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  • 99
    New England Biolabs bamhi bamhi
    Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector <t>pΔAPRT1-NEO5,</t> with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and <t>BamHI-digested</t> genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).
    Bamhi Bamhi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1033 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bamhi digestion
    Characterization of part of the mouse Rad54B genomic locus and generation of mouse ES cells carrying a disrupted mRad54B allele. (A) Part of the mRad54B genomic locus and structure of the targeting construct. Exons 12 to 15 are indicated by black boxes. Shown are the locations of selected restriction sites, EcoRI (E), <t>BamHI</t> (B), BglII (Bg), HindIII (H), and XbaI (X). The positions of two different probes, named A and B, are indicated. (B) DNA blot analysis of G418-resistant ES clones with probe A and EcoRI digested DNA. The wild-type (wt) allele yields a 3.0-kb band, while the disrupted allele results in a 3.6-kb band. Lane 1, wild-type ES cell; lane 2, clone with a randomly integrated targeting construct; lane 3, clone with a homologously integrated targeting construct. (C) RNA blot analysis of mRad54B transcripts in mice carrying the disrupted allele. Total RNA (15 μg) isolated from testes of wild-type, mRad54B +/ − , and mRad54B −/− males was probed with 5′ and 3′ mRad54B <t>cDNA</t> probes. A GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA probe served as a loading control.
    Bamhi Digestion, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 276 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Jena Bioscience bamhi
    Characterization of part of the mouse Rad54B genomic locus and generation of mouse ES cells carrying a disrupted mRad54B allele. (A) Part of the mRad54B genomic locus and structure of the targeting construct. Exons 12 to 15 are indicated by black boxes. Shown are the locations of selected restriction sites, EcoRI (E), <t>BamHI</t> (B), BglII (Bg), HindIII (H), and XbaI (X). The positions of two different probes, named A and B, are indicated. (B) DNA blot analysis of G418-resistant ES clones with probe A and EcoRI digested DNA. The wild-type (wt) allele yields a 3.0-kb band, while the disrupted allele results in a 3.6-kb band. Lane 1, wild-type ES cell; lane 2, clone with a randomly integrated targeting construct; lane 3, clone with a homologously integrated targeting construct. (C) RNA blot analysis of mRad54B transcripts in mice carrying the disrupted allele. Total RNA (15 μg) isolated from testes of wild-type, mRad54B +/ − , and mRad54B −/− males was probed with 5′ and 3′ mRad54B <t>cDNA</t> probes. A GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA probe served as a loading control.
    Bamhi, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bamhi
    Joining of incompatible <t>DNA</t> ends by MRN and DNA ligase IIIα/XRCC1: microhomology-mediated joining of DNA ends with incompatible 5′ overhangs. A , circular plasmid DNA was digested with either KpnI ( left ) and PstI ( right ) to generate noncomplementary 3′ overhangs or EcoRI ( left ) and <t>BamHI</t> ( right ), to generate noncomplementary 5′ overhangs. Joining of the incompatible DNA ends was detected by the PCR using the indicated primers. B , DNA substrate (0.8 pmol) with noncomplementary 3′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. C , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. D , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of either DNA ligase IIIα/XRCC1 or DNA ligase IV/XRCC4 in the absence or presence of an equimolar amount of MRN. E , sequences of the junctions generated by the joining of incompatible 5′ ends by DNA ligase IIIα/XRCC1 in combination with either hMre11 or MRN are shown. Bolded residues indicate microhomologies.
    Bamhi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10444 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs bamhi hf
    Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and <t>BamHI-HF,</t> into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) <t>DNA</t> from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.
    Bamhi Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher fastdigest bamhi
    Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and <t>BamHI-HF,</t> into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) <t>DNA</t> from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.
    Fastdigest Bamhi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bamhi
    Plasmids used for TLS assays. ( A ) Construction of an AP site-containing plasmid. Plasmid pVP15 is linearized by BbrPI digestion. The <t>PstI-BamHI</t> fragment of <t>pVP9</t> is removed by digestion and purification on a microcon YM-30 column. Both linearized plasmids
    Bamhi, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5283 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega bamhi
    Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total <t>DNA</t> (7 µg) was digested with <t>BamHI</t> which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λ BstE II, L. bicolor wild type (Wt) and transgenic strains.
    Bamhi, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 2171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    System Biosciences Inc bamhi
    Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total <t>DNA</t> (7 µg) was digested with <t>BamHI</t> which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λ BstE II, L. bicolor wild type (Wt) and transgenic strains.
    Bamhi, supplied by System Biosciences Inc, used in various techniques. Bioz Stars score: 97/100, based on 315 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Valiant bamhi
    Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total <t>DNA</t> (7 µg) was digested with <t>BamHI</t> which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λ BstE II, L. bicolor wild type (Wt) and transgenic strains.
    Bamhi, supplied by Valiant, used in various techniques. Bioz Stars score: 94/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Boehringer Mannheim bamhi
    Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total <t>DNA</t> (7 µg) was digested with <t>BamHI</t> which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λ BstE II, L. bicolor wild type (Wt) and transgenic strains.
    Bamhi, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 87/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bamhi  (Roche)
    94
    Roche bamhi
    Transformation of a K. marxianus <t>ura3</t> mutant with a linear DNA of S. cerevisiae URA3 (Sc URA3 ). (A) The K. marxianus ura3 mutant was not transformed with the intact Sc URA3 plasmid (pRS316) but with SmaI-digested pRS316 (4.8 kb) and the Sc URA3 PCR fragment (1.7 kb). (B) Southern blot hybridization of chromosomal DNA of S. cerevisiae BY4704 (lane 1), K. marxianus wild-type (WT) DMKU3-1042 (lane 2), ura3 mutant RAK3605 (lane 3), and nine Sc URA3 transformants from strain RAK3605 (lanes 4 to 12). Chromosomal DNA was digested with <t>BamHI,</t> run on a 0.8% agarose gel, transferred to a nylon membrane, and hybridized with a digoxigenin-labeled Sc URA3 probe. The bands indicated by an arrowhead are likely the authentic K. marxianus URA3 that cross-hybridizes with Sc URA3 . M, DNA size marker.
    Bamhi, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 985 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Eurofins bamhi
    Transformation of a K. marxianus <t>ura3</t> mutant with a linear DNA of S. cerevisiae URA3 (Sc URA3 ). (A) The K. marxianus ura3 mutant was not transformed with the intact Sc URA3 plasmid (pRS316) but with SmaI-digested pRS316 (4.8 kb) and the Sc URA3 PCR fragment (1.7 kb). (B) Southern blot hybridization of chromosomal DNA of S. cerevisiae BY4704 (lane 1), K. marxianus wild-type (WT) DMKU3-1042 (lane 2), ura3 mutant RAK3605 (lane 3), and nine Sc URA3 transformants from strain RAK3605 (lanes 4 to 12). Chromosomal DNA was digested with <t>BamHI,</t> run on a 0.8% agarose gel, transferred to a nylon membrane, and hybridized with a digoxigenin-labeled Sc URA3 probe. The bands indicated by an arrowhead are likely the authentic K. marxianus URA3 that cross-hybridizes with Sc URA3 . M, DNA size marker.
    Bamhi, supplied by Eurofins, used in various techniques. Bioz Stars score: 94/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Toyobo bamhi
    The effect of transiently expressed nuclear <t>MxA</t> proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with <t>BamHI.</t> These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).
    Bamhi, supplied by Toyobo, used in various techniques. Bioz Stars score: 85/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa bamhi
    <t>Integron</t> comparisons based on <t>BamHI</t> digestion of plasmids. Genes are denoted by arrows and colours based on the gene function classifications. Shaded areas denote regions with > 99% nucleotide sequence identity, with the exception of > 97% for the comparison of GC aacA4cr in In846 and GC aacA4′-37 in novel In1059. A , Comparison of In37, In843, In62, In1021, In846 and novel In1059 integrons after BamHI digestion. B - a , Comparison of integrons In37, novel In1059 and transposon Tn1696 (DQ310703); B - b , Comparison of integrons In846, novel In1059 and transposon Tn7017 (KJ571202). The novel In1059 integron shares higher sequence identity with integron Tn7017 than with integron Tn1696
    Bamhi, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 5047 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare bamhi
    <t>Integron</t> comparisons based on <t>BamHI</t> digestion of plasmids. Genes are denoted by arrows and colours based on the gene function classifications. Shaded areas denote regions with > 99% nucleotide sequence identity, with the exception of > 97% for the comparison of GC aacA4cr in In846 and GC aacA4′-37 in novel In1059. A , Comparison of In37, In843, In62, In1021, In846 and novel In1059 integrons after BamHI digestion. B - a , Comparison of integrons In37, novel In1059 and transposon Tn1696 (DQ310703); B - b , Comparison of integrons In846, novel In1059 and transposon Tn7017 (KJ571202). The novel In1059 integron shares higher sequence identity with integron Tn7017 than with integron Tn1696
    Bamhi, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 4319 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GenScript bamhi
    <t>Integron</t> comparisons based on <t>BamHI</t> digestion of plasmids. Genes are denoted by arrows and colours based on the gene function classifications. Shaded areas denote regions with > 99% nucleotide sequence identity, with the exception of > 97% for the comparison of GC aacA4cr in In846 and GC aacA4′-37 in novel In1059. A , Comparison of In37, In843, In62, In1021, In846 and novel In1059 integrons after BamHI digestion. B - a , Comparison of integrons In37, novel In1059 and transposon Tn1696 (DQ310703); B - b , Comparison of integrons In846, novel In1059 and transposon Tn7017 (KJ571202). The novel In1059 integron shares higher sequence identity with integron Tn7017 than with integron Tn1696
    Bamhi, supplied by GenScript, used in various techniques. Bioz Stars score: 94/100, based on 405 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa bamhi linker
    <t>Integron</t> comparisons based on <t>BamHI</t> digestion of plasmids. Genes are denoted by arrows and colours based on the gene function classifications. Shaded areas denote regions with > 99% nucleotide sequence identity, with the exception of > 97% for the comparison of GC aacA4cr in In846 and GC aacA4′-37 in novel In1059. A , Comparison of In37, In843, In62, In1021, In846 and novel In1059 integrons after BamHI digestion. B - a , Comparison of integrons In37, novel In1059 and transposon Tn1696 (DQ310703); B - b , Comparison of integrons In846, novel In1059 and transposon Tn7017 (KJ571202). The novel In1059 integron shares higher sequence identity with integron Tn7017 than with integron Tn1696
    Bamhi Linker, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Shanghai Genechem bamhi agei
    <t>Integron</t> comparisons based on <t>BamHI</t> digestion of plasmids. Genes are denoted by arrows and colours based on the gene function classifications. Shaded areas denote regions with > 99% nucleotide sequence identity, with the exception of > 97% for the comparison of GC aacA4cr in In846 and GC aacA4′-37 in novel In1059. A , Comparison of In37, In843, In62, In1021, In846 and novel In1059 integrons after BamHI digestion. B - a , Comparison of integrons In37, novel In1059 and transposon Tn1696 (DQ310703); B - b , Comparison of integrons In846, novel In1059 and transposon Tn7017 (KJ571202). The novel In1059 integron shares higher sequence identity with integron Tn7017 than with integron Tn1696
    Bamhi Agei, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 96/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega bamhi enzyme
    <t>Integron</t> comparisons based on <t>BamHI</t> digestion of plasmids. Genes are denoted by arrows and colours based on the gene function classifications. Shaded areas denote regions with > 99% nucleotide sequence identity, with the exception of > 97% for the comparison of GC aacA4cr in In846 and GC aacA4′-37 in novel In1059. A , Comparison of In37, In843, In62, In1021, In846 and novel In1059 integrons after BamHI digestion. B - a , Comparison of integrons In37, novel In1059 and transposon Tn1696 (DQ310703); B - b , Comparison of integrons In846, novel In1059 and transposon Tn7017 (KJ571202). The novel In1059 integron shares higher sequence identity with integron Tn7017 than with integron Tn1696
    Bamhi Enzyme, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    TaKaRa bamhi hindiii
    <t>Integron</t> comparisons based on <t>BamHI</t> digestion of plasmids. Genes are denoted by arrows and colours based on the gene function classifications. Shaded areas denote regions with > 99% nucleotide sequence identity, with the exception of > 97% for the comparison of GC aacA4cr in In846 and GC aacA4′-37 in novel In1059. A , Comparison of In37, In843, In62, In1021, In846 and novel In1059 integrons after BamHI digestion. B - a , Comparison of integrons In37, novel In1059 and transposon Tn1696 (DQ310703); B - b , Comparison of integrons In846, novel In1059 and transposon Tn7017 (KJ571202). The novel In1059 integron shares higher sequence identity with integron Tn7017 than with integron Tn1696
    Bamhi Hindiii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene bamhi linker
    Targeted disruption of Sept6 . (A) A schematic representation of the targeting strategy. The structure of Sept6 products (protein and mRNA) (top), the wild-type locus, the targeting construct, and the targeted locus are shown. Genomic fragments, including exon 3 (a box with diagonal lines) of Sept6 , that were obtained by the screening of the genomic library and the probes used for a Southern blot analysis are indicated by bold horizontal and dotted lines, respectively. The restriction sites and a converted restriction site are indicated by vertical solid and dotted arrows, respectively. The orientations of the neomycin resistance gene ( Neo ) and the herpes simplex virus thymidine kinase gene ( tk ) are indicated by horizontal arrows. The primers used are indicated by horizontal arrowheads. The location of P-loop motif (P), the second ATG (*), and a breakpoint in fusion with MLL (B.P.) are shown in Sept6 protein and mRNA. B, <t>BamHI;</t> Bs, BstXI; N, NheI; S, SacI; Sp, SphI; X, <t>XhoI.</t> (B) Homologous recombination in the male ES cells by Southern blot analysis. BamHI- or SphI-digested genomic DNA extracted from the ES cells was hybridized with the 5′ probe or the 3′ probe, resulting in the wild-type 5.5- and targeted 6.3-kb bands or the wild-type (Y/+) 7.4- and targeted (Y/−) 5.3-kb bands (data not shown), respectively. (C and D) The expression of Sept6 protein in the brain (C) and mRNA in MEFs (D, upper panel) derived from the wild-type (Y/+) and disrupted (Y/−) mice by Western blot analysis using the anti-Sept6-1 antibody and RT-PCR analysis using primers S6-41S and S6KO-AS-Pr, respectively. The expression levels of β 2 microglobulin (D, lower panel) were used as internal standards of the RT-PCR analysis. M, 100-bp DNA ladder (New England Biolabs).
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    Promega bamhi noti
    Targeted disruption of Sept6 . (A) A schematic representation of the targeting strategy. The structure of Sept6 products (protein and mRNA) (top), the wild-type locus, the targeting construct, and the targeted locus are shown. Genomic fragments, including exon 3 (a box with diagonal lines) of Sept6 , that were obtained by the screening of the genomic library and the probes used for a Southern blot analysis are indicated by bold horizontal and dotted lines, respectively. The restriction sites and a converted restriction site are indicated by vertical solid and dotted arrows, respectively. The orientations of the neomycin resistance gene ( Neo ) and the herpes simplex virus thymidine kinase gene ( tk ) are indicated by horizontal arrows. The primers used are indicated by horizontal arrowheads. The location of P-loop motif (P), the second ATG (*), and a breakpoint in fusion with MLL (B.P.) are shown in Sept6 protein and mRNA. B, <t>BamHI;</t> Bs, BstXI; N, NheI; S, SacI; Sp, SphI; X, <t>XhoI.</t> (B) Homologous recombination in the male ES cells by Southern blot analysis. BamHI- or SphI-digested genomic DNA extracted from the ES cells was hybridized with the 5′ probe or the 3′ probe, resulting in the wild-type 5.5- and targeted 6.3-kb bands or the wild-type (Y/+) 7.4- and targeted (Y/−) 5.3-kb bands (data not shown), respectively. (C and D) The expression of Sept6 protein in the brain (C) and mRNA in MEFs (D, upper panel) derived from the wild-type (Y/+) and disrupted (Y/−) mice by Western blot analysis using the anti-Sept6-1 antibody and RT-PCR analysis using primers S6-41S and S6KO-AS-Pr, respectively. The expression levels of β 2 microglobulin (D, lower panel) were used as internal standards of the RT-PCR analysis. M, 100-bp DNA ladder (New England Biolabs).
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    GenScript bamhi site
    Targeted disruption of Sept6 . (A) A schematic representation of the targeting strategy. The structure of Sept6 products (protein and mRNA) (top), the wild-type locus, the targeting construct, and the targeted locus are shown. Genomic fragments, including exon 3 (a box with diagonal lines) of Sept6 , that were obtained by the screening of the genomic library and the probes used for a Southern blot analysis are indicated by bold horizontal and dotted lines, respectively. The restriction sites and a converted restriction site are indicated by vertical solid and dotted arrows, respectively. The orientations of the neomycin resistance gene ( Neo ) and the herpes simplex virus thymidine kinase gene ( tk ) are indicated by horizontal arrows. The primers used are indicated by horizontal arrowheads. The location of P-loop motif (P), the second ATG (*), and a breakpoint in fusion with MLL (B.P.) are shown in Sept6 protein and mRNA. B, <t>BamHI;</t> Bs, BstXI; N, NheI; S, SacI; Sp, SphI; X, <t>XhoI.</t> (B) Homologous recombination in the male ES cells by Southern blot analysis. BamHI- or SphI-digested genomic DNA extracted from the ES cells was hybridized with the 5′ probe or the 3′ probe, resulting in the wild-type 5.5- and targeted 6.3-kb bands or the wild-type (Y/+) 7.4- and targeted (Y/−) 5.3-kb bands (data not shown), respectively. (C and D) The expression of Sept6 protein in the brain (C) and mRNA in MEFs (D, upper panel) derived from the wild-type (Y/+) and disrupted (Y/−) mice by Western blot analysis using the anti-Sept6-1 antibody and RT-PCR analysis using primers S6-41S and S6KO-AS-Pr, respectively. The expression levels of β 2 microglobulin (D, lower panel) were used as internal standards of the RT-PCR analysis. M, 100-bp DNA ladder (New England Biolabs).
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    TaKaRa ecori bamhi
    Targeted disruption of Sept6 . (A) A schematic representation of the targeting strategy. The structure of Sept6 products (protein and mRNA) (top), the wild-type locus, the targeting construct, and the targeted locus are shown. Genomic fragments, including exon 3 (a box with diagonal lines) of Sept6 , that were obtained by the screening of the genomic library and the probes used for a Southern blot analysis are indicated by bold horizontal and dotted lines, respectively. The restriction sites and a converted restriction site are indicated by vertical solid and dotted arrows, respectively. The orientations of the neomycin resistance gene ( Neo ) and the herpes simplex virus thymidine kinase gene ( tk ) are indicated by horizontal arrows. The primers used are indicated by horizontal arrowheads. The location of P-loop motif (P), the second ATG (*), and a breakpoint in fusion with MLL (B.P.) are shown in Sept6 protein and mRNA. B, <t>BamHI;</t> Bs, BstXI; N, NheI; S, SacI; Sp, SphI; X, <t>XhoI.</t> (B) Homologous recombination in the male ES cells by Southern blot analysis. BamHI- or SphI-digested genomic DNA extracted from the ES cells was hybridized with the 5′ probe or the 3′ probe, resulting in the wild-type 5.5- and targeted 6.3-kb bands or the wild-type (Y/+) 7.4- and targeted (Y/−) 5.3-kb bands (data not shown), respectively. (C and D) The expression of Sept6 protein in the brain (C) and mRNA in MEFs (D, upper panel) derived from the wild-type (Y/+) and disrupted (Y/−) mice by Western blot analysis using the anti-Sept6-1 antibody and RT-PCR analysis using primers S6-41S and S6KO-AS-Pr, respectively. The expression levels of β 2 microglobulin (D, lower panel) were used as internal standards of the RT-PCR analysis. M, 100-bp DNA ladder (New England Biolabs).
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    Thermo Fisher noti bamhi
    Targeted disruption of Sept6 . (A) A schematic representation of the targeting strategy. The structure of Sept6 products (protein and mRNA) (top), the wild-type locus, the targeting construct, and the targeted locus are shown. Genomic fragments, including exon 3 (a box with diagonal lines) of Sept6 , that were obtained by the screening of the genomic library and the probes used for a Southern blot analysis are indicated by bold horizontal and dotted lines, respectively. The restriction sites and a converted restriction site are indicated by vertical solid and dotted arrows, respectively. The orientations of the neomycin resistance gene ( Neo ) and the herpes simplex virus thymidine kinase gene ( tk ) are indicated by horizontal arrows. The primers used are indicated by horizontal arrowheads. The location of P-loop motif (P), the second ATG (*), and a breakpoint in fusion with MLL (B.P.) are shown in Sept6 protein and mRNA. B, <t>BamHI;</t> Bs, BstXI; N, NheI; S, SacI; Sp, SphI; X, <t>XhoI.</t> (B) Homologous recombination in the male ES cells by Southern blot analysis. BamHI- or SphI-digested genomic DNA extracted from the ES cells was hybridized with the 5′ probe or the 3′ probe, resulting in the wild-type 5.5- and targeted 6.3-kb bands or the wild-type (Y/+) 7.4- and targeted (Y/−) 5.3-kb bands (data not shown), respectively. (C and D) The expression of Sept6 protein in the brain (C) and mRNA in MEFs (D, upper panel) derived from the wild-type (Y/+) and disrupted (Y/−) mice by Western blot analysis using the anti-Sept6-1 antibody and RT-PCR analysis using primers S6-41S and S6KO-AS-Pr, respectively. The expression levels of β 2 microglobulin (D, lower panel) were used as internal standards of the RT-PCR analysis. M, 100-bp DNA ladder (New England Biolabs).
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    Thermo Fisher bamhi hindiii
    Targeted disruption of Sept6 . (A) A schematic representation of the targeting strategy. The structure of Sept6 products (protein and mRNA) (top), the wild-type locus, the targeting construct, and the targeted locus are shown. Genomic fragments, including exon 3 (a box with diagonal lines) of Sept6 , that were obtained by the screening of the genomic library and the probes used for a Southern blot analysis are indicated by bold horizontal and dotted lines, respectively. The restriction sites and a converted restriction site are indicated by vertical solid and dotted arrows, respectively. The orientations of the neomycin resistance gene ( Neo ) and the herpes simplex virus thymidine kinase gene ( tk ) are indicated by horizontal arrows. The primers used are indicated by horizontal arrowheads. The location of P-loop motif (P), the second ATG (*), and a breakpoint in fusion with MLL (B.P.) are shown in Sept6 protein and mRNA. B, <t>BamHI;</t> Bs, BstXI; N, NheI; S, SacI; Sp, SphI; X, <t>XhoI.</t> (B) Homologous recombination in the male ES cells by Southern blot analysis. BamHI- or SphI-digested genomic DNA extracted from the ES cells was hybridized with the 5′ probe or the 3′ probe, resulting in the wild-type 5.5- and targeted 6.3-kb bands or the wild-type (Y/+) 7.4- and targeted (Y/−) 5.3-kb bands (data not shown), respectively. (C and D) The expression of Sept6 protein in the brain (C) and mRNA in MEFs (D, upper panel) derived from the wild-type (Y/+) and disrupted (Y/−) mice by Western blot analysis using the anti-Sept6-1 antibody and RT-PCR analysis using primers S6-41S and S6KO-AS-Pr, respectively. The expression levels of β 2 microglobulin (D, lower panel) were used as internal standards of the RT-PCR analysis. M, 100-bp DNA ladder (New England Biolabs).
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    Thermo Fisher bamhi fd
    Targeted disruption of Sept6 . (A) A schematic representation of the targeting strategy. The structure of Sept6 products (protein and mRNA) (top), the wild-type locus, the targeting construct, and the targeted locus are shown. Genomic fragments, including exon 3 (a box with diagonal lines) of Sept6 , that were obtained by the screening of the genomic library and the probes used for a Southern blot analysis are indicated by bold horizontal and dotted lines, respectively. The restriction sites and a converted restriction site are indicated by vertical solid and dotted arrows, respectively. The orientations of the neomycin resistance gene ( Neo ) and the herpes simplex virus thymidine kinase gene ( tk ) are indicated by horizontal arrows. The primers used are indicated by horizontal arrowheads. The location of P-loop motif (P), the second ATG (*), and a breakpoint in fusion with MLL (B.P.) are shown in Sept6 protein and mRNA. B, <t>BamHI;</t> Bs, BstXI; N, NheI; S, SacI; Sp, SphI; X, <t>XhoI.</t> (B) Homologous recombination in the male ES cells by Southern blot analysis. BamHI- or SphI-digested genomic DNA extracted from the ES cells was hybridized with the 5′ probe or the 3′ probe, resulting in the wild-type 5.5- and targeted 6.3-kb bands or the wild-type (Y/+) 7.4- and targeted (Y/−) 5.3-kb bands (data not shown), respectively. (C and D) The expression of Sept6 protein in the brain (C) and mRNA in MEFs (D, upper panel) derived from the wild-type (Y/+) and disrupted (Y/−) mice by Western blot analysis using the anti-Sept6-1 antibody and RT-PCR analysis using primers S6-41S and S6KO-AS-Pr, respectively. The expression levels of β 2 microglobulin (D, lower panel) were used as internal standards of the RT-PCR analysis. M, 100-bp DNA ladder (New England Biolabs).
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    Image Search Results


    Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pΔAPRT1-NEO5, with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).

    Journal: Genes

    Article Title: Resistance to 6-Methylpurine is Conferred by Defective Adenine Phosphoribosyltransferase in Tetrahymena

    doi: 10.3390/genes9040179

    Figure Lengend Snippet: Effect of partial APRT1 knockout on cell sensitivity to 6mp. ( A ) A schematic showing the APRT1 genomic locus (upper), the plasmid vector pΔAPRT1-NEO5, with paromomycin resistance cassette ( NEO5 ) (middle), and after homologous recombination (lower). ( B ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from wild-type cells and partial APRT1 knockout cells. Molecular weight of signals against the probe corresponds to the prediction in ( A ). ( C ) Cell growth curves in the presence of 15 µg/mL 6mp. Points and attached bars correspond to mean measurements from three identical experiments and their standard deviations, respectively. Cells sensitive to 6mp all died by 72 h. ( D ) Southern blot analysis of XhoI- and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after measurement of cell growth rates in 6mp. ( E ) Southern blot analysis of XhoI-and BamHI-digested genomic DNA from partial APRT1 knockout cells before and after the induction of phenotypic assortment with 5 mg/mL paromomycin for 2 months. Molecular weight of signals against the probe corresponds to that predicted in the schematic in ( A ).

    Article Snippet: Amplified forward and reverse target fragments were cloned into the BamHI–BamHI and PstI–PstI I sites, respectively, of pAkRNAi-NEO5 with the NEBuilder HF DNA Assembly Kit (New England BioLabs) to create the hairpin cassette.

    Techniques: Knock-Out, Plasmid Preparation, Homologous Recombination, Southern Blot, Molecular Weight

    Distinct Influences of the Ku80 C-terminus on DNA-PK Activation with Linearized Plasmid DNA. a) DNA-PK kinase stimulation with plasmid DNA linearized with EcoRV generating blunt-ended termini and with KpnI generating 4 base 3’ single stranded overhangs. b) DNA-PK kinase stimulation with plasmid DNA linearized with XhoI and BamHI generating 4 base 5’ single stranded overhangs. DNA substrates are depicted pictorially. DNA termini generated by digestion are depicted below each graph indicating locations of pyrimidines (Py) and purines (Pu). Kinase activity is reported as the mean and SD of pmol of phosphate transferred. Asterisks indicate statistically significant differences compared to wild type (p

    Journal: PLoS ONE

    Article Title: Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit

    doi: 10.1371/journal.pone.0127321

    Figure Lengend Snippet: Distinct Influences of the Ku80 C-terminus on DNA-PK Activation with Linearized Plasmid DNA. a) DNA-PK kinase stimulation with plasmid DNA linearized with EcoRV generating blunt-ended termini and with KpnI generating 4 base 3’ single stranded overhangs. b) DNA-PK kinase stimulation with plasmid DNA linearized with XhoI and BamHI generating 4 base 5’ single stranded overhangs. DNA substrates are depicted pictorially. DNA termini generated by digestion are depicted below each graph indicating locations of pyrimidines (Py) and purines (Pu). Kinase activity is reported as the mean and SD of pmol of phosphate transferred. Asterisks indicate statistically significant differences compared to wild type (p

    Article Snippet: Kinase assays using plasmid DNA substrates were performed with pcDNA3.1 digested with either XhoI, BamHI, EcoRV, and KpnI or pCAG-GFP digested with XbaI or EcoRI (New England Biolabs).

    Techniques: Activation Assay, Plasmid Preparation, Generated, Activity Assay

    BamHI digestion prior to bisulfite sequencing decreases cytosine unconvertion rate. Targeted bisulfite sequencing was used to compare undigested and digested DNA methylation levels at five different regions of the mtDNA from human muscle cells and SKOV3 cells ( N = 3). (A) Drawing displays the mtDNA regions investigated by targeted bisulfite sequencing. (B) Percentage methylation for undigested and digested mtDNA. Full circle represents cytosines in CpG context whereas open circle is cytosines in non-CpG context. Results are presented with a min-max interval and a sign test was used to test for significant methylation differences. D-loop (6–298) P = 2.02E-41 (Lonza) and P = 1.40E-24 (SKOV3); D-loop (279–458): P = 5.00E-12 (Lonza) and P = 8.20E-08 (SKOV3); tRNA-F+12S: P = 9.22E-15 (Lonza) and P = 1.29E-9 (SKOV3); 16S: P = 6.50E-05; ND5: P = 1.70E-05 (Lonza) and P = 9.97E-13 (SKOV3); CYTB: P = 2.96E-09 (Lonza), and P = 6.68E-13 (SKOV3). D-loop (6–298) includes origin of replication and tRNA-F+12S includes heavy strand promoter 2. P H1 : heavy-strand promoter 1; P H2 : Heavy-strand promote 2; P L : Light-strand promoter; OH: Origin of replication from heavy-strand; O L : Origin of replication from light-strand. ND: not determined.

    Journal: Frontiers in Genetics

    Article Title: Evidence Suggesting Absence of Mitochondrial DNA Methylation

    doi: 10.3389/fgene.2017.00166

    Figure Lengend Snippet: BamHI digestion prior to bisulfite sequencing decreases cytosine unconvertion rate. Targeted bisulfite sequencing was used to compare undigested and digested DNA methylation levels at five different regions of the mtDNA from human muscle cells and SKOV3 cells ( N = 3). (A) Drawing displays the mtDNA regions investigated by targeted bisulfite sequencing. (B) Percentage methylation for undigested and digested mtDNA. Full circle represents cytosines in CpG context whereas open circle is cytosines in non-CpG context. Results are presented with a min-max interval and a sign test was used to test for significant methylation differences. D-loop (6–298) P = 2.02E-41 (Lonza) and P = 1.40E-24 (SKOV3); D-loop (279–458): P = 5.00E-12 (Lonza) and P = 8.20E-08 (SKOV3); tRNA-F+12S: P = 9.22E-15 (Lonza) and P = 1.29E-9 (SKOV3); 16S: P = 6.50E-05; ND5: P = 1.70E-05 (Lonza) and P = 9.97E-13 (SKOV3); CYTB: P = 2.96E-09 (Lonza), and P = 6.68E-13 (SKOV3). D-loop (6–298) includes origin of replication and tRNA-F+12S includes heavy strand promoter 2. P H1 : heavy-strand promoter 1; P H2 : Heavy-strand promote 2; P L : Light-strand promoter; OH: Origin of replication from heavy-strand; O L : Origin of replication from light-strand. ND: not determined.

    Article Snippet: Restriction Enzyme Treatment Genomic DNA was either untreated or treated with BamHI restriction enzyme (New England Biolabs, cat # R0136) to obtain circular or linearized mtDNA.

    Techniques: Methylation Sequencing, DNA Methylation Assay, Methylation

    Sperm transfected with E6/E7 plasmid penetrate oocytes. a . Scheme of the recombinant plasmid pIRES2-AcGFP1-E6E7. E6/E7 genes have been amplified (1032 bp) from plasmid p1321 HPV-16 E6/E7 by PCR and subcloned to plasmid pIRES2-AcGFP1 between SalI and BamHI restriction sites. b . PCR for HPV E6/E7 genes from transfected sperm. Lane M: DNA marker (100 bp); 1: sperm transfected with recombinant E6/E7 plasmid; 2: negative control (no template); 3: sperm transfected only with Lipofectamine 2000; 4: positive control (pIRES2-AcGFP1-E6E7 plasmid). c . Mean number of human sperm penetrated per hamster oocyte in control and sperm transfected with HPV-16 E6/E7 plasmid. d . Hamster oocytes penetrated by control sperm and sperm transfected with HPV16 E6/E7 plasmid in bright field (BF, upper panel) and fluorescence (FL, lower panel) using SYBR green DNA stain.

    Journal: PLoS ONE

    Article Title: Mechanism of Human Papillomavirus Binding to Human Spermatozoa and Fertilizing Ability of Infected Spermatozoa

    doi: 10.1371/journal.pone.0015036

    Figure Lengend Snippet: Sperm transfected with E6/E7 plasmid penetrate oocytes. a . Scheme of the recombinant plasmid pIRES2-AcGFP1-E6E7. E6/E7 genes have been amplified (1032 bp) from plasmid p1321 HPV-16 E6/E7 by PCR and subcloned to plasmid pIRES2-AcGFP1 between SalI and BamHI restriction sites. b . PCR for HPV E6/E7 genes from transfected sperm. Lane M: DNA marker (100 bp); 1: sperm transfected with recombinant E6/E7 plasmid; 2: negative control (no template); 3: sperm transfected only with Lipofectamine 2000; 4: positive control (pIRES2-AcGFP1-E6E7 plasmid). c . Mean number of human sperm penetrated per hamster oocyte in control and sperm transfected with HPV-16 E6/E7 plasmid. d . Hamster oocytes penetrated by control sperm and sperm transfected with HPV16 E6/E7 plasmid in bright field (BF, upper panel) and fluorescence (FL, lower panel) using SYBR green DNA stain.

    Article Snippet: The PCR mixture consisted of 5 µL Expand high fidelity buffer with MgCl2 , 1 µL of PCR grade nucleotide mix (10×), 20 pmol of each primer with SalI and BamHI restriction sites (New England Biolabs, Ipswich, MA) including forward: 5′-GTGCGCGTCGACGTGATGCACCAAAAGAGAACTG-3′ and reverse: 5′-GTGCGCGGATCCGTGTGGTTTCTGAGAACAGATG-3′ , 0,75 µL Expand high fidelity enzyme mix (Expand High Fidelity PCR System dNTPack, Roche Applied Science, Mannheim, Germany) and sterile H2 O in a final volume of 50 μL.

    Techniques: Transfection, Plasmid Preparation, Recombinant, Amplification, Polymerase Chain Reaction, Marker, Negative Control, Positive Control, Fluorescence, SYBR Green Assay, Staining

    Map of PCR primer binding sites and restriction enzyme cut sizes for the barcode fragment of COI. BamHI restriction sites are only found in haplotype group A alleles while AflIII and BseYI restriction sites are only found in haplotype group B alleles.

    Journal: Systematic entomology

    Article Title: A tale of two haplotype groups: Evaluating the New World Junonia ring species hypothesis using the distribution of divergent COI haplotypes

    doi: 10.1111/syen.12120

    Figure Lengend Snippet: Map of PCR primer binding sites and restriction enzyme cut sizes for the barcode fragment of COI. BamHI restriction sites are only found in haplotype group A alleles while AflIII and BseYI restriction sites are only found in haplotype group B alleles.

    Article Snippet: PCR products of the correct size were evaluated by a triple digest using BseYI, AflIII, and BamHI restriction endonucleases (New England Biolabs, Ipswich, MA, USA) ( ).

    Techniques: Polymerase Chain Reaction, Binding Assay

    Figure 4. In vivo assembly of synthetic genes by IVOE. Each overlapping region allowed crossover events to occur between fragments giving rise to an autonomously repaired vector containing single and double expression cassettes in the correct orientation. ( A ) and ( B ) Single expression cassettes are constructed in pESC by engineering specific primers containing overhangs to foster in vivo cloning with the linearized plasmid in yeast (pJRoC30 was used as template for Vp or Lac amplifications). ( C ) and ( D ) The pESC constructs obtained in ( A ) and ( B ) were used as scaffolds to assemble Lac and Vp genes under the control of different promoter/terminator pairs. Primers used: (1)-MCS1- Vp / Lac -α-BamHI, (2)-MCS2- Vp -ter-NheI, (3)-MCS2- Lac -ter-NheI, (4)-MCS1- Vp / Lac -α-SpeI, (5)-MCS1- Lac -ter-SacI and (6)-MCS1- Vp -ter-SacI. Black arrows indicate the direction of the transcription process.

    Journal: Bioengineered

    Article Title: Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae

    doi: 10.4161/bioe.29167

    Figure Lengend Snippet: Figure 4. In vivo assembly of synthetic genes by IVOE. Each overlapping region allowed crossover events to occur between fragments giving rise to an autonomously repaired vector containing single and double expression cassettes in the correct orientation. ( A ) and ( B ) Single expression cassettes are constructed in pESC by engineering specific primers containing overhangs to foster in vivo cloning with the linearized plasmid in yeast (pJRoC30 was used as template for Vp or Lac amplifications). ( C ) and ( D ) The pESC constructs obtained in ( A ) and ( B ) were used as scaffolds to assemble Lac and Vp genes under the control of different promoter/terminator pairs. Primers used: (1)-MCS1- Vp / Lac -α-BamHI, (2)-MCS2- Vp -ter-NheI, (3)-MCS2- Lac -ter-NheI, (4)-MCS1- Vp / Lac -α-SpeI, (5)-MCS1- Lac -ter-SacI and (6)-MCS1- Vp -ter-SacI. Black arrows indicate the direction of the transcription process.

    Article Snippet: The ura3 -deficient S. cerevisiae strain BJ5465 ( α ura3–52 trp1 leu2Δ1 his3Δ200 pep4::HIS2 prb1Δ1.6R can1 GAL1 ) was obtained from LGCPromochem, the NucleoSpin Plasmid kit was purchased from Macherey-Nagel, and the restriction enzymes BamHI, NheI, SpeI, SacI, and NotI from New England Biolabs.

    Techniques: In Vivo, Plasmid Preparation, Expressing, Construct, Clone Assay

    TMV-CP DNA fragments (1% agarose gel). ( A ) The whole TMV-CP fragments with BamH I and Xho I restriction enzyme cutting sites which have been cloned in PGEX-6P-1. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a positive control with DNA template. Lane 3 is a negative control without DNA template. ( B ) The whole TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a negative control without DNA template. Lane 3 is a positive control with DNA template. ( C ) The truncation of four amino acids from the C-terminus of TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. Lane 1 is a negative control without DNA template, whereas lane 2 is a positive control with DNA template. Lane 3 is the amplified PCR product that ran at approximately 500 bp compared with the DNA marker (lane M).

    Journal: Virology Journal

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein

    doi: 10.1186/1743-422X-9-279

    Figure Lengend Snippet: TMV-CP DNA fragments (1% agarose gel). ( A ) The whole TMV-CP fragments with BamH I and Xho I restriction enzyme cutting sites which have been cloned in PGEX-6P-1. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a positive control with DNA template. Lane 3 is a negative control without DNA template. ( B ) The whole TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a negative control without DNA template. Lane 3 is a positive control with DNA template. ( C ) The truncation of four amino acids from the C-terminus of TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. Lane 1 is a negative control without DNA template, whereas lane 2 is a positive control with DNA template. Lane 3 is the amplified PCR product that ran at approximately 500 bp compared with the DNA marker (lane M).

    Article Snippet: Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ).

    Techniques: Agarose Gel Electrophoresis, Clone Assay, Amplification, Polymerase Chain Reaction, Marker, Positive Control, Negative Control

    Characterization of part of the mouse Rad54B genomic locus and generation of mouse ES cells carrying a disrupted mRad54B allele. (A) Part of the mRad54B genomic locus and structure of the targeting construct. Exons 12 to 15 are indicated by black boxes. Shown are the locations of selected restriction sites, EcoRI (E), BamHI (B), BglII (Bg), HindIII (H), and XbaI (X). The positions of two different probes, named A and B, are indicated. (B) DNA blot analysis of G418-resistant ES clones with probe A and EcoRI digested DNA. The wild-type (wt) allele yields a 3.0-kb band, while the disrupted allele results in a 3.6-kb band. Lane 1, wild-type ES cell; lane 2, clone with a randomly integrated targeting construct; lane 3, clone with a homologously integrated targeting construct. (C) RNA blot analysis of mRad54B transcripts in mice carrying the disrupted allele. Total RNA (15 μg) isolated from testes of wild-type, mRad54B +/ − , and mRad54B −/− males was probed with 5′ and 3′ mRad54B cDNA probes. A GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA probe served as a loading control.

    Journal: Molecular and Cellular Biology

    Article Title: Differential Contributions of Mammalian Rad54 Paralogs to Recombination, DNA Damage Repair, and Meiosis †

    doi: 10.1128/MCB.26.3.976-989.2006

    Figure Lengend Snippet: Characterization of part of the mouse Rad54B genomic locus and generation of mouse ES cells carrying a disrupted mRad54B allele. (A) Part of the mRad54B genomic locus and structure of the targeting construct. Exons 12 to 15 are indicated by black boxes. Shown are the locations of selected restriction sites, EcoRI (E), BamHI (B), BglII (Bg), HindIII (H), and XbaI (X). The positions of two different probes, named A and B, are indicated. (B) DNA blot analysis of G418-resistant ES clones with probe A and EcoRI digested DNA. The wild-type (wt) allele yields a 3.0-kb band, while the disrupted allele results in a 3.6-kb band. Lane 1, wild-type ES cell; lane 2, clone with a randomly integrated targeting construct; lane 3, clone with a homologously integrated targeting construct. (C) RNA blot analysis of mRad54B transcripts in mice carrying the disrupted allele. Total RNA (15 μg) isolated from testes of wild-type, mRad54B +/ − , and mRad54B −/− males was probed with 5′ and 3′ mRad54B cDNA probes. A GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA probe served as a loading control.

    Article Snippet: The hRAD54B cDNA was subcloned from pUC18 into the BamHI site of pFastBac (Invitrogen).

    Techniques: Construct, Clone Assay, Northern blot, Mouse Assay, Isolation

    Golden Gate-based multisite mutagenesis. A) The FlpO ORF contains two internal BsaI sites. These are mutated by amplifying fragments of the FlpO ORF via PCR. The primers contain flanking BsaI sites that lead to compatible overhangs after restriction digest. The site-directed mutagenesis vector (pGG-sdm) contains BsaI sites for the mutagenesis assembly. Additionally, BamHI and KpnI sites allow the transfer of the mutated DNA assembly to Golden Gate entry vectors. From there, mRNA can be generated using SP6 RNA polymerase. Note that the pGG-sdm vector represents a standard Gateway TM middle entry vector. B) A Golden Gate reaction is used to prepare a dual FRT-based recombination construct in the Gateway TM middle entry vector. An LR reaction was prepared to generate a final expression construct. ISceI sites flank the expression cassette. The dual FRT element is driven by the zebrafish beta actin 2 promoter and a human globin intron with SV40 polyA is used in the 3’ entry vector. C) Zebrafish embryos were injected with the expression construct alone or in combination with FlpO mRNA. Without FlpO mRNA 64% (n=98) of the fish were positively injected and showed eGFP expression and the absence of mCherry. In combination with FlpO mRNA 60% (n=79) of the fish were positively injected and all of them showed the complete absence of green fluorescence and the appearance of red fluorescence, which is indicative for proper FlpO activity.

    Journal: PLoS ONE

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs

    doi: 10.1371/journal.pone.0076117

    Figure Lengend Snippet: Golden Gate-based multisite mutagenesis. A) The FlpO ORF contains two internal BsaI sites. These are mutated by amplifying fragments of the FlpO ORF via PCR. The primers contain flanking BsaI sites that lead to compatible overhangs after restriction digest. The site-directed mutagenesis vector (pGG-sdm) contains BsaI sites for the mutagenesis assembly. Additionally, BamHI and KpnI sites allow the transfer of the mutated DNA assembly to Golden Gate entry vectors. From there, mRNA can be generated using SP6 RNA polymerase. Note that the pGG-sdm vector represents a standard Gateway TM middle entry vector. B) A Golden Gate reaction is used to prepare a dual FRT-based recombination construct in the Gateway TM middle entry vector. An LR reaction was prepared to generate a final expression construct. ISceI sites flank the expression cassette. The dual FRT element is driven by the zebrafish beta actin 2 promoter and a human globin intron with SV40 polyA is used in the 3’ entry vector. C) Zebrafish embryos were injected with the expression construct alone or in combination with FlpO mRNA. Without FlpO mRNA 64% (n=98) of the fish were positively injected and showed eGFP expression and the absence of mCherry. In combination with FlpO mRNA 60% (n=79) of the fish were positively injected and all of them showed the complete absence of green fluorescence and the appearance of red fluorescence, which is indicative for proper FlpO activity.

    Article Snippet: For that, we used standard BamHI, KpnI enzymes from Thermo, Fisher together with the Fastdigest buffer (Thermo, Fisher).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Plasmid Preparation, Generated, Construct, Expressing, Injection, Fluorescence In Situ Hybridization, Fluorescence, Activity Assay

    Golden Gate entry vector design and cloning. Cloning cassette used to fill Golden Gate entry vectors. Inserts can be introduced in two ways. XcmI restriction digest generates overhangs that can be used for TA cloning. Second, BamHI and KpnI sites can be used to clone inserts with different length either via standard ligation or an oligo annealing and cloning procedure. This cassette is flanked with BsaI sites used for the Golden Gate assembly. Each entry vector contains an SP6 promoter and a globin intron and SV40 polyA site flanking the insert for the generation of mRNA.

    Journal: PLoS ONE

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs

    doi: 10.1371/journal.pone.0076117

    Figure Lengend Snippet: Golden Gate entry vector design and cloning. Cloning cassette used to fill Golden Gate entry vectors. Inserts can be introduced in two ways. XcmI restriction digest generates overhangs that can be used for TA cloning. Second, BamHI and KpnI sites can be used to clone inserts with different length either via standard ligation or an oligo annealing and cloning procedure. This cassette is flanked with BsaI sites used for the Golden Gate assembly. Each entry vector contains an SP6 promoter and a globin intron and SV40 polyA site flanking the insert for the generation of mRNA.

    Article Snippet: For that, we used standard BamHI, KpnI enzymes from Thermo, Fisher together with the Fastdigest buffer (Thermo, Fisher).

    Techniques: Plasmid Preparation, Clone Assay, TA Cloning, Ligation

    Southern blot analysis of plasmid DNA from Enterobacter sp. strains 1061 and 1434 and their transconjugants. Plasmid DNA was digested with the SmaI, XhoI, and BamHI endonucleases in lanes 1 to 3, 4 to 6, 7 to 9, and 10 to 12, respectively, and hybridized

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Extended-Spectrum Beta-Lactamases among Enterobacter Isolates Obtained in Tel Aviv, Israel

    doi: 10.1128/AAC.49.3.1150-1156.2005

    Figure Lengend Snippet: Southern blot analysis of plasmid DNA from Enterobacter sp. strains 1061 and 1434 and their transconjugants. Plasmid DNA was digested with the SmaI, XhoI, and BamHI endonucleases in lanes 1 to 3, 4 to 6, 7 to 9, and 10 to 12, respectively, and hybridized

    Article Snippet: Plasmid DNA was digested with SmaI, XhoI, and BamHI endonucleases (MBI Fermentas); and the resulting restriction pattern was visualized in a 1% agarose gel by ethidium bromide staining.

    Techniques: Southern Blot, Plasmid Preparation

    Genotyping of EBVaGC strains . A: PCR -RFLP of the BamHI-F region showing the f variant (presence of two bands of 128 bp and 71 bp) for the control C666-1 cell line (lane 1) and one band of 199 bp for the F variant for cases GC2 to GC6 (lane 2 to 6). B: PCR-RFLP of the BamHI-W1/I1 region. DNA fragments were digested with BamHI giving two bands of 139 and 67 bp (type D) for GC2 to GC6 (lane 2 to 6) or one band of 245 bp (type C) for the control C666-1 cell line (lane 1). C: EBNA3C region. EBV strains of types A and B correspond to DNA fragment of 153 and 246 bp respectively. Case GC6 shows dual infection with both types A and B viruses. B95.8 is a type A virus that was used as a control (lane 1). D: XhoI polymorphism in exon 1 of the BNLF1 gene. PCR product was digested by XhoI to yield two bands of 343 bp and 154 bp in XhoI + variant for GC2 to GC6 (lane 2 to 6). C666-1 cell line is a positive of loss- XhoI variant (lane 1).

    Journal: Virology Journal

    Article Title: Characteristics of epstein barr virus variants associated with gastric carcinoma in Southern Tunisia

    doi: 10.1186/1743-422X-8-500

    Figure Lengend Snippet: Genotyping of EBVaGC strains . A: PCR -RFLP of the BamHI-F region showing the f variant (presence of two bands of 128 bp and 71 bp) for the control C666-1 cell line (lane 1) and one band of 199 bp for the F variant for cases GC2 to GC6 (lane 2 to 6). B: PCR-RFLP of the BamHI-W1/I1 region. DNA fragments were digested with BamHI giving two bands of 139 and 67 bp (type D) for GC2 to GC6 (lane 2 to 6) or one band of 245 bp (type C) for the control C666-1 cell line (lane 1). C: EBNA3C region. EBV strains of types A and B correspond to DNA fragment of 153 and 246 bp respectively. Case GC6 shows dual infection with both types A and B viruses. B95.8 is a type A virus that was used as a control (lane 1). D: XhoI polymorphism in exon 1 of the BNLF1 gene. PCR product was digested by XhoI to yield two bands of 343 bp and 154 bp in XhoI + variant for GC2 to GC6 (lane 2 to 6). C666-1 cell line is a positive of loss- XhoI variant (lane 1).

    Article Snippet: The enzymatic reactions were carried out in a final volume of 20 μl containing 10 μl of PCR product, 1X digestion buffer and 10 units of BamHI enzyme (Fermentas).

    Techniques: Polymerase Chain Reaction, Variant Assay, Infection

    Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by KpnI and BamHI/Lane 2: 100-bp DNA ladder marker

    Journal: Iranian Journal of Parasitology

    Article Title: Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct

    doi:

    Figure Lengend Snippet: Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by KpnI and BamHI/Lane 2: 100-bp DNA ladder marker

    Article Snippet: Construction of the antisense ptr1 gene Recombinant pBluescript containing L. major ptr1 gene (accession no. EF113119) was digested with KpnI and BamHI enzymes and purified using a Fermentas DNA purification kit (Cat. No. k0513) and then subcloned into pcDNA3 digested with KpnI and BamHI.

    Techniques: Electrophoresis, Marker

    Identification of recombinant plasmid pcDNA-rPTR using enzyme digestion. Lane 1:100-bp DNA ladder marker. Lane 2: pcDNA-rPTR digested with KpnI and BamHI

    Journal: Iranian Journal of Parasitology

    Article Title: Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct

    doi:

    Figure Lengend Snippet: Identification of recombinant plasmid pcDNA-rPTR using enzyme digestion. Lane 1:100-bp DNA ladder marker. Lane 2: pcDNA-rPTR digested with KpnI and BamHI

    Article Snippet: Construction of the antisense ptr1 gene Recombinant pBluescript containing L. major ptr1 gene (accession no. EF113119) was digested with KpnI and BamHI enzymes and purified using a Fermentas DNA purification kit (Cat. No. k0513) and then subcloned into pcDNA3 digested with KpnI and BamHI.

    Techniques: Recombinant, Plasmid Preparation, Marker

    Confirmation of Self-Ligated pP15A, kana R by Digestion Lane 2: digestion of pP15A, kana R vector with BamHI ; lane 3, xbaI; Lane 4, double digestion with BamHI-XbaI; Lane 5, undigested pP15A, kana R vector; Lanes 1 and 6, GeneRuler DNA ladder mix (Thermo Scientific, Waltham, MA, USA).

    Journal: Jundishapur Journal of Microbiology

    Article Title: Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance

    doi: 10.5812/jjm.29384

    Figure Lengend Snippet: Confirmation of Self-Ligated pP15A, kana R by Digestion Lane 2: digestion of pP15A, kana R vector with BamHI ; lane 3, xbaI; Lane 4, double digestion with BamHI-XbaI; Lane 5, undigested pP15A, kana R vector; Lanes 1 and 6, GeneRuler DNA ladder mix (Thermo Scientific, Waltham, MA, USA).

    Article Snippet: The pTZ57R plasmid was extracted using a GeneJET™ Plasmid Miniprep Kit (Thermo Scientific, Waltham, MA, USA) and was digested using EcoRI and BamHI restriction enzymes (Thermo Scientific, Waltham, MA, USA).

    Techniques: Plasmid Preparation

    Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the BamH I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.

    Journal: Virology

    Article Title: Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs

    doi: 10.1016/j.virol.2010.03.025

    Figure Lengend Snippet: Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the BamH I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.

    Article Snippet: It was generated by digesting with BamH I and then filling in the sticky ends with DNA polymerase Pfx (Invitrogen) followed by blunt-end re-ligation. pGlucG was derived by inserting the PCR-amplified 12 nt 5′UTR of GRV into EcoR I/Sca I-digested pTUlucTU, in place of the TBTV 5′ UTR.

    Techniques: Luciferase, Activity Assay, Generated, Mutagenesis, Construct, Sequencing

    ). 17 nt CS is shown in bold italic. (B) Genome organization of RSDaV RNA and maps of translation reporter constructs. RSlucRS has both UTRs of RSDaV flanking the luciferase coding sequence (fLUC). RSluc has only the RSDaV 5′UTR. RSlucRSBF differs from RSlucRS only by a 4 nt GAUC insertion (underlined) in the BamH I site. Bases are numbered according to their position in the RSDaV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities are normalized to uncapped RSlucRS (defined as 100%). Error bars indicate standard error.

    Journal: Virology

    Article Title: Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs

    doi: 10.1016/j.virol.2010.03.025

    Figure Lengend Snippet: ). 17 nt CS is shown in bold italic. (B) Genome organization of RSDaV RNA and maps of translation reporter constructs. RSlucRS has both UTRs of RSDaV flanking the luciferase coding sequence (fLUC). RSluc has only the RSDaV 5′UTR. RSlucRSBF differs from RSlucRS only by a 4 nt GAUC insertion (underlined) in the BamH I site. Bases are numbered according to their position in the RSDaV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities are normalized to uncapped RSlucRS (defined as 100%). Error bars indicate standard error.

    Article Snippet: It was generated by digesting with BamH I and then filling in the sticky ends with DNA polymerase Pfx (Invitrogen) followed by blunt-end re-ligation. pGlucG was derived by inserting the PCR-amplified 12 nt 5′UTR of GRV into EcoR I/Sca I-digested pTUlucTU, in place of the TBTV 5′ UTR.

    Techniques: Construct, Luciferase, Sequencing

    Joining of incompatible DNA ends by MRN and DNA ligase IIIα/XRCC1: microhomology-mediated joining of DNA ends with incompatible 5′ overhangs. A , circular plasmid DNA was digested with either KpnI ( left ) and PstI ( right ) to generate noncomplementary 3′ overhangs or EcoRI ( left ) and BamHI ( right ), to generate noncomplementary 5′ overhangs. Joining of the incompatible DNA ends was detected by the PCR using the indicated primers. B , DNA substrate (0.8 pmol) with noncomplementary 3′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. C , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. D , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of either DNA ligase IIIα/XRCC1 or DNA ligase IV/XRCC4 in the absence or presence of an equimolar amount of MRN. E , sequences of the junctions generated by the joining of incompatible 5′ ends by DNA ligase IIIα/XRCC1 in combination with either hMre11 or MRN are shown. Bolded residues indicate microhomologies.

    Journal: The Journal of Biological Chemistry

    Article Title: Human Mre11/Human Rad50/Nbs1 and DNA Ligase III?/XRCC1 Protein Complexes Act Together in an Alternative Nonhomologous End Joining Pathway *

    doi: 10.1074/jbc.M111.274159

    Figure Lengend Snippet: Joining of incompatible DNA ends by MRN and DNA ligase IIIα/XRCC1: microhomology-mediated joining of DNA ends with incompatible 5′ overhangs. A , circular plasmid DNA was digested with either KpnI ( left ) and PstI ( right ) to generate noncomplementary 3′ overhangs or EcoRI ( left ) and BamHI ( right ), to generate noncomplementary 5′ overhangs. Joining of the incompatible DNA ends was detected by the PCR using the indicated primers. B , DNA substrate (0.8 pmol) with noncomplementary 3′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. C , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of DNA ligase IIIα/XRCC1 alone or in combination with equimolar amounts of hMre11 and MRN as indicated. D , DNA substrate (0.8 pmol) with noncomplementary 5′ overhangs was incubated with 0.01 pmol of either DNA ligase IIIα/XRCC1 or DNA ligase IV/XRCC4 in the absence or presence of an equimolar amount of MRN. E , sequences of the junctions generated by the joining of incompatible 5′ ends by DNA ligase IIIα/XRCC1 in combination with either hMre11 or MRN are shown. Bolded residues indicate microhomologies.

    Article Snippet: Linear DNA molecules were generated by digestion of pGADT7 (Invitrogen) with BamHI and EcoRI for the four nucleotide 5′ overhangs and of pcDNA4HisMax C (Invitrogen) with KpnI and PstI for the four nucleotide 3′ overhangs.

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Incubation, Generated

    Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and BamHI-HF, into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) DNA from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.

    Journal: The Journal of Clinical Investigation

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo

    doi: 10.1172/JCI97053

    Figure Lengend Snippet: Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and BamHI-HF, into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) DNA from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.

    Article Snippet: Fifteen micrograms of DNA from each mouse was double digested with BamHI-HF and HindIII-HF (New England BioLabs) and single digested using AflII (New England BioLabs) in a total volume of 100 μl at 37°C overnight.

    Techniques: Transgenic Assay, Mouse Assay, Clone Assay, Plasmid Preparation, Construct, Southern Blot, Western Blot, Isolation, Polymerase Chain Reaction, Expressing, SDS Page, Positive Control

    Physical representation of the first 1 Mbp of pseudochromosome 1 in ‘Hongyang’ genome sequence with mapped reads found by stdGBS and rtGBS methods, all treatments, supported by ≥10 reads. The occurrence of BamH I sites per method and/or plant is denoted by coloured dots: Sites found in all treatments by stdGBS and rtGBS (green dot), found by either one type of library (red and yellow dots), and sites poorly represented by KFE and KFF (pink dot). The extract_aliquot data is denoted by an asterisk, in the following order from top to bottom of each plant_method panel: 2_4, 2_3, 2_2, 2_1, 1_4, 1_3, 1_2, 1_1.

    Journal: PLoS ONE

    Article Title: Random Tagging Genotyping by Sequencing (rtGBS), an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome

    doi: 10.1371/journal.pone.0143193

    Figure Lengend Snippet: Physical representation of the first 1 Mbp of pseudochromosome 1 in ‘Hongyang’ genome sequence with mapped reads found by stdGBS and rtGBS methods, all treatments, supported by ≥10 reads. The occurrence of BamH I sites per method and/or plant is denoted by coloured dots: Sites found in all treatments by stdGBS and rtGBS (green dot), found by either one type of library (red and yellow dots), and sites poorly represented by KFE and KFF (pink dot). The extract_aliquot data is denoted by an asterisk, in the following order from top to bottom of each plant_method panel: 2_4, 2_3, 2_2, 2_1, 1_4, 1_3, 1_2, 1_1.

    Article Snippet: We used 20 units of BamH I- HF with CutSmart reaction buffer (New England Biolabs) for each RE digestion.

    Techniques: Sequencing

    Difference on coverage depth obtained by stdGBS and rtGBS. A) Heatmaps of the mapped BamH I sites covered by ≥10 reads. B) Read coverage depth of the concatenated dataset for each library method. The cut-off value of 10 reads is marked with a red dotted line.

    Journal: PLoS ONE

    Article Title: Random Tagging Genotyping by Sequencing (rtGBS), an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome

    doi: 10.1371/journal.pone.0143193

    Figure Lengend Snippet: Difference on coverage depth obtained by stdGBS and rtGBS. A) Heatmaps of the mapped BamH I sites covered by ≥10 reads. B) Read coverage depth of the concatenated dataset for each library method. The cut-off value of 10 reads is marked with a red dotted line.

    Article Snippet: We used 20 units of BamH I- HF with CutSmart reaction buffer (New England Biolabs) for each RE digestion.

    Techniques:

    Venn diagrams summarizing the BamH I sites supported by ≥10 reads from the combined datasets of all treatments for stdGBS and rtGBS. The combined datasets were constructed by concatenating all plants_extract_aliquot ([KF.][ 12 ][1234]) for both lanes from each library method (green circles). The intersection between the two sets corresponds to the BamH I sites in common by all plants and ‘Hongyang’ denoted as “population” (blue circles).

    Journal: PLoS ONE

    Article Title: Random Tagging Genotyping by Sequencing (rtGBS), an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome

    doi: 10.1371/journal.pone.0143193

    Figure Lengend Snippet: Venn diagrams summarizing the BamH I sites supported by ≥10 reads from the combined datasets of all treatments for stdGBS and rtGBS. The combined datasets were constructed by concatenating all plants_extract_aliquot ([KF.][ 12 ][1234]) for both lanes from each library method (green circles). The intersection between the two sets corresponds to the BamH I sites in common by all plants and ‘Hongyang’ denoted as “population” (blue circles).

    Article Snippet: We used 20 units of BamH I- HF with CutSmart reaction buffer (New England Biolabs) for each RE digestion.

    Techniques: Construct

    Fragment size distribution analysis. A) Semi-logarithmic display of the BamH I fragment size distribution among the ‘Hongyang’ genome (population) and the two library methods. B) Probability distribution plots for the two GBS library methods vs. Hongyang genome RE fragment size distribution.

    Journal: PLoS ONE

    Article Title: Random Tagging Genotyping by Sequencing (rtGBS), an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome

    doi: 10.1371/journal.pone.0143193

    Figure Lengend Snippet: Fragment size distribution analysis. A) Semi-logarithmic display of the BamH I fragment size distribution among the ‘Hongyang’ genome (population) and the two library methods. B) Probability distribution plots for the two GBS library methods vs. Hongyang genome RE fragment size distribution.

    Article Snippet: We used 20 units of BamH I- HF with CutSmart reaction buffer (New England Biolabs) for each RE digestion.

    Techniques:

    Plasmids used for TLS assays. ( A ) Construction of an AP site-containing plasmid. Plasmid pVP15 is linearized by BbrPI digestion. The PstI-BamHI fragment of pVP9 is removed by digestion and purification on a microcon YM-30 column. Both linearized plasmids

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Mutational specificity and genetic control of replicative bypass of an abasic site in yeast

    doi: 10.1073/pnas.0711227105

    Figure Lengend Snippet: Plasmids used for TLS assays. ( A ) Construction of an AP site-containing plasmid. Plasmid pVP15 is linearized by BbrPI digestion. The PstI-BamHI fragment of pVP9 is removed by digestion and purification on a microcon YM-30 column. Both linearized plasmids

    Article Snippet: Monoadducted heteroduplex plasmids were generated as follows: Plasmids pVP9 and pVP10 were digested with BamHI and PstI ( A ), and the vector portion was separated from the 25-bp insert by centrifugation in a YM-30 microcon filter unit (Millipore).

    Techniques: Plasmid Preparation, Purification

    Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total DNA (7 µg) was digested with BamHI which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λ BstE II, L. bicolor wild type (Wt) and transgenic strains.

    Journal: Microbial biotechnology

    Article Title: T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor

    doi: 10.1111/j.1751-7915.2008.00029.x

    Figure Lengend Snippet: Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total DNA (7 µg) was digested with BamHI which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λ BstE II, L. bicolor wild type (Wt) and transgenic strains.

    Article Snippet: Southern blotting Approximately 7 µg of fungal genomic DNA per sample was digested overnight with BamHI (Promega) which cuts once within the T‐DNA, separated in 1% agarose gel and transferred by alkaline capillarity blotting to a Hygrobond‐ N+ nylon membrane (Amershamn Biosciences) according to the manufacturer's protocol.

    Techniques: Southern Blot, Transgenic Assay, Marker

    pHg/pBks rescue plasmid. The binding site for the Post‐RB primer is indicated by an arrow. GPD promoter Agaricus bisporus : glyceraldehide‐3‐phosphate dehydrogenase promoter of Agaricus bisporus . hph : hph gene of E. coli coding for an aminocyclitol phosphotransferase that confers resistance to Hygromycin B and structurally related antibiotics. 35S‐3′: cauliflower mosaic virus 35S terminator. Amp R: bla (ApR) gene of E. coli coding for a β‐lactamase that confers resistance to ampicillin. ORI: replication origin of pBluescript KS+. LB: T‐DNA left border of pCAMBIA1300. RB: T‐DNA right border of pCAMBIA1300. Kan R: aadA gene of E. coli coding for an aminoglycoside phosphotransferase that confers resistance to kanamycin. Relevant restriction sites (SacI, BamHI and XbaI) within the T‐DNA are indicated.

    Journal: Microbial biotechnology

    Article Title: T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor

    doi: 10.1111/j.1751-7915.2008.00029.x

    Figure Lengend Snippet: pHg/pBks rescue plasmid. The binding site for the Post‐RB primer is indicated by an arrow. GPD promoter Agaricus bisporus : glyceraldehide‐3‐phosphate dehydrogenase promoter of Agaricus bisporus . hph : hph gene of E. coli coding for an aminocyclitol phosphotransferase that confers resistance to Hygromycin B and structurally related antibiotics. 35S‐3′: cauliflower mosaic virus 35S terminator. Amp R: bla (ApR) gene of E. coli coding for a β‐lactamase that confers resistance to ampicillin. ORI: replication origin of pBluescript KS+. LB: T‐DNA left border of pCAMBIA1300. RB: T‐DNA right border of pCAMBIA1300. Kan R: aadA gene of E. coli coding for an aminoglycoside phosphotransferase that confers resistance to kanamycin. Relevant restriction sites (SacI, BamHI and XbaI) within the T‐DNA are indicated.

    Article Snippet: Southern blotting Approximately 7 µg of fungal genomic DNA per sample was digested overnight with BamHI (Promega) which cuts once within the T‐DNA, separated in 1% agarose gel and transferred by alkaline capillarity blotting to a Hygrobond‐ N+ nylon membrane (Amershamn Biosciences) according to the manufacturer's protocol.

    Techniques: Plasmid Preparation, Binding Assay

    Transformation of a K. marxianus ura3 mutant with a linear DNA of S. cerevisiae URA3 (Sc URA3 ). (A) The K. marxianus ura3 mutant was not transformed with the intact Sc URA3 plasmid (pRS316) but with SmaI-digested pRS316 (4.8 kb) and the Sc URA3 PCR fragment (1.7 kb). (B) Southern blot hybridization of chromosomal DNA of S. cerevisiae BY4704 (lane 1), K. marxianus wild-type (WT) DMKU3-1042 (lane 2), ura3 mutant RAK3605 (lane 3), and nine Sc URA3 transformants from strain RAK3605 (lanes 4 to 12). Chromosomal DNA was digested with BamHI, run on a 0.8% agarose gel, transferred to a nylon membrane, and hybridized with a digoxigenin-labeled Sc URA3 probe. The bands indicated by an arrowhead are likely the authentic K. marxianus URA3 that cross-hybridizes with Sc URA3 . M, DNA size marker.

    Journal: Applied and Environmental Microbiology

    Article Title: High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042 ▿

    doi: 10.1128/AEM.01854-08

    Figure Lengend Snippet: Transformation of a K. marxianus ura3 mutant with a linear DNA of S. cerevisiae URA3 (Sc URA3 ). (A) The K. marxianus ura3 mutant was not transformed with the intact Sc URA3 plasmid (pRS316) but with SmaI-digested pRS316 (4.8 kb) and the Sc URA3 PCR fragment (1.7 kb). (B) Southern blot hybridization of chromosomal DNA of S. cerevisiae BY4704 (lane 1), K. marxianus wild-type (WT) DMKU3-1042 (lane 2), ura3 mutant RAK3605 (lane 3), and nine Sc URA3 transformants from strain RAK3605 (lanes 4 to 12). Chromosomal DNA was digested with BamHI, run on a 0.8% agarose gel, transferred to a nylon membrane, and hybridized with a digoxigenin-labeled Sc URA3 probe. The bands indicated by an arrowhead are likely the authentic K. marxianus URA3 that cross-hybridizes with Sc URA3 . M, DNA size marker.

    Article Snippet: Chromosomal DNA from BY4704, K. marxianus DMKU3-1042, a K. marxianus ura3 mutant (RAK3605), and Sc URA3 transformants of the ura3 mutant was isolated and digested with BamHI (Roche Diagnostics GmbH, Mannheim, Germany).

    Techniques: Transformation Assay, Mutagenesis, Plasmid Preparation, Polymerase Chain Reaction, Southern Blot, Hybridization, Agarose Gel Electrophoresis, Labeling, Marker

    The effect of transiently expressed nuclear MxA proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).

    Journal: Nucleic Acids Research

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome

    doi: 10.1093/nar/gkh192

    Figure Lengend Snippet: The effect of transiently expressed nuclear MxA proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).

    Article Snippet: The MxA fragment prepared by digestion of pET3a-MxA (a gift from Dr Staeheli) with NdeI (BioLabs) and BamHI (TOYOBO) was once cloned into NdeI- and BamHI-digested pET14b (Novagen).

    Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Standard Deviation, Mutagenesis, Expressing, Amplification, Polymerase Chain Reaction, Derivative Assay, Construct, Clone Assay

    Integron comparisons based on BamHI digestion of plasmids. Genes are denoted by arrows and colours based on the gene function classifications. Shaded areas denote regions with > 99% nucleotide sequence identity, with the exception of > 97% for the comparison of GC aacA4cr in In846 and GC aacA4′-37 in novel In1059. A , Comparison of In37, In843, In62, In1021, In846 and novel In1059 integrons after BamHI digestion. B - a , Comparison of integrons In37, novel In1059 and transposon Tn1696 (DQ310703); B - b , Comparison of integrons In846, novel In1059 and transposon Tn7017 (KJ571202). The novel In1059 integron shares higher sequence identity with integron Tn7017 than with integron Tn1696

    Journal: Antimicrobial Resistance and Infection Control

    Article Title: Characterization of the novel In1059 harbouring VIM gene cassette

    doi: 10.1186/s13756-017-0204-1

    Figure Lengend Snippet: Integron comparisons based on BamHI digestion of plasmids. Genes are denoted by arrows and colours based on the gene function classifications. Shaded areas denote regions with > 99% nucleotide sequence identity, with the exception of > 97% for the comparison of GC aacA4cr in In846 and GC aacA4′-37 in novel In1059. A , Comparison of In37, In843, In62, In1021, In846 and novel In1059 integrons after BamHI digestion. B - a , Comparison of integrons In37, novel In1059 and transposon Tn1696 (DQ310703); B - b , Comparison of integrons In846, novel In1059 and transposon Tn7017 (KJ571202). The novel In1059 integron shares higher sequence identity with integron Tn7017 than with integron Tn1696

    Article Snippet: The plasmids were digested with BamHI (TaKaRa, Dalian, China), and the six integron-harbouring recombinant plasmids were electrophoretically resolved to generate genetic maps.

    Techniques: Sequencing

    Targeted disruption of Sept6 . (A) A schematic representation of the targeting strategy. The structure of Sept6 products (protein and mRNA) (top), the wild-type locus, the targeting construct, and the targeted locus are shown. Genomic fragments, including exon 3 (a box with diagonal lines) of Sept6 , that were obtained by the screening of the genomic library and the probes used for a Southern blot analysis are indicated by bold horizontal and dotted lines, respectively. The restriction sites and a converted restriction site are indicated by vertical solid and dotted arrows, respectively. The orientations of the neomycin resistance gene ( Neo ) and the herpes simplex virus thymidine kinase gene ( tk ) are indicated by horizontal arrows. The primers used are indicated by horizontal arrowheads. The location of P-loop motif (P), the second ATG (*), and a breakpoint in fusion with MLL (B.P.) are shown in Sept6 protein and mRNA. B, BamHI; Bs, BstXI; N, NheI; S, SacI; Sp, SphI; X, XhoI. (B) Homologous recombination in the male ES cells by Southern blot analysis. BamHI- or SphI-digested genomic DNA extracted from the ES cells was hybridized with the 5′ probe or the 3′ probe, resulting in the wild-type 5.5- and targeted 6.3-kb bands or the wild-type (Y/+) 7.4- and targeted (Y/−) 5.3-kb bands (data not shown), respectively. (C and D) The expression of Sept6 protein in the brain (C) and mRNA in MEFs (D, upper panel) derived from the wild-type (Y/+) and disrupted (Y/−) mice by Western blot analysis using the anti-Sept6-1 antibody and RT-PCR analysis using primers S6-41S and S6KO-AS-Pr, respectively. The expression levels of β 2 microglobulin (D, lower panel) were used as internal standards of the RT-PCR analysis. M, 100-bp DNA ladder (New England Biolabs).

    Journal: Molecular and Cellular Biology

    Article Title: Disruption of Sept6, a Fusion Partner Gene of MLL, Does Not Affect Ontogeny, Leukemogenesis Induced by MLL-SEPT6, or Phenotype Induced by the Loss of Sept4

    doi: 10.1128/MCB.25.24.10965-10978.2005

    Figure Lengend Snippet: Targeted disruption of Sept6 . (A) A schematic representation of the targeting strategy. The structure of Sept6 products (protein and mRNA) (top), the wild-type locus, the targeting construct, and the targeted locus are shown. Genomic fragments, including exon 3 (a box with diagonal lines) of Sept6 , that were obtained by the screening of the genomic library and the probes used for a Southern blot analysis are indicated by bold horizontal and dotted lines, respectively. The restriction sites and a converted restriction site are indicated by vertical solid and dotted arrows, respectively. The orientations of the neomycin resistance gene ( Neo ) and the herpes simplex virus thymidine kinase gene ( tk ) are indicated by horizontal arrows. The primers used are indicated by horizontal arrowheads. The location of P-loop motif (P), the second ATG (*), and a breakpoint in fusion with MLL (B.P.) are shown in Sept6 protein and mRNA. B, BamHI; Bs, BstXI; N, NheI; S, SacI; Sp, SphI; X, XhoI. (B) Homologous recombination in the male ES cells by Southern blot analysis. BamHI- or SphI-digested genomic DNA extracted from the ES cells was hybridized with the 5′ probe or the 3′ probe, resulting in the wild-type 5.5- and targeted 6.3-kb bands or the wild-type (Y/+) 7.4- and targeted (Y/−) 5.3-kb bands (data not shown), respectively. (C and D) The expression of Sept6 protein in the brain (C) and mRNA in MEFs (D, upper panel) derived from the wild-type (Y/+) and disrupted (Y/−) mice by Western blot analysis using the anti-Sept6-1 antibody and RT-PCR analysis using primers S6-41S and S6KO-AS-Pr, respectively. The expression levels of β 2 microglobulin (D, lower panel) were used as internal standards of the RT-PCR analysis. M, 100-bp DNA ladder (New England Biolabs).

    Article Snippet: The BstXI site of the genomic clone and the ClaI site of the pBluescript were converted to the XhoI site and the BamHI site by using an XhoI linker and a BamHI linker (Stratagene), respectively.

    Techniques: Construct, Southern Blot, Homologous Recombination, Expressing, Derivative Assay, Mouse Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction