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  • 99
    New England Biolabs restriction enzymes ecor v
    Confirmation of the mutation in PA14Δ hepP . PA14 and PA14Δ hepP were grown in LB broth and the chromosomal DNA was extracted. a <t>PCR</t> analysis to detect the presence of MAR2xT7 within hepP . PCR reactions were run using the chromosomal DNA from each strain as a template and primers corresponding to the DNA sequences 94 bp upstream and 179 bp downstream of the hepP structural gene ( zbdP- For3/ hepP- Rev3, Table 2 ). The expected 1926-bp fragment from PA14 (lane 1) and the 2920-bp fragment (the additional 994 bp from MAR2xT7 ) from PA14Δ hepP (lane 2) were detected. Lane 3 is a no-template control and the molecular size standards are in lane 4. b Restriction analysis of the PCR products. The coding sequence for hepP does not contain an <t>EcoR</t> V restriction enzyme site, while MAR2xT7 contains a single EcoR V site. Digestion of the PCR products with EcoR V failed to reduce the size of the 1926-bp fragment obtained from PA14 (lane 3) but resulted in the cleavage of the product obtained from PA14Δ hepP into the expected 800 bp and 2120 bp fragments (lane 4). Lane 1 contains the molecular size standards; lane 2 was left empty
    Restriction Enzymes Ecor V, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 4 article reviews
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    93
    Millipore ecor v
    Confirmation of the mutation in PA14Δ hepP . PA14 and PA14Δ hepP were grown in LB broth and the chromosomal DNA was extracted. a <t>PCR</t> analysis to detect the presence of MAR2xT7 within hepP . PCR reactions were run using the chromosomal DNA from each strain as a template and primers corresponding to the DNA sequences 94 bp upstream and 179 bp downstream of the hepP structural gene ( zbdP- For3/ hepP- Rev3, Table 2 ). The expected 1926-bp fragment from PA14 (lane 1) and the 2920-bp fragment (the additional 994 bp from MAR2xT7 ) from PA14Δ hepP (lane 2) were detected. Lane 3 is a no-template control and the molecular size standards are in lane 4. b Restriction analysis of the PCR products. The coding sequence for hepP does not contain an <t>EcoR</t> V restriction enzyme site, while MAR2xT7 contains a single EcoR V site. Digestion of the PCR products with EcoR V failed to reduce the size of the 1926-bp fragment obtained from PA14 (lane 3) but resulted in the cleavage of the product obtained from PA14Δ hepP into the expected 800 bp and 2120 bp fragments (lane 4). Lane 1 contains the molecular size standards; lane 2 was left empty
    Ecor V, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    TaKaRa ecor v
    Electrophoresis of PCR product and pUCm-T-ZNRD1 digested with enzymes. Lane 1: PCR product; Lane 2, 3: Marker (200 bp); Lane 4: recombinant of pUCm-T-ZNRD1 cleaved by <t>EcoR</t> V and Xba I; Lane 5: recombinant of pUCm-T-ZNRD1.
    Ecor V, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega ecor v
    Analysis of the CSAD gene knockout. (a) Schematic diagram of pGT01xf insertion into the CSAD gene. Rectangles represent exons and open rectangles represent, untranslated regions. “A” shows the location of the VIS amplicon which is produced from the wild type chromosome and “B” shows the location of the β -geo amplicon which is produced from the gene trap vector inserted between exons 8 and 9. SA: splicing acceptor; pA: polyadenylation sequence; EV: <t>EcoR</t> V sites. (b) Southern analysis of genomic <t>DNA</t> digested with EcoR V and hybridized using [ 32 P]-dCTP labeled CSAD cDNA. The wild type CSAD EcoR V fragment is 5.9 kb and that of disrupted fragment is 7.1 kb. “+/+”: WT; “+/−”: CSAD +/− ; “−/−”: CSAD −/− . (c) PCR products (1038 bp and 682 bp) with VIS and β -geo primer sets. β -geo product is absent in WT and VIS PCR product is absent in CSAD −/− . Both PCR products are present in CSAD +/− . (d) Northern blot analysis of total RNA from liver with [ 32 P]-dCTP labeled cDNA and α -actin as a control probe. (e) Western blot analysis of kidney homogenates probed with an anti-CSAD antibody and detected with an alkaline phosphatase labeled goat anti-rabbit antibody. CSAD (51 kD) was not detected in CSAD −/− homogenates.
    Ecor V, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Roche ecor v
    Analysis of the CSAD gene knockout. (a) Schematic diagram of pGT01xf insertion into the CSAD gene. Rectangles represent exons and open rectangles represent, untranslated regions. “A” shows the location of the VIS amplicon which is produced from the wild type chromosome and “B” shows the location of the β -geo amplicon which is produced from the gene trap vector inserted between exons 8 and 9. SA: splicing acceptor; pA: polyadenylation sequence; EV: <t>EcoR</t> V sites. (b) Southern analysis of genomic <t>DNA</t> digested with EcoR V and hybridized using [ 32 P]-dCTP labeled CSAD cDNA. The wild type CSAD EcoR V fragment is 5.9 kb and that of disrupted fragment is 7.1 kb. “+/+”: WT; “+/−”: CSAD +/− ; “−/−”: CSAD −/− . (c) PCR products (1038 bp and 682 bp) with VIS and β -geo primer sets. β -geo product is absent in WT and VIS PCR product is absent in CSAD −/− . Both PCR products are present in CSAD +/− . (d) Northern blot analysis of total RNA from liver with [ 32 P]-dCTP labeled cDNA and α -actin as a control probe. (e) Western blot analysis of kidney homogenates probed with an anti-CSAD antibody and detected with an alkaline phosphatase labeled goat anti-rabbit antibody. CSAD (51 kD) was not detected in CSAD −/− homogenates.
    Ecor V, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher ecor v
