with a Cre-recombinase approach where the same intervening static sequence is removed through the recombination between two loxP sites. (d) In the Cre-based designs we utilize a combination of two mutant loxP sites; loxP-JT15 and loxP-JTZ17 which promote superior Cre-induced recombination compared to wild-type loxP sites as the resulting double-mutant loxp-JT15/JTZ17 has lost the binding capacity of the Cre-recombinase making the recombination a unidirectional event. (e) The loxP-JT15/JTZ17 combination resulted in 79%, 81% and 89% recombined product with 30 minute, 60 minute and overnight Cre-recombination respectively. With restriction enzyme digestion (MluI, which cuts inside the 3′ domain) of the remaining un-recombined product, the remaining fraction of un-truncated plasmid could be removed (last three columns in E). The expected bands are 1007 bp and 464 bp respectively. (f) In the third and final design, we generated two libraries (3 4) where the design is reversed to that the fragment together with the barcode is excised into a mini-plasmid after Cre-recombinase exposure with the fragment and barcode in close proximity.

    Journal: Scientific Reports

    Article Title: A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing

    doi: 10.1038/srep37563

    Figure Lengend Snippet: Design and validation of three alternative approaches for sequence truncation prior to library sequencing. (a) In library 1 we utilized a sticky-end restriction enzyme (SalI) digestion and T4 ligation to remove the static sequence separating the variable genomic fragment of interest with the degenerate DNA barcode (BC) sequence. (b) When the library plasmid was truncated, the barcode could be sequenced together with the variable genetic fragment to generate a look-up table (LUT) using the Ion Torrent sequencing platform. However, the sequencing results from library 1 displayed extensive recombination between barcode and fragment (left in B). This was confirmed to not have been originating from the cloning process through the use of PCR free sequencing using the PacBio sequencer on the non-digested plasmid (centre in B). Using the Cre-recombinase based approach in C, this recombination could be significantly reduced (right in B). (c) In library 2 we replaced the restriction enzyme approach with a Cre-recombinase approach where the same intervening static sequence is removed through the recombination between two loxP sites. (d) In the Cre-based designs we utilize a combination of two mutant loxP sites; loxP-JT15 and loxP-JTZ17 which promote superior Cre-induced recombination compared to wild-type loxP sites as the resulting double-mutant loxp-JT15/JTZ17 has lost the binding capacity of the Cre-recombinase making the recombination a unidirectional event. (e) The loxP-JT15/JTZ17 combination resulted in 79%, 81% and 89% recombined product with 30 minute, 60 minute and overnight Cre-recombination respectively. With restriction enzyme digestion (MluI, which cuts inside the 3′ domain) of the remaining un-recombined product, the remaining fraction of un-truncated plasmid could be removed (last three columns in E). The expected bands are 1007 bp and 464 bp respectively. (f) In the third and final design, we generated two libraries (3 4) where the design is reversed to that the fragment together with the barcode is excised into a mini-plasmid after Cre-recombinase exposure with the fragment and barcode in close proximity.

    Article Snippet: 750 ng DNA was incubated with 3 U Cre-recombinase (NEB), 10X buffer and water and incubated at 37 °C for 90 minutes.

    Techniques: Sequencing, Ligation, Plasmid Preparation, Clone Assay, Polymerase Chain Reaction, Mutagenesis, Binding Assay, Generated

    Development and characterization of an optimized emulsion PCR protocol. (a) An optimized, large scale, emulsion PCR protocol was developed to reduce template switching in the generation of amplicons for Ion Torrent and Illumina sequencing. A 50 μl PCR reaction (Phusion Hot Start with green buffer) was mixed with mineral oil and surfactant. (b) The mix was converted into a large scale emulsion using a Fast Prep homogenizer in 5 minutes. (c) To evaluate the stability and size distribution of the formed micelles, three separate PCR reactions were prepared and labels with different water soluble fluorophores (DyLight 488, 549 and 650 respectively) and made into three emulsion reactions. After mixing together the three emulsions through repeated pipetting the reaction was imaged and quantified using laser-scanning confocal microscopy emulsion. Quantification resulted in a mean diameter of the micelles of 3.7 ± 2.3 μm and a total micelle count of 2.6 × 10 8 per individual reaction. (d) The emulsion was then divided into 6 individual PCR tubes and covered with mineral oil, which remain stable after PCR. (e) Isobutanol was then used to break the emulsion. (f) The compartmentalization of a PCR reaction by emPCR enables even amplification of an equimolar mixture of two oligonucleotides (126 and 150 bp long) with identical flanking sequences but with known difference in PCR efficiency. (g) Formation of chimeras due to template switching was analysed through a comparative experiment utilizing the long amplicon library 3, containing a 1 kb long stretch of constitutive backbone which can be removed using Cre-recombinase ( Fig. 3c ). With regular PCR, this library displayed extensive template switching regardless of PCR protocol or length of constitutive sequence. (h) Using emPCR on the other hand, the template switching could be significantly reduced to a level where it became insensitive to both changes in PCR protocol and constitutive sequence length.

    Journal: Scientific Reports

    Article Title: A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing

    doi: 10.1038/srep37563

    Figure Lengend Snippet: Development and characterization of an optimized emulsion PCR protocol. (a) An optimized, large scale, emulsion PCR protocol was developed to reduce template switching in the generation of amplicons for Ion Torrent and Illumina sequencing. A 50 μl PCR reaction (Phusion Hot Start with green buffer) was mixed with mineral oil and surfactant. (b) The mix was converted into a large scale emulsion using a Fast Prep homogenizer in 5 minutes. (c) To evaluate the stability and size distribution of the formed micelles, three separate PCR reactions were prepared and labels with different water soluble fluorophores (DyLight 488, 549 and 650 respectively) and made into three emulsion reactions. After mixing together the three emulsions through repeated pipetting the reaction was imaged and quantified using laser-scanning confocal microscopy emulsion. Quantification resulted in a mean diameter of the micelles of 3.7 ± 2.3 μm and a total micelle count of 2.6 × 10 8 per individual reaction. (d) The emulsion was then divided into 6 individual PCR tubes and covered with mineral oil, which remain stable after PCR. (e) Isobutanol was then used to break the emulsion. (f) The compartmentalization of a PCR reaction by emPCR enables even amplification of an equimolar mixture of two oligonucleotides (126 and 150 bp long) with identical flanking sequences but with known difference in PCR efficiency. (g) Formation of chimeras due to template switching was analysed through a comparative experiment utilizing the long amplicon library 3, containing a 1 kb long stretch of constitutive backbone which can be removed using Cre-recombinase ( Fig. 3c ). With regular PCR, this library displayed extensive template switching regardless of PCR protocol or length of constitutive sequence. (h) Using emPCR on the other hand, the template switching could be significantly reduced to a level where it became insensitive to both changes in PCR protocol and constitutive sequence length.

    Article Snippet: 750 ng DNA was incubated with 3 U Cre-recombinase (NEB), 10X buffer and water and incubated at 37 °C for 90 minutes.

    Techniques: Polymerase Chain Reaction, Sequencing, Confocal Microscopy, Amplification

    PFGE-based clustering of C. coli strains investigated in this study. All 68 strains were analyzed by PFGE with SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Two strains (1004 A

    Journal: Applied and Environmental Microbiology

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys ▿

    doi: 10.1128/AEM.02346-06

    Figure Lengend Snippet: PFGE-based clustering of C. coli strains investigated in this study. All 68 strains were analyzed by PFGE with SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Two strains (1004 A

    Article Snippet: PFGE was performed using SmaI and KpnI (New England Biolabs) by following the protocol described by Ribot et al. ( ) with a few minor modifications; specifically, the prerestriction step was eliminated and the gels were electrophoresed for 22 h. TIFF images of banding patterns resulting from PFGE and fla typing were analyzed by BioNumerics (version 4.6; Applied Maths).

    Techniques:

    PFGE profiles of group I strains. Nineteen MDR strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. The line labeled “a” indicates

    Journal: Applied and Environmental Microbiology

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys ▿

    doi: 10.1128/AEM.02346-06

    Figure Lengend Snippet: PFGE profiles of group I strains. Nineteen MDR strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. The line labeled “a” indicates

    Article Snippet: PFGE was performed using SmaI and KpnI (New England Biolabs) by following the protocol described by Ribot et al. ( ) with a few minor modifications; specifically, the prerestriction step was eliminated and the gels were electrophoresed for 22 h. TIFF images of banding patterns resulting from PFGE and fla typing were analyzed by BioNumerics (version 4.6; Applied Maths).

    Techniques: Labeling

    PFGE profiles of group II strains. Forty-two strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Strains which were not resistant to all six antibiotics

    Journal: Applied and Environmental Microbiology

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys ▿

    doi: 10.1128/AEM.02346-06

    Figure Lengend Snippet: PFGE profiles of group II strains. Forty-two strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Strains which were not resistant to all six antibiotics

    Article Snippet: PFGE was performed using SmaI and KpnI (New England Biolabs) by following the protocol described by Ribot et al. ( ) with a few minor modifications; specifically, the prerestriction step was eliminated and the gels were electrophoresed for 22 h. TIFF images of banding patterns resulting from PFGE and fla typing were analyzed by BioNumerics (version 4.6; Applied Maths).

    Techniques:

    PFGE profiles of 13 strains with various STs but a conserved PFGE profile (type a ). Cluster analysis of the SmaI and KpnI patterns was performed by BioNumerics as described in Materials and Methods.

    Journal: Applied and Environmental Microbiology

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys ▿

    doi: 10.1128/AEM.02346-06

    Figure Lengend Snippet: PFGE profiles of 13 strains with various STs but a conserved PFGE profile (type a ). Cluster analysis of the SmaI and KpnI patterns was performed by BioNumerics as described in Materials and Methods.

    Article Snippet: PFGE was performed using SmaI and KpnI (New England Biolabs) by following the protocol described by Ribot et al. ( ) with a few minor modifications; specifically, the prerestriction step was eliminated and the gels were electrophoresed for 22 h. TIFF images of banding patterns resulting from PFGE and fla typing were analyzed by BioNumerics (version 4.6; Applied Maths).

    Techniques:

    A comparison of bla KPC-2 -carrying plasmids originated from two K. pneumoniae clones and two E. coli clones isolated in the same time period. Plasmid restriction analysis of transformants carrying these plasmids showed identity between the K. pneumoniae plasmids (K) and the E. coli plasmids (E). Plasmids from both organisms were digested with BglII (lanes 2 to 5), EcoRV (lanes 6 to 9), and SmaI (lanes 10 to 13) prior to electrophoresis. GeneRuler 1-kb DNA ladder (Fermentas Life Sciences), lane 1 (M); E. coli 386, lanes 2, 6, and 10; K. pneumoniae 523 PFGE type R, lanes 3, 7, and 11; E. coli 547, lanes 4, 8, and 12); K. pneumoniae 531, PFGE type, lanes 5, 9, and 13.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Molecular Epidemiology, Sequence Types, and Plasmid Analyses of KPC-Producing Klebsiella pneumoniae Strains in Israel ▿

    doi: 10.1128/AAC.01818-09

    Figure Lengend Snippet: A comparison of bla KPC-2 -carrying plasmids originated from two K. pneumoniae clones and two E. coli clones isolated in the same time period. Plasmid restriction analysis of transformants carrying these plasmids showed identity between the K. pneumoniae plasmids (K) and the E. coli plasmids (E). Plasmids from both organisms were digested with BglII (lanes 2 to 5), EcoRV (lanes 6 to 9), and SmaI (lanes 10 to 13) prior to electrophoresis. GeneRuler 1-kb DNA ladder (Fermentas Life Sciences), lane 1 (M); E. coli 386, lanes 2, 6, and 10; K. pneumoniae 523 PFGE type R, lanes 3, 7, and 11; E. coli 547, lanes 4, 8, and 12); K. pneumoniae 531, PFGE type, lanes 5, 9, and 13.

    Article Snippet: Plasmids isolated from clinical isolates of K. pneumoniae and E. coli strains and their transformants were digested with different restriction endonucleases, such as BglII, SmaI, and EcoRV (New England Biolabs, Boston, MA), and their restriction patterns were compared.

    Techniques: Clone Assay, Isolation, Plasmid Preparation, Electrophoresis