puc19 Thermo Fisher Search Results


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  • 90
    Thermo Fisher puc19
    Comparison of piggyBac , TcBuster , and Tol2 at varying transposase: transposon ratios. HEK-293 cells in 6-well plates were transfected in triplicate with the indicated amount of transposase helper plasmid (+) or <t>pUC19</t> (−) and plasmids carrying the neomycin-resistance transposon for each system. The amount of transposon plasmid transfected was 200 ng ( a ), 500 ng ( b ), or 900 ng ( c ). In c , the piggyBac (+) transposase plates had approximately 1000 colonies, too many to count (TMTC). Cells were diluted 1∶750 onto 10 cm plates in media containing geneticin for selection and incubated for two weeks to allow drug-resistant cells to form colonies that were fixed, stained and counted. Error bars represent the SEM (n = 3).
    Puc19, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1013 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher cloneamp puc19 system
    Comparison of piggyBac , TcBuster , and Tol2 at varying transposase: transposon ratios. HEK-293 cells in 6-well plates were transfected in triplicate with the indicated amount of transposase helper plasmid (+) or <t>pUC19</t> (−) and plasmids carrying the neomycin-resistance transposon for each system. The amount of transposon plasmid transfected was 200 ng ( a ), 500 ng ( b ), or 900 ng ( c ). In c , the piggyBac (+) transposase plates had approximately 1000 colonies, too many to count (TMTC). Cells were diluted 1∶750 onto 10 cm plates in media containing geneticin for selection and incubated for two weeks to allow drug-resistant cells to form colonies that were fixed, stained and counted. Error bars represent the SEM (n = 3).
    Cloneamp Puc19 System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher xbai double digested puc19
    Comparison of piggyBac , TcBuster , and Tol2 at varying transposase: transposon ratios. HEK-293 cells in 6-well plates were transfected in triplicate with the indicated amount of transposase helper plasmid (+) or <t>pUC19</t> (−) and plasmids carrying the neomycin-resistance transposon for each system. The amount of transposon plasmid transfected was 200 ng ( a ), 500 ng ( b ), or 900 ng ( c ). In c , the piggyBac (+) transposase plates had approximately 1000 colonies, too many to count (TMTC). Cells were diluted 1∶750 onto 10 cm plates in media containing geneticin for selection and incubated for two weeks to allow drug-resistant cells to form colonies that were fixed, stained and counted. Error bars represent the SEM (n = 3).
    Xbai Double Digested Puc19, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher plasmid puc19
    Agarose gel electrophoresis of PCR products amplified from <t>pUC19/IRES</t> DNAs using (T7/IRES-AUG) primers (as previously described in the “Experimental Section”). Lane MW: Smart DNA ladder 200 lanes (Eurogentec); lane (-): a negative control for the PCR reaction; lane (Wt): CVB3 wild-type IRES; lane (S3): CVB3 Sabin3-like IRES.
    Plasmid Puc19, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher bamhi psti digested puc19
    Agarose gel electrophoresis of PCR products amplified from <t>pUC19/IRES</t> DNAs using (T7/IRES-AUG) primers (as previously described in the “Experimental Section”). Lane MW: Smart DNA ladder 200 lanes (Eurogentec); lane (-): a negative control for the PCR reaction; lane (Wt): CVB3 wild-type IRES; lane (S3): CVB3 Sabin3-like IRES.
    Bamhi Psti Digested Puc19, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher puc19 ampr
    Agarose gel electrophoresis of PCR products amplified from <t>pUC19/IRES</t> DNAs using (T7/IRES-AUG) primers (as previously described in the “Experimental Section”). Lane MW: Smart DNA ladder 200 lanes (Eurogentec); lane (-): a negative control for the PCR reaction; lane (Wt): CVB3 wild-type IRES; lane (S3): CVB3 Sabin3-like IRES.
    Puc19 Ampr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher puc19 vsr dna
    SDS-PAGE analysis of purified <t>DNA</t> polymerase I, DNA ligase I, and the <t>Vsr</t> endonuclease. Purified proteins (3 μg) were resolved on an 11% polyacrylamide gel run in the presence of SDS and stained with Coomassie Blue. Lane 1 , molecular mass standards
    Puc19 Vsr Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dsdna
    <t>mRNA</t> or ssDNA but not <t>dsDNA</t> transfections lead to stress granule assembly in puromycin-treated cells. ( A ) 1 μg of 2Luc mRNA synthesized in vitro was transfected in NRK cells using lipofectamine in the presence of 2.5 μg/ml puromycin (see ‘Materials and Methods’ section). Anti-HuR and anti-YB-1 labeling show the formation of stress granules 3 h after transfection. ( B ) NRK cells were transfected using lipofectamine (Lipo) with 1 μg of α-globin mRNA, M13 ssDNA, or linearized pUC19 dsDNA in the presence or absence of puromycin for 3 h. Benzonase treatment was performed before the formation of lipoplexes. The statistical analysis was obtained on three different samples. Results are mean ± SD ( n = 3). Both mRNA and ssDNA lead to a significant formation of stress granules in puromycin-treated cells. Anti-YB-1 labeling shows the formation of stress granules 3 h after transfection.
    Dsdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2088 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher pr4 gus puc19
    <t>mRNA</t> or ssDNA but not <t>dsDNA</t> transfections lead to stress granule assembly in puromycin-treated cells. ( A ) 1 μg of 2Luc mRNA synthesized in vitro was transfected in NRK cells using lipofectamine in the presence of 2.5 μg/ml puromycin (see ‘Materials and Methods’ section). Anti-HuR and anti-YB-1 labeling show the formation of stress granules 3 h after transfection. ( B ) NRK cells were transfected using lipofectamine (Lipo) with 1 μg of α-globin mRNA, M13 ssDNA, or linearized pUC19 dsDNA in the presence or absence of puromycin for 3 h. Benzonase treatment was performed before the formation of lipoplexes. The statistical analysis was obtained on three different samples. Results are mean ± SD ( n = 3). Both mRNA and ssDNA lead to a significant formation of stress granules in puromycin-treated cells. Anti-YB-1 labeling shows the formation of stress granules 3 h after transfection.
    Pr4 Gus Puc19, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher bamhi linearized puc19 vectors
    <t>mRNA</t> or ssDNA but not <t>dsDNA</t> transfections lead to stress granule assembly in puromycin-treated cells. ( A ) 1 μg of 2Luc mRNA synthesized in vitro was transfected in NRK cells using lipofectamine in the presence of 2.5 μg/ml puromycin (see ‘Materials and Methods’ section). Anti-HuR and anti-YB-1 labeling show the formation of stress granules 3 h after transfection. ( B ) NRK cells were transfected using lipofectamine (Lipo) with 1 μg of α-globin mRNA, M13 ssDNA, or linearized pUC19 dsDNA in the presence or absence of puromycin for 3 h. Benzonase treatment was performed before the formation of lipoplexes. The statistical analysis was obtained on three different samples. Results are mean ± SD ( n = 3). Both mRNA and ssDNA lead to a significant formation of stress granules in puromycin-treated cells. Anti-YB-1 labeling shows the formation of stress granules 3 h after transfection.
    Bamhi Linearized Puc19 Vectors, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher hin diii digested puc19
    <t>mRNA</t> or ssDNA but not <t>dsDNA</t> transfections lead to stress granule assembly in puromycin-treated cells. ( A ) 1 μg of 2Luc mRNA synthesized in vitro was transfected in NRK cells using lipofectamine in the presence of 2.5 μg/ml puromycin (see ‘Materials and Methods’ section). Anti-HuR and anti-YB-1 labeling show the formation of stress granules 3 h after transfection. ( B ) NRK cells were transfected using lipofectamine (Lipo) with 1 μg of α-globin mRNA, M13 ssDNA, or linearized pUC19 dsDNA in the presence or absence of puromycin for 3 h. Benzonase treatment was performed before the formation of lipoplexes. The statistical analysis was obtained on three different samples. Results are mean ± SD ( n = 3). Both mRNA and ssDNA lead to a significant formation of stress granules in puromycin-treated cells. Anti-YB-1 labeling shows the formation of stress granules 3 h after transfection.
    Hin Diii Digested Puc19, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Thermo Fisher cloning vector puc19
    In vivo functionality of E. coli CD (CodA, in pQE70 expression vector) and the DNA fragments that were selected from four metagenomic libraries: KANOS, URA4, URA3, and Vcz (in <t>pUC19</t> vector). Empty pQE70 vector was used as a negative control. Minimal medium (M9) was supplemented 100 mg/L ampicillin, 15 mg/L kanamycin, 0.1 mM IPTG, and either with 20 mg/L cytosine (M9 + C), isocytosine (M9 + isoC) or uracil (M9 + U, positive control).
    Cloning Vector Puc19, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher supercoiled puc19 plasmid
    Bsu Ku binds circular DNA. ( A ) Left panel , Bsu Ku binding to covalently closed <t>pUC19</t> plasmid. The assay was performed as described in Materials and Methods incubating the indicated amounts of Bsu Ku with 100 ng of <t>supercoiled</t> plasmid pUC19. Products were analysed on 0.7% agarose electrophoresis. One half of each reaction mixture was loaded directly onto the agarose gel (direct loading lanes), while the other half was being treated with Proteinase K. After proteolytic digestion, those samples were loaded in the same agarose gel (Proteinase K treatment lanes). The electrophoretical mobility of the pUC19 plasmid in each case is indicated. In lane c, DNA was incubated with 50 ng of BSA. Right panel , Bsu Ku binding to supercoiled and relaxed plasmid DNA. The assay was performed essentially as described above. ( B ) Bsu Ku AP lyase activity can act on circular substrates. The assay was performed as described in Materials and Methods, incubating either 142 nM of Bsu Ku with 100 ng of plasmid without AP sites (pcDNA3.1) or 18, 36, 72 and 142 nM of Bsu Ku with 100 ng of plasmid containing AP sites (pcDNA3.1-AP) (left panel). The absence and presence of the AP sites in pcDNA3.1 and pcDNA3.1-AP, respectively, was confirmed after digestion with h APE1. As a control of the linear form, plasmids were also digested with EcoRI. NC : nicked circles; SC : supercoiled; L : linear. The assay shown in the right panel was performed by incubating 100 ng of plasmid without (pUC19) or with (pUC19-AP) AP sites with either h APE1 or 142 nM of Bsu Ku, as indicated. Figure 5B , right panel is a composite image made from different parts of the same experiment.
    Supercoiled Puc19 Plasmid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Thermo Fisher puc19 dna mspi hpaii marker 23
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Puc19 Dna Mspi Hpaii Marker 23, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher puc19 standard puc19
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Puc19 Standard Puc19, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher vector puc19 smai
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Vector Puc19 Smai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher kpni xhoi digested puc19 vector
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Kpni Xhoi Digested Puc19 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher dephosphorylated puc19 vector
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Dephosphorylated Puc19 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher xbai psti cut puc19 plasmid
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Xbai Psti Cut Puc19 Plasmid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher kpni digested puc19 vector
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Kpni Digested Puc19 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher plasmid puc19 dna
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Plasmid Puc19 Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher lambda hindiii puc19 taqi sau3a1 molecular weight markers
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Lambda Hindiii Puc19 Taqi Sau3a1 Molecular Weight Markers, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher puc19 expression vector backbone
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Puc19 Expression Vector Backbone, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher empty vector puc19
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Empty Vector Puc19, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Thermo Fisher ecori hindiii digested puc19
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Ecori Hindiii Digested Puc19, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher suicide vector puc19
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Suicide Vector Puc19, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher puc19 rna ladder
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Puc19 Rna Ladder, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher cip treated plasmid puc19
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Cip Treated Plasmid Puc19, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher non specific puc19 vector
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
    Non Specific Puc19 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher bamhi
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
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    Thermo Fisher heat denaturated puc19 dna mspi
    Electrophoresis of HBV genotypes. M: <t>pUC19</t> <t>DNA/</t> Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.
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    Image Search Results


    Comparison of piggyBac , TcBuster , and Tol2 at varying transposase: transposon ratios. HEK-293 cells in 6-well plates were transfected in triplicate with the indicated amount of transposase helper plasmid (+) or pUC19 (−) and plasmids carrying the neomycin-resistance transposon for each system. The amount of transposon plasmid transfected was 200 ng ( a ), 500 ng ( b ), or 900 ng ( c ). In c , the piggyBac (+) transposase plates had approximately 1000 colonies, too many to count (TMTC). Cells were diluted 1∶750 onto 10 cm plates in media containing geneticin for selection and incubated for two weeks to allow drug-resistant cells to form colonies that were fixed, stained and counted. Error bars represent the SEM (n = 3).

    Journal: PLoS ONE

    Article Title: Comparative Analysis of the Recently Discovered hAT Transposon TcBuster in Human Cells

    doi: 10.1371/journal.pone.0042666

    Figure Lengend Snippet: Comparison of piggyBac , TcBuster , and Tol2 at varying transposase: transposon ratios. HEK-293 cells in 6-well plates were transfected in triplicate with the indicated amount of transposase helper plasmid (+) or pUC19 (−) and plasmids carrying the neomycin-resistance transposon for each system. The amount of transposon plasmid transfected was 200 ng ( a ), 500 ng ( b ), or 900 ng ( c ). In c , the piggyBac (+) transposase plates had approximately 1000 colonies, too many to count (TMTC). Cells were diluted 1∶750 onto 10 cm plates in media containing geneticin for selection and incubated for two weeks to allow drug-resistant cells to form colonies that were fixed, stained and counted. Error bars represent the SEM (n = 3).

    Article Snippet: Plasmid constructs All plasmids were prepared for assays by endotoxin-free maxiprep (Qiagen, Valencia, CA). pUC19 (Invitrogen, Carlsbad, CA) was used as filler plasmid DNA for the FuGene6 transfections.

    Techniques: Transfection, Plasmid Preparation, Selection, Incubation, Staining

    The effect of transposon and transposase plasmid dose on the number of drug-resistant colonies formed. ( a ) HEK-293 cells in 6-well plates were transfected in triplicate with either 12.5 ng (light grey bars), 50 ng (dark grey bars), or 500 ng (black bars) of pTcBNeo carrying the neomycin-resistance transposon and 0 ng, 100 ng, 250 ng, or 500 ng of pCMV- TcBuster expressing the transposase. ( b ) HEK-293 cells in 6-well plates were transfected in triplicate with 500 ng of pTcBNeo plasmid carrying the neomycin-resistance transposon and the indicated amount of pCMV- TcBuster (0.5 ng, 1 ng, 5 ng, 10 ng, 25 ng, 50 ng, 100 ng, 250 ng, or 500 ng). In both a and b , pUC19 was used as filler DNA to increase the total amount of DNA transfected to 1 µg. Cells were diluted 1∶750 in selection media and grown for two weeks to allow drug-resistant cells to multiply and form colonies. The colonies were fixed, stained, and counted. The mean and standard error of the mean (SEM; n = 3) are shown.

    Journal: PLoS ONE

    Article Title: Comparative Analysis of the Recently Discovered hAT Transposon TcBuster in Human Cells

    doi: 10.1371/journal.pone.0042666

    Figure Lengend Snippet: The effect of transposon and transposase plasmid dose on the number of drug-resistant colonies formed. ( a ) HEK-293 cells in 6-well plates were transfected in triplicate with either 12.5 ng (light grey bars), 50 ng (dark grey bars), or 500 ng (black bars) of pTcBNeo carrying the neomycin-resistance transposon and 0 ng, 100 ng, 250 ng, or 500 ng of pCMV- TcBuster expressing the transposase. ( b ) HEK-293 cells in 6-well plates were transfected in triplicate with 500 ng of pTcBNeo plasmid carrying the neomycin-resistance transposon and the indicated amount of pCMV- TcBuster (0.5 ng, 1 ng, 5 ng, 10 ng, 25 ng, 50 ng, 100 ng, 250 ng, or 500 ng). In both a and b , pUC19 was used as filler DNA to increase the total amount of DNA transfected to 1 µg. Cells were diluted 1∶750 in selection media and grown for two weeks to allow drug-resistant cells to multiply and form colonies. The colonies were fixed, stained, and counted. The mean and standard error of the mean (SEM; n = 3) are shown.

    Article Snippet: Plasmid constructs All plasmids were prepared for assays by endotoxin-free maxiprep (Qiagen, Valencia, CA). pUC19 (Invitrogen, Carlsbad, CA) was used as filler plasmid DNA for the FuGene6 transfections.

    Techniques: Plasmid Preparation, Transfection, Expressing, Selection, Staining

    Intracellular localization of HBcAg (magnification, ×400). Immunofluorescence staining of HBcAg in HuH-7 cells transfected with (a) pUC19-HBV/E wild-type replicon and (c) pUC19-HBV/E replacement replicon. (b and d) Nuclear staining for the same

    Journal: Journal of Virology

    Article Title: Novel Type of Hepatitis B Virus Mutation: Replacement Mutation Involving a Hepatocyte Nuclear Factor 1 Binding Site Tandem Repeat in Chronic Hepatitis B Virus Genotype E

    doi: 10.1128/JVI.79.22.14404-14410.2005

    Figure Lengend Snippet: Intracellular localization of HBcAg (magnification, ×400). Immunofluorescence staining of HBcAg in HuH-7 cells transfected with (a) pUC19-HBV/E wild-type replicon and (c) pUC19-HBV/E replacement replicon. (b and d) Nuclear staining for the same

    Article Snippet: Finally, the fragments C-B-HindIII-AvrII and pGEM-fragA-C, cut with AvrII and SacI, were cloned into pUC19 without promoters (Invitrogen) cut with HindIII and SacI, and a pUC19-HBV/E wild-type replicon encoding a replication-competent 1.35-unit-length HBV genome was produced.

    Techniques: Immunofluorescence, Staining, Transfection

    (A) Southern blot analysis of intracellular replication competence of replacement mutation with HNF1 site. Double-stranded (DS) DNA and single-stranded (SS) DNA are indicated by arrows. The pUC19-HBV/E replacement replicon (lane 2) replicates at a much

    Journal: Journal of Virology

    Article Title: Novel Type of Hepatitis B Virus Mutation: Replacement Mutation Involving a Hepatocyte Nuclear Factor 1 Binding Site Tandem Repeat in Chronic Hepatitis B Virus Genotype E

    doi: 10.1128/JVI.79.22.14404-14410.2005

    Figure Lengend Snippet: (A) Southern blot analysis of intracellular replication competence of replacement mutation with HNF1 site. Double-stranded (DS) DNA and single-stranded (SS) DNA are indicated by arrows. The pUC19-HBV/E replacement replicon (lane 2) replicates at a much

    Article Snippet: Finally, the fragments C-B-HindIII-AvrII and pGEM-fragA-C, cut with AvrII and SacI, were cloned into pUC19 without promoters (Invitrogen) cut with HindIII and SacI, and a pUC19-HBV/E wild-type replicon encoding a replication-competent 1.35-unit-length HBV genome was produced.

    Techniques: Southern Blot, Mutagenesis

    ( A ) A representative molecule of pUC19 equilibrated in 2D conformation on a freshly cleaved mica with 10 mM Mg 2+ . ( B ) The centerline of the DNA molecule is obtained by tracking the brightest point along the DNA contour. Each point on the centerline is separated by 8 nm. ( C ) Plot of as a function of the separation L . The closed circles are the experimental data obtained by averaging at least 30 molecules. The experimental data are fitted using equation 1 , shown by the red line. The fit gives a persistence length of 56 nm.

    Journal: Nucleic Acids Research

    Article Title: Time-dependent bending rigidity and helical twist of DNA by rearrangement of bound HU protein

    doi: 10.1093/nar/gkt593

    Figure Lengend Snippet: ( A ) A representative molecule of pUC19 equilibrated in 2D conformation on a freshly cleaved mica with 10 mM Mg 2+ . ( B ) The centerline of the DNA molecule is obtained by tracking the brightest point along the DNA contour. Each point on the centerline is separated by 8 nm. ( C ) Plot of as a function of the separation L . The closed circles are the experimental data obtained by averaging at least 30 molecules. The experimental data are fitted using equation 1 , shown by the red line. The fit gives a persistence length of 56 nm.

    Article Snippet: Sample preparation DNA fragments of 1000 bp and pUC19 (2686 bp) were purchased from Thermoscientific (Vilnius, Lithuania).

    Techniques:

    ( A ) Closed circular relaxed pUC19 after treatment with Topo 1. ( B ) Circular DNA incubated with HU in the ratio 1 dimer: 1 bp and imaged after 45 min and ( C ) 2 h. The scale bars denote 500 nm.

    Journal: Nucleic Acids Research

    Article Title: Time-dependent bending rigidity and helical twist of DNA by rearrangement of bound HU protein

    doi: 10.1093/nar/gkt593

    Figure Lengend Snippet: ( A ) Closed circular relaxed pUC19 after treatment with Topo 1. ( B ) Circular DNA incubated with HU in the ratio 1 dimer: 1 bp and imaged after 45 min and ( C ) 2 h. The scale bars denote 500 nm.

    Article Snippet: Sample preparation DNA fragments of 1000 bp and pUC19 (2686 bp) were purchased from Thermoscientific (Vilnius, Lithuania).

    Techniques: Incubation

    ( A ) Supercoiled pUC19. ( B ) Closed circular relaxed pUC19 after treatment of supercoiled pUC19 with Topo 1. ( C ) Supercoil after incubation of relaxed pUC19 with HU for 2 h. The scale bars denote 100 nm.

    Journal: Nucleic Acids Research

    Article Title: Time-dependent bending rigidity and helical twist of DNA by rearrangement of bound HU protein

    doi: 10.1093/nar/gkt593

    Figure Lengend Snippet: ( A ) Supercoiled pUC19. ( B ) Closed circular relaxed pUC19 after treatment of supercoiled pUC19 with Topo 1. ( C ) Supercoil after incubation of relaxed pUC19 with HU for 2 h. The scale bars denote 100 nm.

    Article Snippet: Sample preparation DNA fragments of 1000 bp and pUC19 (2686 bp) were purchased from Thermoscientific (Vilnius, Lithuania).

    Techniques: Incubation

    Independent labeling of two hemilineages in the larval ventral nervous system. (A) Schematic of a CRM-GAL4 transgene inserted into an attP site. Red-shaded boxes indicate sequences that originate from the attB -containing CRM-GAL4 vector. Yellow-shaded boxes indicate sequences from the attP -containing P -element. Gray boxes indicate the surrounding genomic region. attR and attL are the hybrid sites that result from phiC31-mediated recombination between attP and attB . Arrows indicate direction of transcription. The area above the brace indicates the sequences that vary in this study. For simplicity, we show only these elements in subsequent figures. Abbreviations: P, P -element sequence; CRM, cis -regulatory module; DSCP, Drosophila synthetic core promoter; white , mini- white used to score for integrations; pUC19, plasmid backbone from the CRM-GAL4 vector containing bacterial origin of replication and ampicillin resistance gene. (B–D) Maximum confocal z projection of the thoracic region of a wandering third instar larval central nervous system of the same genotype as in Figure 2A [w; P{UAS-mCD8 :: mRFP.LG} /R24B02-LexA( attP40 ); R58G08-GAL4( attP2 )/ P{LexAop-CD2 :: GFP} ]. R24B02-LexA expression is shown in B and in green in D. It is expressed in hemilineage 12A in segments S2 to T2, and in A1. Hemilineage 12A’s cell bodies are located relatively medially, and its neurite bundle projects dorsally. In T1 and T2, the 12A bundle splits into a medial and dorsal branch (arrowheads). In T2, the dorsal branch of 12A projects across the midline. R58G08-GAL4 is shown in C and in magenta in D. It is expressed in hemilineage 3A in segments T1–T3. The cell bodies of 3A are located more laterally than those of 12A. The 3A bundle initially projects dorsally, but then turns laterally and projects to the dorsal region of the leg neuropil (arrows). The white box indicates the area shown in Figure 2A . Background staining of trachea is indicated in C.

    Journal: Genetics

    Article Title: Transvection Is Common Throughout the Drosophila Genome

    doi: 10.1534/genetics.112.140475

    Figure Lengend Snippet: Independent labeling of two hemilineages in the larval ventral nervous system. (A) Schematic of a CRM-GAL4 transgene inserted into an attP site. Red-shaded boxes indicate sequences that originate from the attB -containing CRM-GAL4 vector. Yellow-shaded boxes indicate sequences from the attP -containing P -element. Gray boxes indicate the surrounding genomic region. attR and attL are the hybrid sites that result from phiC31-mediated recombination between attP and attB . Arrows indicate direction of transcription. The area above the brace indicates the sequences that vary in this study. For simplicity, we show only these elements in subsequent figures. Abbreviations: P, P -element sequence; CRM, cis -regulatory module; DSCP, Drosophila synthetic core promoter; white , mini- white used to score for integrations; pUC19, plasmid backbone from the CRM-GAL4 vector containing bacterial origin of replication and ampicillin resistance gene. (B–D) Maximum confocal z projection of the thoracic region of a wandering third instar larval central nervous system of the same genotype as in Figure 2A [w; P{UAS-mCD8 :: mRFP.LG} /R24B02-LexA( attP40 ); R58G08-GAL4( attP2 )/ P{LexAop-CD2 :: GFP} ]. R24B02-LexA expression is shown in B and in green in D. It is expressed in hemilineage 12A in segments S2 to T2, and in A1. Hemilineage 12A’s cell bodies are located relatively medially, and its neurite bundle projects dorsally. In T1 and T2, the 12A bundle splits into a medial and dorsal branch (arrowheads). In T2, the dorsal branch of 12A projects across the midline. R58G08-GAL4 is shown in C and in magenta in D. It is expressed in hemilineage 3A in segments T1–T3. The cell bodies of 3A are located more laterally than those of 12A. The 3A bundle initially projects dorsally, but then turns laterally and projects to the dorsal region of the leg neuropil (arrows). The white box indicates the area shown in Figure 2A . Background staining of trachea is indicated in C.

    Article Snippet: When a CRM-GAL4 is inserted into an attP site, the final result is a locus that is flanked by 5′ and 3′ P -element sequences and contains: (1) a copy of the yellow gene (the positive marker for P -element transformants), (2) one each of attL and attR sites (the outcome of recombination between attP and attB ), (3) mini-white (a positive marker for phiC31 transformants), (4) the CRM-GAL4, and (5) the pUC19-derived backbone (Invitrogen) of the vector used to introduce the CRM-GAL4.

    Techniques: Labeling, Plasmid Preparation, Sequencing, Expressing, Staining

    Agarose gel electrophoresis of PCR products amplified from pUC19/IRES DNAs using (T7/IRES-AUG) primers (as previously described in the “Experimental Section”). Lane MW: Smart DNA ladder 200 lanes (Eurogentec); lane (-): a negative control for the PCR reaction; lane (Wt): CVB3 wild-type IRES; lane (S3): CVB3 Sabin3-like IRES.

    Journal: International Journal of Molecular Sciences

    Article Title: Ribosomal Initiation Complex Assembly within the Wild-Strain of Coxsackievirus B3 and Live-Attenuated Sabin3-like IRESes during the Initiation of Translation

    doi: 10.3390/ijms14034400

    Figure Lengend Snippet: Agarose gel electrophoresis of PCR products amplified from pUC19/IRES DNAs using (T7/IRES-AUG) primers (as previously described in the “Experimental Section”). Lane MW: Smart DNA ladder 200 lanes (Eurogentec); lane (-): a negative control for the PCR reaction; lane (Wt): CVB3 wild-type IRES; lane (S3): CVB3 Sabin3-like IRES.

    Article Snippet: Cloning of the IRES in the pUC19 Vector Amplicons encoding CVB3 IRESes were double-digested with EcoRI and BamHI (Roche Applied Science) (both restriction sites were added by PCR), then purified and inserted into the pUC19 plasmid (Invitrogen) to be digested with the same enzymes.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Negative Control

    Start codon upstream sequences of plasmid borne reporter genes. The sequence starts with the Pst I site of the pRH3 or pUC19 vector (shown in italics). The stop codon at the start of the upstream sequence is underlined and the start codon of nucB or hMGFP is in boldface. A and B: nucB constructs with added (A) or removed (B) SD sequences. C: start codon upstream sequences containing SD sequences that are involved in formation of mRNA secondary structures with different stability. The sequences starting with mg were used in E. coli to translate the MGFP. D: Start codon upstream sequences of plasmid pRH3 borne nucB constructs containing SD sequence length of 10, 8, and 6 nucleotides. E: nucB upstream sequence used to asses the effect of secondary structure 20 bp upstream of the start codon. The secondary structure element was inserted into construct 0 from C after the designated adenosine.

    Journal: PLoS ONE

    Article Title: Inability of Prevotella bryantii to Form a Functional Shine-Dalgarno Interaction Reflects Unique Evolution of Ribosome Binding Sites in Bacteroidetes

    doi: 10.1371/journal.pone.0022914

    Figure Lengend Snippet: Start codon upstream sequences of plasmid borne reporter genes. The sequence starts with the Pst I site of the pRH3 or pUC19 vector (shown in italics). The stop codon at the start of the upstream sequence is underlined and the start codon of nucB or hMGFP is in boldface. A and B: nucB constructs with added (A) or removed (B) SD sequences. C: start codon upstream sequences containing SD sequences that are involved in formation of mRNA secondary structures with different stability. The sequences starting with mg were used in E. coli to translate the MGFP. D: Start codon upstream sequences of plasmid pRH3 borne nucB constructs containing SD sequence length of 10, 8, and 6 nucleotides. E: nucB upstream sequence used to asses the effect of secondary structure 20 bp upstream of the start codon. The secondary structure element was inserted into construct 0 from C after the designated adenosine.

    Article Snippet: The Escherichia coli TOP10 and pUC19 vector were from Invitrogen (USA).

    Techniques: Plasmid Preparation, Sequencing, Construct

    Overview of the microarray strategy . A library of chromosomal DNA fragments of P. syringae pv. phaseolicola NPS3121 (Psp NPS3121) was constructed in the pUC19 vector and introduced into the E. coli Top10 strain. 30% (2880 clones) of the genomic library was sequenced, aligned and annotated against the complete genome of P. syringae pv. phaseolicola 1448A. This strategy allowed selection of 1911 clones that provided approximately 1× coverage of the genome. The fragments of 1911 clones were amplified by PCR reaction, and the products were printed on a microarray slide. This microarray was used to identify genes that are differentially expressed when bean leaf or pod extracts and apoplastic fluid were added to the growth medium.

    Journal: BMC Microbiology

    Article Title: Transcriptional profile of Pseudomonas syringae pv. phaseolicola NPS3121 in response to tissue extracts from a susceptible Phaseolus vulgaris L. cultivar

    doi: 10.1186/1471-2180-9-257

    Figure Lengend Snippet: Overview of the microarray strategy . A library of chromosomal DNA fragments of P. syringae pv. phaseolicola NPS3121 (Psp NPS3121) was constructed in the pUC19 vector and introduced into the E. coli Top10 strain. 30% (2880 clones) of the genomic library was sequenced, aligned and annotated against the complete genome of P. syringae pv. phaseolicola 1448A. This strategy allowed selection of 1911 clones that provided approximately 1× coverage of the genome. The fragments of 1911 clones were amplified by PCR reaction, and the products were printed on a microarray slide. This microarray was used to identify genes that are differentially expressed when bean leaf or pod extracts and apoplastic fluid were added to the growth medium.

    Article Snippet: The genomic fragments were ligated into the plasmid vector pUC19 (Invitrogen, California, USA) previously digested with BamHI, and the ligation mixture was used to transform Escherichia coli TOP10 cells (Invitrogen, California, USA).

    Techniques: Microarray, Construct, Plasmid Preparation, Selection, Clone Assay, Amplification, Polymerase Chain Reaction

    Non-sequence-specific endonucleolytic activity of the integrases. Supercoiled (CC) pUC19 DNA (200 ng) was incubated as described in Materials and Methods with the different INs (5 pmol) for 2 h at 37°C. The products were separated on 1%

    Journal: Nucleic Acids Research

    Article Title: Biochemical and random mutagenesis analysis of the region carrying the catalytic E152 amino acid of HIV-1 integrase

    doi: 10.1093/nar/gkh298

    Figure Lengend Snippet: Non-sequence-specific endonucleolytic activity of the integrases. Supercoiled (CC) pUC19 DNA (200 ng) was incubated as described in Materials and Methods with the different INs (5 pmol) for 2 h at 37°C. The products were separated on 1%

    Article Snippet: The pUC19 vector substrate of the endonucleolytic IN activity was purchased (Gibco) and maintained in E.coli DH5α bacterial strain.

    Techniques: Sequencing, Activity Assay, Incubation

    SDS-PAGE analysis of purified DNA polymerase I, DNA ligase I, and the Vsr endonuclease. Purified proteins (3 μg) were resolved on an 11% polyacrylamide gel run in the presence of SDS and stained with Coomassie Blue. Lane 1 , molecular mass standards

    Journal: The Journal of Biological Chemistry

    Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *

    doi: 10.1074/jbc.M112.384321

    Figure Lengend Snippet: SDS-PAGE analysis of purified DNA polymerase I, DNA ligase I, and the Vsr endonuclease. Purified proteins (3 μg) were resolved on an 11% polyacrylamide gel run in the presence of SDS and stained with Coomassie Blue. Lane 1 , molecular mass standards

    Article Snippet: The phosphorylated oligonucleotides were annealed into the gapped pUC19-VSR DNA as follows: annealing reaction mixtures (47 μl) contained 30 μl of gapped pUC19-VSR DNA, 30.6 pmol of phosphorylated oligonucleotide, 50 m m Tris-HCl (pH 7.5), 10 m m MgOAc, 5 m m dithiothreitol, 50 m m NaCl and were heated to 80 °C in a GeneAmp PCR System 2400 Thermocycler (Applied Biosystems).

    Techniques: SDS Page, Purification, Staining

    DNA polymerase I and the Vsr endonuclease are sufficient to repair a T:G mismatch. The VSP repair reaction contained 50 ng of covalently closed heteroduplex substrate, 52 n m Vsr endonuclease, and a titration of DNA polymerase I from 467 to 0.2 n m as described

    Journal: The Journal of Biological Chemistry

    Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *

    doi: 10.1074/jbc.M112.384321

    Figure Lengend Snippet: DNA polymerase I and the Vsr endonuclease are sufficient to repair a T:G mismatch. The VSP repair reaction contained 50 ng of covalently closed heteroduplex substrate, 52 n m Vsr endonuclease, and a titration of DNA polymerase I from 467 to 0.2 n m as described

    Article Snippet: The phosphorylated oligonucleotides were annealed into the gapped pUC19-VSR DNA as follows: annealing reaction mixtures (47 μl) contained 30 μl of gapped pUC19-VSR DNA, 30.6 pmol of phosphorylated oligonucleotide, 50 m m Tris-HCl (pH 7.5), 10 m m MgOAc, 5 m m dithiothreitol, 50 m m NaCl and were heated to 80 °C in a GeneAmp PCR System 2400 Thermocycler (Applied Biosystems).

    Techniques: Titration

    DNA ligase I seals the nick created by DNA polymerase I nick translation. Repair and ligation reactions were conducted as described under “Materials and Methods” using 200 ng of pUC19-VSR heteroduplex DNA (∼1.2 n m molecules), 10

    Journal: The Journal of Biological Chemistry

    Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *

    doi: 10.1074/jbc.M112.384321

    Figure Lengend Snippet: DNA ligase I seals the nick created by DNA polymerase I nick translation. Repair and ligation reactions were conducted as described under “Materials and Methods” using 200 ng of pUC19-VSR heteroduplex DNA (∼1.2 n m molecules), 10

    Article Snippet: The phosphorylated oligonucleotides were annealed into the gapped pUC19-VSR DNA as follows: annealing reaction mixtures (47 μl) contained 30 μl of gapped pUC19-VSR DNA, 30.6 pmol of phosphorylated oligonucleotide, 50 m m Tris-HCl (pH 7.5), 10 m m MgOAc, 5 m m dithiothreitol, 50 m m NaCl and were heated to 80 °C in a GeneAmp PCR System 2400 Thermocycler (Applied Biosystems).

    Techniques: Nick Translation, Ligation

    Vsr-catalyzed nicking of a covalently closed circular DNA substrate. Vsr-dependent nicking reactions were as described under “Materials and Methods.” A , the titration of the Vsr endonuclease was from 153 to 0 n m with each DNA substrate,

    Journal: The Journal of Biological Chemistry

    Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *

    doi: 10.1074/jbc.M112.384321

    Figure Lengend Snippet: Vsr-catalyzed nicking of a covalently closed circular DNA substrate. Vsr-dependent nicking reactions were as described under “Materials and Methods.” A , the titration of the Vsr endonuclease was from 153 to 0 n m with each DNA substrate,

    Article Snippet: The phosphorylated oligonucleotides were annealed into the gapped pUC19-VSR DNA as follows: annealing reaction mixtures (47 μl) contained 30 μl of gapped pUC19-VSR DNA, 30.6 pmol of phosphorylated oligonucleotide, 50 m m Tris-HCl (pH 7.5), 10 m m MgOAc, 5 m m dithiothreitol, 50 m m NaCl and were heated to 80 °C in a GeneAmp PCR System 2400 Thermocycler (Applied Biosystems).

    Techniques: Titration

    The impact of DNA ligase I concentration on repair track length. Ligation and repair reactions were as described under “Materials and Methods” using 200 ng of covalently closed pUC19-VSR heteroduplex DNA (∼1.2 n m molecules), 10

    Journal: The Journal of Biological Chemistry

    Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *

    doi: 10.1074/jbc.M112.384321

    Figure Lengend Snippet: The impact of DNA ligase I concentration on repair track length. Ligation and repair reactions were as described under “Materials and Methods” using 200 ng of covalently closed pUC19-VSR heteroduplex DNA (∼1.2 n m molecules), 10

    Article Snippet: The phosphorylated oligonucleotides were annealed into the gapped pUC19-VSR DNA as follows: annealing reaction mixtures (47 μl) contained 30 μl of gapped pUC19-VSR DNA, 30.6 pmol of phosphorylated oligonucleotide, 50 m m Tris-HCl (pH 7.5), 10 m m MgOAc, 5 m m dithiothreitol, 50 m m NaCl and were heated to 80 °C in a GeneAmp PCR System 2400 Thermocycler (Applied Biosystems).

    Techniques: Concentration Assay, Ligation

    The Vsr endonuclease incises DNA immediately 5′ to the mismatched thymidine. Vsr nicking reactions were as described under “Materials and Methods.” The Vsr endonuclease was incubated with covalently closed pUC19-VSR heteroduplex

    Journal: The Journal of Biological Chemistry

    Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *

    doi: 10.1074/jbc.M112.384321

    Figure Lengend Snippet: The Vsr endonuclease incises DNA immediately 5′ to the mismatched thymidine. Vsr nicking reactions were as described under “Materials and Methods.” The Vsr endonuclease was incubated with covalently closed pUC19-VSR heteroduplex

    Article Snippet: The phosphorylated oligonucleotides were annealed into the gapped pUC19-VSR DNA as follows: annealing reaction mixtures (47 μl) contained 30 μl of gapped pUC19-VSR DNA, 30.6 pmol of phosphorylated oligonucleotide, 50 m m Tris-HCl (pH 7.5), 10 m m MgOAc, 5 m m dithiothreitol, 50 m m NaCl and were heated to 80 °C in a GeneAmp PCR System 2400 Thermocycler (Applied Biosystems).

    Techniques: Incubation

    mRNA or ssDNA but not dsDNA transfections lead to stress granule assembly in puromycin-treated cells. ( A ) 1 μg of 2Luc mRNA synthesized in vitro was transfected in NRK cells using lipofectamine in the presence of 2.5 μg/ml puromycin (see ‘Materials and Methods’ section). Anti-HuR and anti-YB-1 labeling show the formation of stress granules 3 h after transfection. ( B ) NRK cells were transfected using lipofectamine (Lipo) with 1 μg of α-globin mRNA, M13 ssDNA, or linearized pUC19 dsDNA in the presence or absence of puromycin for 3 h. Benzonase treatment was performed before the formation of lipoplexes. The statistical analysis was obtained on three different samples. Results are mean ± SD ( n = 3). Both mRNA and ssDNA lead to a significant formation of stress granules in puromycin-treated cells. Anti-YB-1 labeling shows the formation of stress granules 3 h after transfection.

    Journal: Nucleic Acids Research

    Article Title: Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules

    doi: 10.1093/nar/gku582

    Figure Lengend Snippet: mRNA or ssDNA but not dsDNA transfections lead to stress granule assembly in puromycin-treated cells. ( A ) 1 μg of 2Luc mRNA synthesized in vitro was transfected in NRK cells using lipofectamine in the presence of 2.5 μg/ml puromycin (see ‘Materials and Methods’ section). Anti-HuR and anti-YB-1 labeling show the formation of stress granules 3 h after transfection. ( B ) NRK cells were transfected using lipofectamine (Lipo) with 1 μg of α-globin mRNA, M13 ssDNA, or linearized pUC19 dsDNA in the presence or absence of puromycin for 3 h. Benzonase treatment was performed before the formation of lipoplexes. The statistical analysis was obtained on three different samples. Results are mean ± SD ( n = 3). Both mRNA and ssDNA lead to a significant formation of stress granules in puromycin-treated cells. Anti-YB-1 labeling shows the formation of stress granules 3 h after transfection.

    Article Snippet: mRNA and ssDNA, and dsDNA transfection One microgram of mRNA (α-globin, and 2Luc mRNAs, fluorescent or not), ssDNA (M13, Invitrogen) or dsDNA (pUC19, Invitrogen) were incubated with 1 μl lipofectamine™ 2000 (Invitrogen) for 20 min in 100 μl of DMEM.

    Techniques: Transfection, Synthesized, In Vitro, Labeling

    In vivo functionality of E. coli CD (CodA, in pQE70 expression vector) and the DNA fragments that were selected from four metagenomic libraries: KANOS, URA4, URA3, and Vcz (in pUC19 vector). Empty pQE70 vector was used as a negative control. Minimal medium (M9) was supplemented 100 mg/L ampicillin, 15 mg/L kanamycin, 0.1 mM IPTG, and either with 20 mg/L cytosine (M9 + C), isocytosine (M9 + isoC) or uracil (M9 + U, positive control).

    Journal: Frontiers in Microbiology

    Article Title: Discovery of Bacterial Deaminases That Convert 5-Fluoroisocytosine Into 5-Fluorouracil

    doi: 10.3389/fmicb.2018.02375

    Figure Lengend Snippet: In vivo functionality of E. coli CD (CodA, in pQE70 expression vector) and the DNA fragments that were selected from four metagenomic libraries: KANOS, URA4, URA3, and Vcz (in pUC19 vector). Empty pQE70 vector was used as a negative control. Minimal medium (M9) was supplemented 100 mg/L ampicillin, 15 mg/L kanamycin, 0.1 mM IPTG, and either with 20 mg/L cytosine (M9 + C), isocytosine (M9 + isoC) or uracil (M9 + U, positive control).

    Article Snippet: High copy number cloning vector pUC19 (Thermo Fisher Scientific) was used for the preparation of metagenomic libraries ( ).

    Techniques: In Vivo, Expressing, Plasmid Preparation, Negative Control, Positive Control

    Bsu Ku binds circular DNA. ( A ) Left panel , Bsu Ku binding to covalently closed pUC19 plasmid. The assay was performed as described in Materials and Methods incubating the indicated amounts of Bsu Ku with 100 ng of supercoiled plasmid pUC19. Products were analysed on 0.7% agarose electrophoresis. One half of each reaction mixture was loaded directly onto the agarose gel (direct loading lanes), while the other half was being treated with Proteinase K. After proteolytic digestion, those samples were loaded in the same agarose gel (Proteinase K treatment lanes). The electrophoretical mobility of the pUC19 plasmid in each case is indicated. In lane c, DNA was incubated with 50 ng of BSA. Right panel , Bsu Ku binding to supercoiled and relaxed plasmid DNA. The assay was performed essentially as described above. ( B ) Bsu Ku AP lyase activity can act on circular substrates. The assay was performed as described in Materials and Methods, incubating either 142 nM of Bsu Ku with 100 ng of plasmid without AP sites (pcDNA3.1) or 18, 36, 72 and 142 nM of Bsu Ku with 100 ng of plasmid containing AP sites (pcDNA3.1-AP) (left panel). The absence and presence of the AP sites in pcDNA3.1 and pcDNA3.1-AP, respectively, was confirmed after digestion with h APE1. As a control of the linear form, plasmids were also digested with EcoRI. NC : nicked circles; SC : supercoiled; L : linear. The assay shown in the right panel was performed by incubating 100 ng of plasmid without (pUC19) or with (pUC19-AP) AP sites with either h APE1 or 142 nM of Bsu Ku, as indicated. Figure 5B , right panel is a composite image made from different parts of the same experiment.

    Journal: Nucleic Acids Research

    Article Title: Efficient processing of abasic sites by bacterial nonhomologous end-joining Ku proteins

    doi: 10.1093/nar/gku1029

    Figure Lengend Snippet: Bsu Ku binds circular DNA. ( A ) Left panel , Bsu Ku binding to covalently closed pUC19 plasmid. The assay was performed as described in Materials and Methods incubating the indicated amounts of Bsu Ku with 100 ng of supercoiled plasmid pUC19. Products were analysed on 0.7% agarose electrophoresis. One half of each reaction mixture was loaded directly onto the agarose gel (direct loading lanes), while the other half was being treated with Proteinase K. After proteolytic digestion, those samples were loaded in the same agarose gel (Proteinase K treatment lanes). The electrophoretical mobility of the pUC19 plasmid in each case is indicated. In lane c, DNA was incubated with 50 ng of BSA. Right panel , Bsu Ku binding to supercoiled and relaxed plasmid DNA. The assay was performed essentially as described above. ( B ) Bsu Ku AP lyase activity can act on circular substrates. The assay was performed as described in Materials and Methods, incubating either 142 nM of Bsu Ku with 100 ng of plasmid without AP sites (pcDNA3.1) or 18, 36, 72 and 142 nM of Bsu Ku with 100 ng of plasmid containing AP sites (pcDNA3.1-AP) (left panel). The absence and presence of the AP sites in pcDNA3.1 and pcDNA3.1-AP, respectively, was confirmed after digestion with h APE1. As a control of the linear form, plasmids were also digested with EcoRI. NC : nicked circles; SC : supercoiled; L : linear. The assay shown in the right panel was performed by incubating 100 ng of plasmid without (pUC19) or with (pUC19-AP) AP sites with either h APE1 or 142 nM of Bsu Ku, as indicated. Figure 5B , right panel is a composite image made from different parts of the same experiment.

    Article Snippet: Agarose gel retardation Two micrograms of supercoiled pUC19 plasmid (Fermentas) were incubated with 10 U of the nickase Nt.BStNBI to obtain nicked pUC19.

    Techniques: Binding Assay, Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis, Incubation, Activity Assay, Activated Clotting Time Assay

    Electrophoresis of HBV genotypes. M: pUC19 DNA/ Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Influence of HLA-DRB1 alleles and HBV genotypes on interferon-α therapy for chronic hepatitis B

    doi: 10.3748/wjg.v11.i30.4753

    Figure Lengend Snippet: Electrophoresis of HBV genotypes. M: pUC19 DNA/ Msp I ( Hpa II) marker 23; lane 1: genotype B (281 bp); lane 2: genotype C (122 bp); lane 3: genotype B+C; lane 4: negative control.

    Article Snippet: Taq DNA polyenzyme and dNTPs were also purchased from Shanghai Branch, Canadian Sangon Company. pUC19 DNA/MspI (HpaII) marker 23 was purchased from MBI Fermentas.

    Techniques: Electrophoresis, Marker, Negative Control

    Electrophoresis of HLA-DRB1*03,*07,*09,*12,*15 alleles. M: pUC19 DNA/ Msp I ( Hpa II) marker 23; 1: DRB1*0701/0702, 232 bp; 2: DRB1*0901, 236 bp; 3: DRB1*1201/1202, 248 bp; 4: DRB1*1501/1502, 197 bp; 5: DRB1*0301/ 0302, 151 bp.

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Influence of HLA-DRB1 alleles and HBV genotypes on interferon-α therapy for chronic hepatitis B

    doi: 10.3748/wjg.v11.i30.4753

    Figure Lengend Snippet: Electrophoresis of HLA-DRB1*03,*07,*09,*12,*15 alleles. M: pUC19 DNA/ Msp I ( Hpa II) marker 23; 1: DRB1*0701/0702, 232 bp; 2: DRB1*0901, 236 bp; 3: DRB1*1201/1202, 248 bp; 4: DRB1*1501/1502, 197 bp; 5: DRB1*0301/ 0302, 151 bp.

    Article Snippet: Taq DNA polyenzyme and dNTPs were also purchased from Shanghai Branch, Canadian Sangon Company. pUC19 DNA/MspI (HpaII) marker 23 was purchased from MBI Fermentas.

    Techniques: Electrophoresis, Marker