    Aggf1 haploinsufficiency results in vascular defects and increases vascular permeability in Aggf1 +/− KO mice. ( A ) Strategy for developing a classical KO mouse line targeting Aggf1 by homologous recombination. The wild type allele represents the genomic organization of the mouse Aggf1 gene. The vertical gray colour-filled boxes indicate the exons of Aggf1 . EV, E1, and H1 stand for restriction enzymes EcoR V, EcoR 1 and Hind 3, respectively. The targeting vector was built on pGT-N38 with insertions of the E1-H3 and the E1-E1 (3-end) fragments as the left and right arm, respectively. The horizontal black line (a) represents a probe used for Southern blotting analysis. The targeted allele shows the genomic organization of Aggf1 after exons 2-11 are replaced with Neo . PCR analysis was later developed for genotyping KO mice. The positions of PCR primers for genotyping are indicated as P1, P2 and P3, P4. ( B ) Southern blot analysis was performed to detect the correctly targeted ES cell clones. The genomic <t>DNA</t> from the targeted ES cells produces two bands of 23.4 kb and 9.1 kb, whereas non-targeted ES cells produce a single band of 23.4 kb. ( C ) Western blotting analysis using an antibody against AGGF1 revealed about 50% reduction of AGGF1 expression in heterozygous Aggf1 +/− embryos (E12.5). ( D ) Representative images showing defective vasculature in Aggf1 +/− yolk sacs and embryos. Haemorrhages were also seen in 35% of developing Aggf1 +/− embryos. ( E ) Detection of haemorrhages in the brain, lung and spleen of 35% of Aggf1 +/− mice at 50 to 60 weeks of age. ( F ) Significantly increased vascular permeability in Aggf1 +/− mice at 50 to 60 weeks of age compared to that of age-matched Aggf1 +/+ controls. ( G ) Matrigel-based endothelial tube formation assays revealed a decreased angiogenesis by microvascular ECs isolated from lungs of Aggf1 +/− mice as compared to that from Aggf1 +/+ control mice at the age of 20 to 30 weeks. Scale bar = 50 μm.
    Ecor V, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 272 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher ecor v enzyme
    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or <t>Bsa</t> I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the <t>EcoR</t> V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).
    Ecor V Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Stratagene ecor v digested plasmid m13
    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or <t>Bsa</t> I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the <t>EcoR</t> V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).
    Ecor V Digested Plasmid M13, supplied by Stratagene, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa ecor v linearized pcdna3 1z vector
    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or <t>Bsa</t> I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the <t>EcoR</t> V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).
    Ecor V Linearized Pcdna3 1z Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher restriction enzyme ecor v
    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or <t>Bsa</t> I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the <t>EcoR</t> V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).
    Restriction Enzyme Ecor V, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher restriction enzymes ecor v
    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or <t>Bsa</t> I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the <t>EcoR</t> V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).
    Restriction Enzymes Ecor V, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TaKaRa ecor v restriction enzymes
    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or <t>Bsa</t> I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the <t>EcoR</t> V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).
    Ecor V Restriction Enzymes, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ecor v nco i digested pet 41
    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or <t>Bsa</t> I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the <t>EcoR</t> V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).
    Ecor V Nco I Digested Pet 41, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs ecor v hf
    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or <t>Bsa</t> I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the <t>EcoR</t> V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).
    Ecor V Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa psimple 18 ecor v bap vector
    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or <t>Bsa</t> I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the <t>EcoR</t> V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).
    Psimple 18 Ecor V Bap Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Confirmation of the mutation in PA14Δ hepP . PA14 and PA14Δ hepP were grown in LB broth and the chromosomal DNA was extracted. a PCR analysis to detect the presence of MAR2xT7 within hepP . PCR reactions were run using the chromosomal DNA from each strain as a template and primers corresponding to the DNA sequences 94 bp upstream and 179 bp downstream of the hepP structural gene ( zbdP- For3/ hepP- Rev3, Table 2 ). The expected 1926-bp fragment from PA14 (lane 1) and the 2920-bp fragment (the additional 994 bp from MAR2xT7 ) from PA14Δ hepP (lane 2) were detected. Lane 3 is a no-template control and the molecular size standards are in lane 4. b Restriction analysis of the PCR products. The coding sequence for hepP does not contain an EcoR V restriction enzyme site, while MAR2xT7 contains a single EcoR V site. Digestion of the PCR products with EcoR V failed to reduce the size of the 1926-bp fragment obtained from PA14 (lane 3) but resulted in the cleavage of the product obtained from PA14Δ hepP into the expected 800 bp and 2120 bp fragments (lane 4). Lane 1 contains the molecular size standards; lane 2 was left empty

    Journal: BMC Microbiology

    Article Title: Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase

    doi: 10.1186/s12866-017-1141-0

    Figure Lengend Snippet: Confirmation of the mutation in PA14Δ hepP . PA14 and PA14Δ hepP were grown in LB broth and the chromosomal DNA was extracted. a PCR analysis to detect the presence of MAR2xT7 within hepP . PCR reactions were run using the chromosomal DNA from each strain as a template and primers corresponding to the DNA sequences 94 bp upstream and 179 bp downstream of the hepP structural gene ( zbdP- For3/ hepP- Rev3, Table 2 ). The expected 1926-bp fragment from PA14 (lane 1) and the 2920-bp fragment (the additional 994 bp from MAR2xT7 ) from PA14Δ hepP (lane 2) were detected. Lane 3 is a no-template control and the molecular size standards are in lane 4. b Restriction analysis of the PCR products. The coding sequence for hepP does not contain an EcoR V restriction enzyme site, while MAR2xT7 contains a single EcoR V site. Digestion of the PCR products with EcoR V failed to reduce the size of the 1926-bp fragment obtained from PA14 (lane 3) but resulted in the cleavage of the product obtained from PA14Δ hepP into the expected 800 bp and 2120 bp fragments (lane 4). Lane 1 contains the molecular size standards; lane 2 was left empty

    Article Snippet: Therefore, to further confirm the mutation of hepP in PA14ΔhepP , we performed restriction enzyme digestion on the PCR products with EcoR V (New England Biolabs).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Sequencing

    Electrophoresis of PCR product and pUCm-T-ZNRD1 digested with enzymes. Lane 1: PCR product; Lane 2, 3: Marker (200 bp); Lane 4: recombinant of pUCm-T-ZNRD1 cleaved by EcoR V and Xba I; Lane 5: recombinant of pUCm-T-ZNRD1.

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Effect of ZNRD1 gene antisense RNA on drug resistant gastric cancer cells

    doi: 10.3748/wjg.v9.i5.894

    Figure Lengend Snippet: Electrophoresis of PCR product and pUCm-T-ZNRD1 digested with enzymes. Lane 1: PCR product; Lane 2, 3: Marker (200 bp); Lane 4: recombinant of pUCm-T-ZNRD1 cleaved by EcoR V and Xba I; Lane 5: recombinant of pUCm-T-ZNRD1.

    Article Snippet: EcoR V, Xba I, BamH I, cloning vector pUCm-T and T4 DNA ligase were purchased from Takara; MTT, DEPC from Sigma.

    Techniques: Electrophoresis, Polymerase Chain Reaction, Marker, Recombinant

    Electrophoresis identification of pcDNA3.1-anti ZNRD1. Lane 1: pcDNA3.1-anti ZNRD1/ EcoR V + BamH I; Lane 2: pcDNA3.1-anti ZNRD1; Lane 3: Marker (200 bp).

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Effect of ZNRD1 gene antisense RNA on drug resistant gastric cancer cells

    doi: 10.3748/wjg.v9.i5.894

    Figure Lengend Snippet: Electrophoresis identification of pcDNA3.1-anti ZNRD1. Lane 1: pcDNA3.1-anti ZNRD1/ EcoR V + BamH I; Lane 2: pcDNA3.1-anti ZNRD1; Lane 3: Marker (200 bp).

    Article Snippet: EcoR V, Xba I, BamH I, cloning vector pUCm-T and T4 DNA ligase were purchased from Takara; MTT, DEPC from Sigma.

    Techniques: Electrophoresis, Marker

    Conserved Domains in Karma . (A) Karma structure. Open boxes depict the two open reading frames of Karma (ORF1 and ORF2). ORF2 contains the endonuclease ( en ) and reverse transcriptase ( rt ) domains. Black boxes depict Cys-rich motifs. Vertical lines above the boxes depict the sites of HpaII and MspI cleavage, and vertical lines below the boxes depict the sites of EcoRV cleavage. Hatched boxes depict probes A (1317 bp) and B (1498 bp) used in DNA gel blot analysis. Bar = 1 kb. (B) Alignment of the conserved reverse transcriptase domain. Gray boxes indicate residues conserved in at least seven of the eight sequences depicted. I to VII denote the seven domains characteristic of reverse transcriptases. Retrotransposon sequences are from rice Karma , maize cin4 , lily del2 , Arabidopsis Ta11-1 , Cannabis LINE-CS, human LINE-1, frog Tx1 , and fruit fly R2Dm .

    Journal: The Plant Cell

    Article Title: Two-Step Regulation and Continuous Retrotransposition of the Rice LINE-Type Retrotransposon Karma

    doi: 10.1105/tpc.011809

    Figure Lengend Snippet: Conserved Domains in Karma . (A) Karma structure. Open boxes depict the two open reading frames of Karma (ORF1 and ORF2). ORF2 contains the endonuclease ( en ) and reverse transcriptase ( rt ) domains. Black boxes depict Cys-rich motifs. Vertical lines above the boxes depict the sites of HpaII and MspI cleavage, and vertical lines below the boxes depict the sites of EcoRV cleavage. Hatched boxes depict probes A (1317 bp) and B (1498 bp) used in DNA gel blot analysis. Bar = 1 kb. (B) Alignment of the conserved reverse transcriptase domain. Gray boxes indicate residues conserved in at least seven of the eight sequences depicted. I to VII denote the seven domains characteristic of reverse transcriptases. Retrotransposon sequences are from rice Karma , maize cin4 , lily del2 , Arabidopsis Ta11-1 , Cannabis LINE-CS, human LINE-1, frog Tx1 , and fruit fly R2Dm .

    Article Snippet: Two micrograms of genomic DNA extracted from leaves was digested with EcoRV and self-ligated using the DNA Ligation Kit Version 2 (Takara Bio, Otsu, Japan).

    Techniques: Western Blot

    Analysis of the CSAD gene knockout. (a) Schematic diagram of pGT01xf insertion into the CSAD gene. Rectangles represent exons and open rectangles represent, untranslated regions. “A” shows the location of the VIS amplicon which is produced from the wild type chromosome and “B” shows the location of the β -geo amplicon which is produced from the gene trap vector inserted between exons 8 and 9. SA: splicing acceptor; pA: polyadenylation sequence; EV: EcoR V sites. (b) Southern analysis of genomic DNA digested with EcoR V and hybridized using [ 32 P]-dCTP labeled CSAD cDNA. The wild type CSAD EcoR V fragment is 5.9 kb and that of disrupted fragment is 7.1 kb. “+/+”: WT; “+/−”: CSAD +/− ; “−/−”: CSAD −/− . (c) PCR products (1038 bp and 682 bp) with VIS and β -geo primer sets. β -geo product is absent in WT and VIS PCR product is absent in CSAD −/− . Both PCR products are present in CSAD +/− . (d) Northern blot analysis of total RNA from liver with [ 32 P]-dCTP labeled cDNA and α -actin as a control probe. (e) Western blot analysis of kidney homogenates probed with an anti-CSAD antibody and detected with an alkaline phosphatase labeled goat anti-rabbit antibody. CSAD (51 kD) was not detected in CSAD −/− homogenates.

    Journal: Journal of Amino Acids

    Article Title: Development of a Novel Cysteine Sulfinic Acid Decarboxylase Knockout Mouse: Dietary Taurine Reduces Neonatal Mortality

    doi: 10.1155/2014/346809

    Figure Lengend Snippet: Analysis of the CSAD gene knockout. (a) Schematic diagram of pGT01xf insertion into the CSAD gene. Rectangles represent exons and open rectangles represent, untranslated regions. “A” shows the location of the VIS amplicon which is produced from the wild type chromosome and “B” shows the location of the β -geo amplicon which is produced from the gene trap vector inserted between exons 8 and 9. SA: splicing acceptor; pA: polyadenylation sequence; EV: EcoR V sites. (b) Southern analysis of genomic DNA digested with EcoR V and hybridized using [ 32 P]-dCTP labeled CSAD cDNA. The wild type CSAD EcoR V fragment is 5.9 kb and that of disrupted fragment is 7.1 kb. “+/+”: WT; “+/−”: CSAD +/− ; “−/−”: CSAD −/− . (c) PCR products (1038 bp and 682 bp) with VIS and β -geo primer sets. β -geo product is absent in WT and VIS PCR product is absent in CSAD −/− . Both PCR products are present in CSAD +/− . (d) Northern blot analysis of total RNA from liver with [ 32 P]-dCTP labeled cDNA and α -actin as a control probe. (e) Western blot analysis of kidney homogenates probed with an anti-CSAD antibody and detected with an alkaline phosphatase labeled goat anti-rabbit antibody. CSAD (51 kD) was not detected in CSAD −/− homogenates.

    Article Snippet: DNA was digested with EcoR V (Promega), fractionated by agarose gel electrophoresis, and transferred to a nylon membrane (Bio-Rad).

    Techniques: Gene Knockout, Amplification, Produced, Plasmid Preparation, Sequencing, Labeling, Polymerase Chain Reaction, Northern Blot, Western Blot

    Aggf1 haploinsufficiency results in vascular defects and increases vascular permeability in Aggf1 +/− KO mice. ( A ) Strategy for developing a classical KO mouse line targeting Aggf1 by homologous recombination. The wild type allele represents the genomic organization of the mouse Aggf1 gene. The vertical gray colour-filled boxes indicate the exons of Aggf1 . EV, E1, and H1 stand for restriction enzymes EcoR V, EcoR 1 and Hind 3, respectively. The targeting vector was built on pGT-N38 with insertions of the E1-H3 and the E1-E1 (3-end) fragments as the left and right arm, respectively. The horizontal black line (a) represents a probe used for Southern blotting analysis. The targeted allele shows the genomic organization of Aggf1 after exons 2-11 are replaced with Neo . PCR analysis was later developed for genotyping KO mice. The positions of PCR primers for genotyping are indicated as P1, P2 and P3, P4. ( B ) Southern blot analysis was performed to detect the correctly targeted ES cell clones. The genomic DNA from the targeted ES cells produces two bands of 23.4 kb and 9.1 kb, whereas non-targeted ES cells produce a single band of 23.4 kb. ( C ) Western blotting analysis using an antibody against AGGF1 revealed about 50% reduction of AGGF1 expression in heterozygous Aggf1 +/− embryos (E12.5). ( D ) Representative images showing defective vasculature in Aggf1 +/− yolk sacs and embryos. Haemorrhages were also seen in 35% of developing Aggf1 +/− embryos. ( E ) Detection of haemorrhages in the brain, lung and spleen of 35% of Aggf1 +/− mice at 50 to 60 weeks of age. ( F ) Significantly increased vascular permeability in Aggf1 +/− mice at 50 to 60 weeks of age compared to that of age-matched Aggf1 +/+ controls. ( G ) Matrigel-based endothelial tube formation assays revealed a decreased angiogenesis by microvascular ECs isolated from lungs of Aggf1 +/− mice as compared to that from Aggf1 +/+ control mice at the age of 20 to 30 weeks. Scale bar = 50 μm.

    Journal: Human Molecular Genetics

    Article Title: Haploinsufficiency of Klippel-Trenaunay syndrome gene Aggf1 inhibits developmental and pathological angiogenesis by inactivating PI3K and AKT and disrupts vascular integrity by activating VE-cadherin

    doi: 10.1093/hmg/ddw273

    Figure Lengend Snippet: Aggf1 haploinsufficiency results in vascular defects and increases vascular permeability in Aggf1 +/− KO mice. ( A ) Strategy for developing a classical KO mouse line targeting Aggf1 by homologous recombination. The wild type allele represents the genomic organization of the mouse Aggf1 gene. The vertical gray colour-filled boxes indicate the exons of Aggf1 . EV, E1, and H1 stand for restriction enzymes EcoR V, EcoR 1 and Hind 3, respectively. The targeting vector was built on pGT-N38 with insertions of the E1-H3 and the E1-E1 (3-end) fragments as the left and right arm, respectively. The horizontal black line (a) represents a probe used for Southern blotting analysis. The targeted allele shows the genomic organization of Aggf1 after exons 2-11 are replaced with Neo . PCR analysis was later developed for genotyping KO mice. The positions of PCR primers for genotyping are indicated as P1, P2 and P3, P4. ( B ) Southern blot analysis was performed to detect the correctly targeted ES cell clones. The genomic DNA from the targeted ES cells produces two bands of 23.4 kb and 9.1 kb, whereas non-targeted ES cells produce a single band of 23.4 kb. ( C ) Western blotting analysis using an antibody against AGGF1 revealed about 50% reduction of AGGF1 expression in heterozygous Aggf1 +/− embryos (E12.5). ( D ) Representative images showing defective vasculature in Aggf1 +/− yolk sacs and embryos. Haemorrhages were also seen in 35% of developing Aggf1 +/− embryos. ( E ) Detection of haemorrhages in the brain, lung and spleen of 35% of Aggf1 +/− mice at 50 to 60 weeks of age. ( F ) Significantly increased vascular permeability in Aggf1 +/− mice at 50 to 60 weeks of age compared to that of age-matched Aggf1 +/+ controls. ( G ) Matrigel-based endothelial tube formation assays revealed a decreased angiogenesis by microvascular ECs isolated from lungs of Aggf1 +/− mice as compared to that from Aggf1 +/+ control mice at the age of 20 to 30 weeks. Scale bar = 50 μm.

    Article Snippet: In brief, ES cell DNA samples were digested with EcoR V (Invitrogen), separated on a 0.8% agarose gel and transferred to a Hybond N+ membrane (Amersham).

    Techniques: Permeability, Mouse Assay, Homologous Recombination, Plasmid Preparation, Southern Blot, Polymerase Chain Reaction, Clone Assay, Western Blot, Expressing, Isolation

    Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or Bsa I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the EcoR V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).

    Journal: PLoS ONE

    Article Title: Efficient Generation of Recombinant Influenza A Viruses Employing a New Approach to Overcome the Genetic Instability of HA Segments

    doi: 10.1371/journal.pone.0116917

    Figure Lengend Snippet: Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or Bsa I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the EcoR V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).

    Article Snippet: Since the cloned HA-segments of H9N2/SA showed instability in the genetic backbone of pCR2.1 sequencing vector, the full-length RT-PCR products of HA segments amplified with primers containing EcoRV and Bsa I sites were digested with EcoR V enzyme (Thermo Scientific, USA) for 1 h at 37°C to produce blunt-ends to allow the ligation into the EcoRV -linearized pSMART-LC-Kan (Lucigen, USA).

    Techniques: Plasmid Preparation, Clone Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction