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  • 95
    New England Biolabs ecori
    Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of <t>Fpg-sensitive</t> sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb <t>EcoRI</t> lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).
    Ecori, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 8899 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore ecorv site
    Disruption of the FOR3 gene in F. oxysporum f. sp. nicotianae . (A) Schematic view of the FOR3 gene disruption strategy. The <t>DNA</t> inserts of p FOR3 and p for3 ::HYG are shown. The FOR3 coding region is indicated by the gray striped box and noncoding regions by the gray line. The vector sequences are indicated by the black line. Restriction sites on the cloning vector are indicated in black, and those in FOR3 are indicated in gray. The sizes of cloned DNA fragments (double-headed arrows), as well as the probes used for Southern (dotted line) or Northern (dashed line) blot analyses, are indicated. Restriction site abbreviations are as follows: E, <t>EcoRV;</t> E1, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SalI; Xb, XbaI; Xh, XhoI. (B) Southern blot of restriction-digested genomic DNA of the wild-type ( FOR3 ) and Δ for3 mutant strains. Restriction site abbreviations are as in panel A. (C) Northern blot of total RNA of the wild-type ( FOR3 ) and Δ for3 mutant strains with a FOR3 probe ( FOR3 ) and the corresponding ethidium bromide signal (RNA) shown.
    Ecorv Site, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher ecori sites
    Construction and expression analysis of Pvs25H in Pichia pastoris . (a) The 5′- and 3′-coding regions of the Pvs25 gene sequence with its predicted amino acid sequence (Ala 23 to Leu 195 ). The Pvs25 gene fused to a hexahistidine tag at its C terminus was inserted into the <t>SnaBI</t> and <t>EcoRI</t> sites of pPIC9K, and the gene was integrated into the chromosomal DNA of P. pastoris strain GS115 by homologous recombination. (b) After selection of a high-producer clone that secreted the recombinant protein, nickel-affinity chromatography-purified Pvs25H was analyzed by SDS-PAGE (lane 1). M, molecular marker. (c) The affinity-purified Pvs25H was fractionated by size exclusion chromatography from which at least five chromatographic peaks were observed. (d) Size exclusion chromatography fractions 14-26 were subjected to SDS-PAGE (15% acrylamide). Based on the apparent molecular mass of each protein band, the fractions 16, 17, 18-19, 21-22, and 22-23 were defined as the tetramer, trimer, dimer, monomer B, and monomer A, respectively. (e) Selected chromatography peaks were subjected to 5 to 20% acrylamide gradient SDS-PAGE under nonreducing or reducing (10% 2-ME and boiling) conditions. (f) Hydrophobic interaction chromatography of the Pvs25H-A and Pvs25H-B monomeric isoforms. Elution fractions (A) are eluates of 2 M NH 4 SO 4 (Pvs25H-A) and elution fractions (B) are eluates of PBS (Pvs25H-B). (g) Each isoform (M, a mixture of dimer, trimer and tetramer; B, B isoform; A, A isoform) was subjected to 15% acrylamide SDS-PAGE under various conditions, as indicated. (h) The N-terminal protein sequence of each isoform. Positively numbered amino acid residues (Ala 1 and the following) are residues of the Pvs25 protein; negatively numbered amino acids are derivatives of the pPIC9K α-factor secretion signal. The A monomer revealed a single, unique sequence, but the B monomer and the multimers showed a mixture of different N termini.
    Ecori Sites, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 215 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher ecori
    One-Step Cloning and Heterologous Expression of the <t>Conglobatin</t> Gene Cluster (A) A 41-kbp <t>XhoI-EcoRI</t> DNA fragment (black) containing the five genes congA–E is generated by XhoI and EcoRI digestion of total genomic DNA. A 5.3-kbp pSET152 fragment was obtained by PCR amplification using as template the pSET152-derived plasmid pIB139. The resulting linear vector fragment had 39- and 41-bp flanking regions, respectively, identical to the termini of the target DNA (see Experimental Procedures ). (B) Gibson assembly leads to specific cloning of the target fragment, to give the bifunctional E. coli - Streptomyces plasmid pYJ24. The deduced open reading frame functions in the fragment are given in Table S1 . (C) Heterologous expression in S. coelicolor M1154 is confirmed by HPLC-MS and comparison with authentic compound produced by S. conglobatus (see also Figure S1 ). The mass extraction of m / z 499–500 is used to display the data. The y axis scale of S. conglobatus is 20 times larger than that of pYJ24/M1154 or M1154.
    Ecori, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 9791 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher buffer ecori
    Confirmation of glpD disruption. A ) Southern blot analysis confirms the disruption of glpD . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with <t>BamHI</t> and blotted on a nylon membrane. Digoxygenein-11-dUTP labeled bb0243 probe was utilized to visualize glpD . Migration positions of DNA molecular size markers are indicated on the left. B ) glpD disruption occurred via by a double crossover insertion of flgB - aadA . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI or <t>EcoRI,</t> which would be specific for the proximal or distal bb0243 flanking chromosomal regions, respectively, and blotted to a nylon membrane. Blots were developed with a digoxygenein-11-dUTP-labeled aadA probe. A schematic diagram indicates the sizes of the fragments expected to contain flgB - aadA . Migration positions of DNA molecular size markers are indicated on the left of each panel.
    Buffer Ecori, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare ecori
    Generation and Molecular Analysis of Transgenic Alfalfa Altered in Expression of IOMT. (A) Binary vector constructs that harbor IOMT sequences in sense and antisense orientations (see Methods for details). B L , left border; B R , right border; Kan r , kanamycin resistance gene; ori, origin of replication; TEV, tobacco etch virus leader sequence. (B) <t>DNA</t> gel blot analysis of empty vector (V), untransformed control (C), and a range of IOMT sense transformants. Genomic DNA was digested with <t>EcoRI,</t> which cuts twice within the sense construct, and hybridized with an 800-bp IOMT8 probe. (C) DNA gel blot border analysis of the same DNA samples shown in (B) but after digestion of DNA with HindIII and hybridization with the T-DNA left border sequence. (D) Protein gel blot analysis of leaf extracts from a range of IOMT sense transgene-expressing, empty vector (V), and untransformed control (C) alfalfa plants. Blots were probed with anti-(alfalfa IOMT) polyclonal antiserum. (E) Enzymatic activity of IOMT in leaf tissue from untransformed (C) and empty vector–transformed (V) controls and from a range of IOMT8 sense transformants.
    Ecori, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 2966 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega ecori
    Cloning of the HBV S gene and 3'-UTR in the rapid cloning vector pCR2.1 . (A) An image of an agarose gel showing the MS-1-MS-2 PCR product. The * indicates the position of the PCR product. (B) An image of an agarose gel showing the supercoiled pCR2.1-HBsAg(S) and the * indicates the position of the plasmid. (C) An image of an agarose gel showing the <t>KpnI/EcoRI</t> digestion products of pCR2.1-HBsAg(S). The * indicates the position of the linearized plasmid and the ** indicates the position of the dropped insert. The left lane in each gel contains 1 kb DNA ladder (Promega, USA); the sizes of the respective bands is indicated on the left side of each panel.
    Ecori, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 2734 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ecori  (Roche)
    99
    Roche ecori
    The fAFLP pattern generated with <t>EcoRI-G</t> and <t>MseI-G</t> primers from different biocontrol, environmental and clinical P. agglomerans isolates . ( A ) C9-1, ( B ) CIP 82.100, ( C ) P10c, ( D ) Eh325, ( E ) EM21cb, ( F ) EM22cb, ( G ) CIP A181. Biocontrol strain P. agglomerans C9-1 showed a completely divergent fAFLP pattern with respect to the other isolates of the species. The 490-bp band which was prevalent in biocontrol and environmental isolates, but was absent from clinical isolates and from strain C9-1 is indicated by the arrow.
    Ecori, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 1104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Toyobo ecori
    The effect of transiently expressed nuclear <t>MxA</t> proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with <t>EcoRI</t> followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).
    Ecori, supplied by Toyobo, used in various techniques. Bioz Stars score: 99/100, based on 159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Jena Bioscience ecori
    The effect of transiently expressed nuclear <t>MxA</t> proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with <t>EcoRI</t> followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).
    Ecori, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 89/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Addgene inc ecori
    The effect of transiently expressed nuclear <t>MxA</t> proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with <t>EcoRI</t> followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).
    Ecori, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 1155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Stratagene ecori
    Structure and targeting strategy for the mouse Ereg gene. Four genes for the EGF family of ligands, Epgn , Ereg , Areg , and Btc , are located in chromosome 5 from Mb 90.7 to 91.0. (a) The exons of Ereg are labeled 1 to 5. The sizes of exons are given under each exon, and the sizes of introns (introns 1 to 4) are given in parentheses. The empty square at exons 1 and 5 indicates 5′ and 3′ nontranslated region, respectively. The Ereg wild-type allele, targeting vector, and targeted Ereg tm1Dwt allele are shown in b. RI, RV, and N denote <t>EcoRI,</t> EcoRV, and NdeI sites, respectively. The vector region flanking the 5′ homology arm of the targeting construct is not included in this schematic, and nls, SV40pA, and neo represent the nuclear localization signal-tagged lacZ reporter gene, a simian virus 40 polyadenylation signal, and a neomycin transphosphorylase expression cassette, respectively. The genomic <t>DNA</t> fragment between NsiI and EcoRI, represented by a solid line, was used as a probe for Southern blot hybridizations. Arrows indicate primer sets used for genotyping wild-type and targeted alleles.
    Ecori, supplied by Stratagene, used in various techniques. Bioz Stars score: 96/100, based on 2283 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Genechem ecori
    Structure and targeting strategy for the mouse Ereg gene. Four genes for the EGF family of ligands, Epgn , Ereg , Areg , and Btc , are located in chromosome 5 from Mb 90.7 to 91.0. (a) The exons of Ereg are labeled 1 to 5. The sizes of exons are given under each exon, and the sizes of introns (introns 1 to 4) are given in parentheses. The empty square at exons 1 and 5 indicates 5′ and 3′ nontranslated region, respectively. The Ereg wild-type allele, targeting vector, and targeted Ereg tm1Dwt allele are shown in b. RI, RV, and N denote <t>EcoRI,</t> EcoRV, and NdeI sites, respectively. The vector region flanking the 5′ homology arm of the targeting construct is not included in this schematic, and nls, SV40pA, and neo represent the nuclear localization signal-tagged lacZ reporter gene, a simian virus 40 polyadenylation signal, and a neomycin transphosphorylase expression cassette, respectively. The genomic <t>DNA</t> fragment between NsiI and EcoRI, represented by a solid line, was used as a probe for Southern blot hybridizations. Arrows indicate primer sets used for genotyping wild-type and targeted alleles.
    Ecori, supplied by Genechem, used in various techniques. Bioz Stars score: 99/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Sangon Biotech ecori
    Structure and targeting strategy for the mouse Ereg gene. Four genes for the EGF family of ligands, Epgn , Ereg , Areg , and Btc , are located in chromosome 5 from Mb 90.7 to 91.0. (a) The exons of Ereg are labeled 1 to 5. The sizes of exons are given under each exon, and the sizes of introns (introns 1 to 4) are given in parentheses. The empty square at exons 1 and 5 indicates 5′ and 3′ nontranslated region, respectively. The Ereg wild-type allele, targeting vector, and targeted Ereg tm1Dwt allele are shown in b. RI, RV, and N denote <t>EcoRI,</t> EcoRV, and NdeI sites, respectively. The vector region flanking the 5′ homology arm of the targeting construct is not included in this schematic, and nls, SV40pA, and neo represent the nuclear localization signal-tagged lacZ reporter gene, a simian virus 40 polyadenylation signal, and a neomycin transphosphorylase expression cassette, respectively. The genomic <t>DNA</t> fragment between NsiI and EcoRI, represented by a solid line, was used as a probe for Southern blot hybridizations. Arrows indicate primer sets used for genotyping wild-type and targeted alleles.
    Ecori, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 95/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GenScript ecori
    Structure and targeting strategy for the mouse Ereg gene. Four genes for the EGF family of ligands, Epgn , Ereg , Areg , and Btc , are located in chromosome 5 from Mb 90.7 to 91.0. (a) The exons of Ereg are labeled 1 to 5. The sizes of exons are given under each exon, and the sizes of introns (introns 1 to 4) are given in parentheses. The empty square at exons 1 and 5 indicates 5′ and 3′ nontranslated region, respectively. The Ereg wild-type allele, targeting vector, and targeted Ereg tm1Dwt allele are shown in b. RI, RV, and N denote <t>EcoRI,</t> EcoRV, and NdeI sites, respectively. The vector region flanking the 5′ homology arm of the targeting construct is not included in this schematic, and nls, SV40pA, and neo represent the nuclear localization signal-tagged lacZ reporter gene, a simian virus 40 polyadenylation signal, and a neomycin transphosphorylase expression cassette, respectively. The genomic <t>DNA</t> fragment between NsiI and EcoRI, represented by a solid line, was used as a probe for Southern blot hybridizations. Arrows indicate primer sets used for genotyping wild-type and targeted alleles.
    Ecori, supplied by GenScript, used in various techniques. Bioz Stars score: 99/100, based on 180 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    SibEnzyme ecori
    Restriction endonuclease <t>EcoRI</t> cleavage protection by 23-nt chimeric TFOs in models A , B and C . Incubation of labelled DNA duplexes in restriction buffer containing 30 mM MgCl 2 for 1 h at <t>25°C</t> alone (lane 1), with EcoRI (lane 2) and with EcoRI and a 20-fold excess of chimeric TFOs (lane 3). Electrophoresis was done with 20% polyacrylamide gel in 7 M urea and 0.1 M TBE at 25°C. Alpha nucleotides are shown in lower case italics.
    Ecori, supplied by SibEnzyme, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Agilent technologies ecori
    Restriction endonuclease <t>EcoRI</t> cleavage protection by 23-nt chimeric TFOs in models A , B and C . Incubation of labelled DNA duplexes in restriction buffer containing 30 mM MgCl 2 for 1 h at <t>25°C</t> alone (lane 1), with EcoRI (lane 2) and with EcoRI and a 20-fold excess of chimeric TFOs (lane 3). Electrophoresis was done with 20% polyacrylamide gel in 7 M urea and 0.1 M TBE at 25°C. Alpha nucleotides are shown in lower case italics.
    Ecori, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs ecori hf
    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with <t>PstI</t> and <t>EcoRI</t> resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
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    New England Biolabs eco ri mtase
    Continuous monitoring of methyltransferases. To perform the methyltransferase assay, a 100 μl reaction typically contained the following assay components: 3.5 μg purified recombinant Mtn, 1 μg XOD (10 units/mg), 0.2 μg HRP (300 units/mg), 0.1 mM Amplex Red, 5 mM MgCl 2 , 0.5 mM SAM (32 mM stock), 1 mM salicylic acid or ~14 nM lambda <t>DNA</t> (0.5 mg/ml), 1-3 μg SA <t>MTase</t> or 80 units Eco RI MTase, and 50 mM Tris-HCl (pH 7.5) or 50 mM potassium phosphate buffer (pH 7.5). For SA MTase, the mixture was additionally supplemented with 1 mM KCl to stimulate activity. Reactions were initiated either with addition of the substrate or enzyme. Fluorescence output was monitored in 96-well microplates at 590 nm with excitation set at 530 nm. The reactions were incubated at 30°C without shaking for up to 60 mins. (A) Methyltransferase activity of Eco RI MTase in the presence of lambda DNA and SAM. (B) Confirmation of lambda DNA methylation by agarose gel electrophoresis. (C) Methyltransferase activity of SA MTase in the presence of salicylic acid (SA) and SAM. (D) Confirmation of the synthesis of methyl salicylate by HPLC.
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    77
    GE Healthcare puc18 ecori
    Continuous monitoring of methyltransferases. To perform the methyltransferase assay, a 100 μl reaction typically contained the following assay components: 3.5 μg purified recombinant Mtn, 1 μg XOD (10 units/mg), 0.2 μg HRP (300 units/mg), 0.1 mM Amplex Red, 5 mM MgCl 2 , 0.5 mM SAM (32 mM stock), 1 mM salicylic acid or ~14 nM lambda <t>DNA</t> (0.5 mg/ml), 1-3 μg SA <t>MTase</t> or 80 units Eco RI MTase, and 50 mM Tris-HCl (pH 7.5) or 50 mM potassium phosphate buffer (pH 7.5). For SA MTase, the mixture was additionally supplemented with 1 mM KCl to stimulate activity. Reactions were initiated either with addition of the substrate or enzyme. Fluorescence output was monitored in 96-well microplates at 590 nm with excitation set at 530 nm. The reactions were incubated at 30°C without shaking for up to 60 mins. (A) Methyltransferase activity of Eco RI MTase in the presence of lambda DNA and SAM. (B) Confirmation of lambda DNA methylation by agarose gel electrophoresis. (C) Methyltransferase activity of SA MTase in the presence of salicylic acid (SA) and SAM. (D) Confirmation of the synthesis of methyl salicylate by HPLC.
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    81
    Millipore ecori clai
    Continuous monitoring of methyltransferases. To perform the methyltransferase assay, a 100 μl reaction typically contained the following assay components: 3.5 μg purified recombinant Mtn, 1 μg XOD (10 units/mg), 0.2 μg HRP (300 units/mg), 0.1 mM Amplex Red, 5 mM MgCl 2 , 0.5 mM SAM (32 mM stock), 1 mM salicylic acid or ~14 nM lambda <t>DNA</t> (0.5 mg/ml), 1-3 μg SA <t>MTase</t> or 80 units Eco RI MTase, and 50 mM Tris-HCl (pH 7.5) or 50 mM potassium phosphate buffer (pH 7.5). For SA MTase, the mixture was additionally supplemented with 1 mM KCl to stimulate activity. Reactions were initiated either with addition of the substrate or enzyme. Fluorescence output was monitored in 96-well microplates at 590 nm with excitation set at 530 nm. The reactions were incubated at 30°C without shaking for up to 60 mins. (A) Methyltransferase activity of Eco RI MTase in the presence of lambda DNA and SAM. (B) Confirmation of lambda DNA methylation by agarose gel electrophoresis. (C) Methyltransferase activity of SA MTase in the presence of salicylic acid (SA) and SAM. (D) Confirmation of the synthesis of methyl salicylate by HPLC.
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    79
    Roche xhoi ecori
    Continuous monitoring of methyltransferases. To perform the methyltransferase assay, a 100 μl reaction typically contained the following assay components: 3.5 μg purified recombinant Mtn, 1 μg XOD (10 units/mg), 0.2 μg HRP (300 units/mg), 0.1 mM Amplex Red, 5 mM MgCl 2 , 0.5 mM SAM (32 mM stock), 1 mM salicylic acid or ~14 nM lambda <t>DNA</t> (0.5 mg/ml), 1-3 μg SA <t>MTase</t> or 80 units Eco RI MTase, and 50 mM Tris-HCl (pH 7.5) or 50 mM potassium phosphate buffer (pH 7.5). For SA MTase, the mixture was additionally supplemented with 1 mM KCl to stimulate activity. Reactions were initiated either with addition of the substrate or enzyme. Fluorescence output was monitored in 96-well microplates at 590 nm with excitation set at 530 nm. The reactions were incubated at 30°C without shaking for up to 60 mins. (A) Methyltransferase activity of Eco RI MTase in the presence of lambda DNA and SAM. (B) Confirmation of lambda DNA methylation by agarose gel electrophoresis. (C) Methyltransferase activity of SA MTase in the presence of salicylic acid (SA) and SAM. (D) Confirmation of the synthesis of methyl salicylate by HPLC.
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    84
    TaKaRa ecori blni
    Continuous monitoring of methyltransferases. To perform the methyltransferase assay, a 100 μl reaction typically contained the following assay components: 3.5 μg purified recombinant Mtn, 1 μg XOD (10 units/mg), 0.2 μg HRP (300 units/mg), 0.1 mM Amplex Red, 5 mM MgCl 2 , 0.5 mM SAM (32 mM stock), 1 mM salicylic acid or ~14 nM lambda <t>DNA</t> (0.5 mg/ml), 1-3 μg SA <t>MTase</t> or 80 units Eco RI MTase, and 50 mM Tris-HCl (pH 7.5) or 50 mM potassium phosphate buffer (pH 7.5). For SA MTase, the mixture was additionally supplemented with 1 mM KCl to stimulate activity. Reactions were initiated either with addition of the substrate or enzyme. Fluorescence output was monitored in 96-well microplates at 590 nm with excitation set at 530 nm. The reactions were incubated at 30°C without shaking for up to 60 mins. (A) Methyltransferase activity of Eco RI MTase in the presence of lambda DNA and SAM. (B) Confirmation of lambda DNA methylation by agarose gel electrophoresis. (C) Methyltransferase activity of SA MTase in the presence of salicylic acid (SA) and SAM. (D) Confirmation of the synthesis of methyl salicylate by HPLC.
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    92
    TaKaRa ecori xhoi
    Continuous monitoring of methyltransferases. To perform the methyltransferase assay, a 100 μl reaction typically contained the following assay components: 3.5 μg purified recombinant Mtn, 1 μg XOD (10 units/mg), 0.2 μg HRP (300 units/mg), 0.1 mM Amplex Red, 5 mM MgCl 2 , 0.5 mM SAM (32 mM stock), 1 mM salicylic acid or ~14 nM lambda <t>DNA</t> (0.5 mg/ml), 1-3 μg SA <t>MTase</t> or 80 units Eco RI MTase, and 50 mM Tris-HCl (pH 7.5) or 50 mM potassium phosphate buffer (pH 7.5). For SA MTase, the mixture was additionally supplemented with 1 mM KCl to stimulate activity. Reactions were initiated either with addition of the substrate or enzyme. Fluorescence output was monitored in 96-well microplates at 590 nm with excitation set at 530 nm. The reactions were incubated at 30°C without shaking for up to 60 mins. (A) Methyltransferase activity of Eco RI MTase in the presence of lambda DNA and SAM. (B) Confirmation of lambda DNA methylation by agarose gel electrophoresis. (C) Methyltransferase activity of SA MTase in the presence of salicylic acid (SA) and SAM. (D) Confirmation of the synthesis of methyl salicylate by HPLC.
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    93
    TaKaRa ecori endonucleases
    Continuous monitoring of methyltransferases. To perform the methyltransferase assay, a 100 μl reaction typically contained the following assay components: 3.5 μg purified recombinant Mtn, 1 μg XOD (10 units/mg), 0.2 μg HRP (300 units/mg), 0.1 mM Amplex Red, 5 mM MgCl 2 , 0.5 mM SAM (32 mM stock), 1 mM salicylic acid or ~14 nM lambda <t>DNA</t> (0.5 mg/ml), 1-3 μg SA <t>MTase</t> or 80 units Eco RI MTase, and 50 mM Tris-HCl (pH 7.5) or 50 mM potassium phosphate buffer (pH 7.5). For SA MTase, the mixture was additionally supplemented with 1 mM KCl to stimulate activity. Reactions were initiated either with addition of the substrate or enzyme. Fluorescence output was monitored in 96-well microplates at 590 nm with excitation set at 530 nm. The reactions were incubated at 30°C without shaking for up to 60 mins. (A) Methyltransferase activity of Eco RI MTase in the presence of lambda DNA and SAM. (B) Confirmation of lambda DNA methylation by agarose gel electrophoresis. (C) Methyltransferase activity of SA MTase in the presence of salicylic acid (SA) and SAM. (D) Confirmation of the synthesis of methyl salicylate by HPLC.
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    Thermo Fisher ecori noti
    Continuous monitoring of methyltransferases. To perform the methyltransferase assay, a 100 μl reaction typically contained the following assay components: 3.5 μg purified recombinant Mtn, 1 μg XOD (10 units/mg), 0.2 μg HRP (300 units/mg), 0.1 mM Amplex Red, 5 mM MgCl 2 , 0.5 mM SAM (32 mM stock), 1 mM salicylic acid or ~14 nM lambda <t>DNA</t> (0.5 mg/ml), 1-3 μg SA <t>MTase</t> or 80 units Eco RI MTase, and 50 mM Tris-HCl (pH 7.5) or 50 mM potassium phosphate buffer (pH 7.5). For SA MTase, the mixture was additionally supplemented with 1 mM KCl to stimulate activity. Reactions were initiated either with addition of the substrate or enzyme. Fluorescence output was monitored in 96-well microplates at 590 nm with excitation set at 530 nm. The reactions were incubated at 30°C without shaking for up to 60 mins. (A) Methyltransferase activity of Eco RI MTase in the presence of lambda DNA and SAM. (B) Confirmation of lambda DNA methylation by agarose gel electrophoresis. (C) Methyltransferase activity of SA MTase in the presence of salicylic acid (SA) and SAM. (D) Confirmation of the synthesis of methyl salicylate by HPLC.
    Ecori Noti, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ecori xhoi
    Continuous monitoring of methyltransferases. To perform the methyltransferase assay, a 100 μl reaction typically contained the following assay components: 3.5 μg purified recombinant Mtn, 1 μg XOD (10 units/mg), 0.2 μg HRP (300 units/mg), 0.1 mM Amplex Red, 5 mM MgCl 2 , 0.5 mM SAM (32 mM stock), 1 mM salicylic acid or ~14 nM lambda <t>DNA</t> (0.5 mg/ml), 1-3 μg SA <t>MTase</t> or 80 units Eco RI MTase, and 50 mM Tris-HCl (pH 7.5) or 50 mM potassium phosphate buffer (pH 7.5). For SA MTase, the mixture was additionally supplemented with 1 mM KCl to stimulate activity. Reactions were initiated either with addition of the substrate or enzyme. Fluorescence output was monitored in 96-well microplates at 590 nm with excitation set at 530 nm. The reactions were incubated at 30°C without shaking for up to 60 mins. (A) Methyltransferase activity of Eco RI MTase in the presence of lambda DNA and SAM. (B) Confirmation of lambda DNA methylation by agarose gel electrophoresis. (C) Methyltransferase activity of SA MTase in the presence of salicylic acid (SA) and SAM. (D) Confirmation of the synthesis of methyl salicylate by HPLC.
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    Thermo Fisher hindiii ecori
    Continuous monitoring of methyltransferases. To perform the methyltransferase assay, a 100 μl reaction typically contained the following assay components: 3.5 μg purified recombinant Mtn, 1 μg XOD (10 units/mg), 0.2 μg HRP (300 units/mg), 0.1 mM Amplex Red, 5 mM MgCl 2 , 0.5 mM SAM (32 mM stock), 1 mM salicylic acid or ~14 nM lambda <t>DNA</t> (0.5 mg/ml), 1-3 μg SA <t>MTase</t> or 80 units Eco RI MTase, and 50 mM Tris-HCl (pH 7.5) or 50 mM potassium phosphate buffer (pH 7.5). For SA MTase, the mixture was additionally supplemented with 1 mM KCl to stimulate activity. Reactions were initiated either with addition of the substrate or enzyme. Fluorescence output was monitored in 96-well microplates at 590 nm with excitation set at 530 nm. The reactions were incubated at 30°C without shaking for up to 60 mins. (A) Methyltransferase activity of Eco RI MTase in the presence of lambda DNA and SAM. (B) Confirmation of lambda DNA methylation by agarose gel electrophoresis. (C) Methyltransferase activity of SA MTase in the presence of salicylic acid (SA) and SAM. (D) Confirmation of the synthesis of methyl salicylate by HPLC.
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    Image Search Results


    Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Journal: Mutagenesis

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine

    doi: 10.1093/mutage/get027

    Figure Lengend Snippet: Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Article Snippet: Prior to treatment of Ad DNA with either T4 pyrimidine-DNA glycosylase [T4pdg; New England Biolabs (NEB) M0308S] or formamidopyrimidine (Fapy)-DNA glycosylase (Fpg; NEB M02040), all samples were digested overnight by 40 units of EcoRI (NEB R0101) in a total reaction volume of 50 µl in 1× NEB buffer 1.

    Techniques: Southern Blot, Incubation, Generated

    Disruption of the FOR3 gene in F. oxysporum f. sp. nicotianae . (A) Schematic view of the FOR3 gene disruption strategy. The DNA inserts of p FOR3 and p for3 ::HYG are shown. The FOR3 coding region is indicated by the gray striped box and noncoding regions by the gray line. The vector sequences are indicated by the black line. Restriction sites on the cloning vector are indicated in black, and those in FOR3 are indicated in gray. The sizes of cloned DNA fragments (double-headed arrows), as well as the probes used for Southern (dotted line) or Northern (dashed line) blot analyses, are indicated. Restriction site abbreviations are as follows: E, EcoRV; E1, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SalI; Xb, XbaI; Xh, XhoI. (B) Southern blot of restriction-digested genomic DNA of the wild-type ( FOR3 ) and Δ for3 mutant strains. Restriction site abbreviations are as in panel A. (C) Northern blot of total RNA of the wild-type ( FOR3 ) and Δ for3 mutant strains with a FOR3 probe ( FOR3 ) and the corresponding ethidium bromide signal (RNA) shown.

    Journal: Eukaryotic Cell

    Article Title: Use of the Plant Defense Protein Osmotin To Identify Fusarium oxysporum Genes That Control Cell Wall Properties ▿ Genes That Control Cell Wall Properties ▿ ‡

    doi: 10.1128/EC.00316-09

    Figure Lengend Snippet: Disruption of the FOR3 gene in F. oxysporum f. sp. nicotianae . (A) Schematic view of the FOR3 gene disruption strategy. The DNA inserts of p FOR3 and p for3 ::HYG are shown. The FOR3 coding region is indicated by the gray striped box and noncoding regions by the gray line. The vector sequences are indicated by the black line. Restriction sites on the cloning vector are indicated in black, and those in FOR3 are indicated in gray. The sizes of cloned DNA fragments (double-headed arrows), as well as the probes used for Southern (dotted line) or Northern (dashed line) blot analyses, are indicated. Restriction site abbreviations are as follows: E, EcoRV; E1, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SalI; Xb, XbaI; Xh, XhoI. (B) Southern blot of restriction-digested genomic DNA of the wild-type ( FOR3 ) and Δ for3 mutant strains. Restriction site abbreviations are as in panel A. (C) Northern blot of total RNA of the wild-type ( FOR3 ) and Δ for3 mutant strains with a FOR3 probe ( FOR3 ) and the corresponding ethidium bromide signal (RNA) shown.

    Article Snippet: To construct p for3 ::HYG, a 0.7-kb fragment containing the 3′ part of the FOR3 ORF (+3205 bp to +3941 bp) was amplified from F. oxysporum genomic DNA by PCR using the primer pair 5′-ATCGAGTCTTGCCGACGAT-3′/5′-TGAGATCCGTCTTCAGGATC-3′ and inserted into the EcoRV site of the pSTblue-1 vector (Novagen, Madison, WI).

    Techniques: Plasmid Preparation, Clone Assay, Northern Blot, Southern Blot, Mutagenesis

    Construction and expression analysis of Pvs25H in Pichia pastoris . (a) The 5′- and 3′-coding regions of the Pvs25 gene sequence with its predicted amino acid sequence (Ala 23 to Leu 195 ). The Pvs25 gene fused to a hexahistidine tag at its C terminus was inserted into the SnaBI and EcoRI sites of pPIC9K, and the gene was integrated into the chromosomal DNA of P. pastoris strain GS115 by homologous recombination. (b) After selection of a high-producer clone that secreted the recombinant protein, nickel-affinity chromatography-purified Pvs25H was analyzed by SDS-PAGE (lane 1). M, molecular marker. (c) The affinity-purified Pvs25H was fractionated by size exclusion chromatography from which at least five chromatographic peaks were observed. (d) Size exclusion chromatography fractions 14-26 were subjected to SDS-PAGE (15% acrylamide). Based on the apparent molecular mass of each protein band, the fractions 16, 17, 18-19, 21-22, and 22-23 were defined as the tetramer, trimer, dimer, monomer B, and monomer A, respectively. (e) Selected chromatography peaks were subjected to 5 to 20% acrylamide gradient SDS-PAGE under nonreducing or reducing (10% 2-ME and boiling) conditions. (f) Hydrophobic interaction chromatography of the Pvs25H-A and Pvs25H-B monomeric isoforms. Elution fractions (A) are eluates of 2 M NH 4 SO 4 (Pvs25H-A) and elution fractions (B) are eluates of PBS (Pvs25H-B). (g) Each isoform (M, a mixture of dimer, trimer and tetramer; B, B isoform; A, A isoform) was subjected to 15% acrylamide SDS-PAGE under various conditions, as indicated. (h) The N-terminal protein sequence of each isoform. Positively numbered amino acid residues (Ala 1 and the following) are residues of the Pvs25 protein; negatively numbered amino acids are derivatives of the pPIC9K α-factor secretion signal. The A monomer revealed a single, unique sequence, but the B monomer and the multimers showed a mixture of different N termini.

    Journal: Infection and Immunity

    Article Title: Plasmodium vivax Ookinete Surface Protein Pvs25 Linked to Cholera Toxin B Subunit Induces Potent Transmission-Blocking Immunity by Intranasal as Well as Subcutaneous Immunization ▿

    doi: 10.1128/IAI.00306-10

    Figure Lengend Snippet: Construction and expression analysis of Pvs25H in Pichia pastoris . (a) The 5′- and 3′-coding regions of the Pvs25 gene sequence with its predicted amino acid sequence (Ala 23 to Leu 195 ). The Pvs25 gene fused to a hexahistidine tag at its C terminus was inserted into the SnaBI and EcoRI sites of pPIC9K, and the gene was integrated into the chromosomal DNA of P. pastoris strain GS115 by homologous recombination. (b) After selection of a high-producer clone that secreted the recombinant protein, nickel-affinity chromatography-purified Pvs25H was analyzed by SDS-PAGE (lane 1). M, molecular marker. (c) The affinity-purified Pvs25H was fractionated by size exclusion chromatography from which at least five chromatographic peaks were observed. (d) Size exclusion chromatography fractions 14-26 were subjected to SDS-PAGE (15% acrylamide). Based on the apparent molecular mass of each protein band, the fractions 16, 17, 18-19, 21-22, and 22-23 were defined as the tetramer, trimer, dimer, monomer B, and monomer A, respectively. (e) Selected chromatography peaks were subjected to 5 to 20% acrylamide gradient SDS-PAGE under nonreducing or reducing (10% 2-ME and boiling) conditions. (f) Hydrophobic interaction chromatography of the Pvs25H-A and Pvs25H-B monomeric isoforms. Elution fractions (A) are eluates of 2 M NH 4 SO 4 (Pvs25H-A) and elution fractions (B) are eluates of PBS (Pvs25H-B). (g) Each isoform (M, a mixture of dimer, trimer and tetramer; B, B isoform; A, A isoform) was subjected to 15% acrylamide SDS-PAGE under various conditions, as indicated. (h) The N-terminal protein sequence of each isoform. Positively numbered amino acid residues (Ala 1 and the following) are residues of the Pvs25 protein; negatively numbered amino acids are derivatives of the pPIC9K α-factor secretion signal. The A monomer revealed a single, unique sequence, but the B monomer and the multimers showed a mixture of different N termini.

    Article Snippet: The fragment was subcloned into the SnaBI and EcoRI sites of the P. pastoris expression vector pPIC9K (Life Technologies, Carlsbad, CA) to construct the plasmid pPvs25H, which was designed to express an α-factor signal-Pvs25-hexahistidine fusion protein (see Fig. ).

    Techniques: Expressing, Sequencing, Homologous Recombination, Selection, Recombinant, Affinity Chromatography, Purification, SDS Page, Marker, Affinity Purification, Size-exclusion Chromatography, Chromatography, Hydrophobic Interaction Chromatography

    Analysis of DNA fractions obtained by SRD and MND treatment of sperm nuclei. All DNA samples were resolved on 1.8% agarose gels. (Lane 1 ) A 0.4–10-kb DNA ladder. (Lane 2 ) Total (unfractionated) DNA (NF). (Lane 3 ) The soluble DNA released after extraction of sperm nuclei with 0.65 M NaCl followed by digestion with BamHI and EcoRI (SRDS). (Lane 5 ) The corresponding insoluble pellet (SRDI). (Lane 4 ) DNA released from sperm after MNase digestion (MNDS). (Lane 6 ) The corresponding insoluble pellet (MNDI).

    Journal: Genome Research

    Article Title: Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences

    doi: 10.1101/gr.094953.109

    Figure Lengend Snippet: Analysis of DNA fractions obtained by SRD and MND treatment of sperm nuclei. All DNA samples were resolved on 1.8% agarose gels. (Lane 1 ) A 0.4–10-kb DNA ladder. (Lane 2 ) Total (unfractionated) DNA (NF). (Lane 3 ) The soluble DNA released after extraction of sperm nuclei with 0.65 M NaCl followed by digestion with BamHI and EcoRI (SRDS). (Lane 5 ) The corresponding insoluble pellet (SRDI). (Lane 4 ) DNA released from sperm after MNase digestion (MNDS). (Lane 6 ) The corresponding insoluble pellet (MNDI).

    Article Snippet: The pellets containing ∼5 × 108 nuclei were then resuspended in BamHI buffer and digested with 2000 U each of EcoRI and BamHI (Invitrogen) for 90 min at 37°C with gentle agitation, allowing digested DNA to leach out of the nuclei.

    Techniques:

    One-Step Cloning and Heterologous Expression of the Conglobatin Gene Cluster (A) A 41-kbp XhoI-EcoRI DNA fragment (black) containing the five genes congA–E is generated by XhoI and EcoRI digestion of total genomic DNA. A 5.3-kbp pSET152 fragment was obtained by PCR amplification using as template the pSET152-derived plasmid pIB139. The resulting linear vector fragment had 39- and 41-bp flanking regions, respectively, identical to the termini of the target DNA (see Experimental Procedures ). (B) Gibson assembly leads to specific cloning of the target fragment, to give the bifunctional E. coli - Streptomyces plasmid pYJ24. The deduced open reading frame functions in the fragment are given in Table S1 . (C) Heterologous expression in S. coelicolor M1154 is confirmed by HPLC-MS and comparison with authentic compound produced by S. conglobatus (see also Figure S1 ). The mass extraction of m / z 499–500 is used to display the data. The y axis scale of S. conglobatus is 20 times larger than that of pYJ24/M1154 or M1154.

    Journal: Chemistry & Biology

    Article Title: Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin

    doi: 10.1016/j.chembiol.2015.05.010

    Figure Lengend Snippet: One-Step Cloning and Heterologous Expression of the Conglobatin Gene Cluster (A) A 41-kbp XhoI-EcoRI DNA fragment (black) containing the five genes congA–E is generated by XhoI and EcoRI digestion of total genomic DNA. A 5.3-kbp pSET152 fragment was obtained by PCR amplification using as template the pSET152-derived plasmid pIB139. The resulting linear vector fragment had 39- and 41-bp flanking regions, respectively, identical to the termini of the target DNA (see Experimental Procedures ). (B) Gibson assembly leads to specific cloning of the target fragment, to give the bifunctional E. coli - Streptomyces plasmid pYJ24. The deduced open reading frame functions in the fragment are given in Table S1 . (C) Heterologous expression in S. coelicolor M1154 is confirmed by HPLC-MS and comparison with authentic compound produced by S. conglobatus (see also Figure S1 ). The mass extraction of m / z 499–500 is used to display the data. The y axis scale of S. conglobatus is 20 times larger than that of pYJ24/M1154 or M1154.

    Article Snippet: Single-Step Cloning of the Conglobatin Gene Cluster S. conglobatus genomic DNA was digested with XhoI and EcoRI (FastDigest, Thermo Scientific) at 37°C for 3.5 hr.

    Techniques: Clone Assay, Expressing, Generated, Polymerase Chain Reaction, Amplification, Derivative Assay, Plasmid Preparation, High Performance Liquid Chromatography, Mass Spectrometry, Produced

    Confirmation of glpD disruption. A ) Southern blot analysis confirms the disruption of glpD . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI and blotted on a nylon membrane. Digoxygenein-11-dUTP labeled bb0243 probe was utilized to visualize glpD . Migration positions of DNA molecular size markers are indicated on the left. B ) glpD disruption occurred via by a double crossover insertion of flgB - aadA . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI or EcoRI, which would be specific for the proximal or distal bb0243 flanking chromosomal regions, respectively, and blotted to a nylon membrane. Blots were developed with a digoxygenein-11-dUTP-labeled aadA probe. A schematic diagram indicates the sizes of the fragments expected to contain flgB - aadA . Migration positions of DNA molecular size markers are indicated on the left of each panel.

    Journal: PLoS Pathogens

    Article Title: Borrelia burgdorferi Requires Glycerol for Maximum Fitness During The Tick Phase of the Enzootic Cycle

    doi: 10.1371/journal.ppat.1002102

    Figure Lengend Snippet: Confirmation of glpD disruption. A ) Southern blot analysis confirms the disruption of glpD . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI and blotted on a nylon membrane. Digoxygenein-11-dUTP labeled bb0243 probe was utilized to visualize glpD . Migration positions of DNA molecular size markers are indicated on the left. B ) glpD disruption occurred via by a double crossover insertion of flgB - aadA . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI or EcoRI, which would be specific for the proximal or distal bb0243 flanking chromosomal regions, respectively, and blotted to a nylon membrane. Blots were developed with a digoxygenein-11-dUTP-labeled aadA probe. A schematic diagram indicates the sizes of the fragments expected to contain flgB - aadA . Migration positions of DNA molecular size markers are indicated on the left of each panel.

    Article Snippet: B. burgdorferi DNA was fragmented by incubation with 4.5 units of BamHI in 1× buffer B (Fermentas, Glen Burnie, MD) or EcoRI in buffer EcoRI (Fermentas) overnight at 37°C.

    Techniques: Southern Blot, Labeling, Migration

    Generation and Molecular Analysis of Transgenic Alfalfa Altered in Expression of IOMT. (A) Binary vector constructs that harbor IOMT sequences in sense and antisense orientations (see Methods for details). B L , left border; B R , right border; Kan r , kanamycin resistance gene; ori, origin of replication; TEV, tobacco etch virus leader sequence. (B) DNA gel blot analysis of empty vector (V), untransformed control (C), and a range of IOMT sense transformants. Genomic DNA was digested with EcoRI, which cuts twice within the sense construct, and hybridized with an 800-bp IOMT8 probe. (C) DNA gel blot border analysis of the same DNA samples shown in (B) but after digestion of DNA with HindIII and hybridization with the T-DNA left border sequence. (D) Protein gel blot analysis of leaf extracts from a range of IOMT sense transgene-expressing, empty vector (V), and untransformed control (C) alfalfa plants. Blots were probed with anti-(alfalfa IOMT) polyclonal antiserum. (E) Enzymatic activity of IOMT in leaf tissue from untransformed (C) and empty vector–transformed (V) controls and from a range of IOMT8 sense transformants.

    Journal: The Plant Cell

    Article Title: Genetic Manipulation of Isoflavone 7-O-Methyltransferase Enhances Biosynthesis of 4?-O-Methylated Isoflavonoid Phytoalexins and Disease Resistance in Alfalfa

    doi:

    Figure Lengend Snippet: Generation and Molecular Analysis of Transgenic Alfalfa Altered in Expression of IOMT. (A) Binary vector constructs that harbor IOMT sequences in sense and antisense orientations (see Methods for details). B L , left border; B R , right border; Kan r , kanamycin resistance gene; ori, origin of replication; TEV, tobacco etch virus leader sequence. (B) DNA gel blot analysis of empty vector (V), untransformed control (C), and a range of IOMT sense transformants. Genomic DNA was digested with EcoRI, which cuts twice within the sense construct, and hybridized with an 800-bp IOMT8 probe. (C) DNA gel blot border analysis of the same DNA samples shown in (B) but after digestion of DNA with HindIII and hybridization with the T-DNA left border sequence. (D) Protein gel blot analysis of leaf extracts from a range of IOMT sense transgene-expressing, empty vector (V), and untransformed control (C) alfalfa plants. Blots were probed with anti-(alfalfa IOMT) polyclonal antiserum. (E) Enzymatic activity of IOMT in leaf tissue from untransformed (C) and empty vector–transformed (V) controls and from a range of IOMT8 sense transformants.

    Article Snippet: Genomic DNA was isolated from leaf tissues with a Nucleon Phytopure plant DNA extraction kit (Amersham, Piscataway, NJ), digested with EcoRI, subjected to electrophoresis through an 0.8% agarose gel, and transferred to a Hybond-N membrane (Amersham) by capillary blotting.

    Techniques: Transgenic Assay, Expressing, Plasmid Preparation, Construct, Sequencing, Western Blot, Hybridization, Activity Assay, Transformation Assay

    Cloning of the HBV S gene and 3'-UTR in the rapid cloning vector pCR2.1 . (A) An image of an agarose gel showing the MS-1-MS-2 PCR product. The * indicates the position of the PCR product. (B) An image of an agarose gel showing the supercoiled pCR2.1-HBsAg(S) and the * indicates the position of the plasmid. (C) An image of an agarose gel showing the KpnI/EcoRI digestion products of pCR2.1-HBsAg(S). The * indicates the position of the linearized plasmid and the ** indicates the position of the dropped insert. The left lane in each gel contains 1 kb DNA ladder (Promega, USA); the sizes of the respective bands is indicated on the left side of each panel.

    Journal: BMC Research Notes

    Article Title: Expression and purification of hepatitis B surface antigen S from Escherichia coli; a new simple method

    doi: 10.1186/1756-0500-5-125

    Figure Lengend Snippet: Cloning of the HBV S gene and 3'-UTR in the rapid cloning vector pCR2.1 . (A) An image of an agarose gel showing the MS-1-MS-2 PCR product. The * indicates the position of the PCR product. (B) An image of an agarose gel showing the supercoiled pCR2.1-HBsAg(S) and the * indicates the position of the plasmid. (C) An image of an agarose gel showing the KpnI/EcoRI digestion products of pCR2.1-HBsAg(S). The * indicates the position of the linearized plasmid and the ** indicates the position of the dropped insert. The left lane in each gel contains 1 kb DNA ladder (Promega, USA); the sizes of the respective bands is indicated on the left side of each panel.

    Article Snippet: Sub-cloning of the S gene and the HBV-3 ' -UTR in pRP265 The insert cloned in pCR2.1-HBsAg(S) as described above was digested out using the restriction enzymes KpnI and EcoRI (Promega, USA), gel purified, and ligated into the expression vector pRP265 that was restricted using the same enzymes, gel purified and treated with shrimp alkaline phosphatase (Promega, USA).

    Techniques: Clone Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Mass Spectrometry, Polymerase Chain Reaction

    Sub-cloning of the HBV S gene and 3'-UTR in the expression vector pRP265 . (A) An image of an agarose gel showing the supercoiled pRP265 and the * indicates the position of the plasmid. (B) An image of an agarose gel showing the KpnI/EcoRI digestion products of pRP265 and the * indicates the position of the linearized plasmid. (C) An image of an agarose gel showing the supercoiled pRP-HBsAg(S)-GST and the * indicates the position of the plasmid. (D) An image of an agarose gel showing the KpnI/EcoRI digestion products of pRP-HBsAg(S)-GST. The * indicates the position of the linearized plasmid and the ** indicates the position of the dropped insert. The left lane in each gel contains 1 kb DNA ladder (Promega, USA); the sizes of the respective bands is indicated on the left side of each panel.

    Journal: BMC Research Notes

    Article Title: Expression and purification of hepatitis B surface antigen S from Escherichia coli; a new simple method

    doi: 10.1186/1756-0500-5-125

    Figure Lengend Snippet: Sub-cloning of the HBV S gene and 3'-UTR in the expression vector pRP265 . (A) An image of an agarose gel showing the supercoiled pRP265 and the * indicates the position of the plasmid. (B) An image of an agarose gel showing the KpnI/EcoRI digestion products of pRP265 and the * indicates the position of the linearized plasmid. (C) An image of an agarose gel showing the supercoiled pRP-HBsAg(S)-GST and the * indicates the position of the plasmid. (D) An image of an agarose gel showing the KpnI/EcoRI digestion products of pRP-HBsAg(S)-GST. The * indicates the position of the linearized plasmid and the ** indicates the position of the dropped insert. The left lane in each gel contains 1 kb DNA ladder (Promega, USA); the sizes of the respective bands is indicated on the left side of each panel.

    Article Snippet: Sub-cloning of the S gene and the HBV-3 ' -UTR in pRP265 The insert cloned in pCR2.1-HBsAg(S) as described above was digested out using the restriction enzymes KpnI and EcoRI (Promega, USA), gel purified, and ligated into the expression vector pRP265 that was restricted using the same enzymes, gel purified and treated with shrimp alkaline phosphatase (Promega, USA).

    Techniques: Subcloning, Expressing, Plasmid Preparation, Agarose Gel Electrophoresis

    The fAFLP pattern generated with EcoRI-G and MseI-G primers from different biocontrol, environmental and clinical P. agglomerans isolates . ( A ) C9-1, ( B ) CIP 82.100, ( C ) P10c, ( D ) Eh325, ( E ) EM21cb, ( F ) EM22cb, ( G ) CIP A181. Biocontrol strain P. agglomerans C9-1 showed a completely divergent fAFLP pattern with respect to the other isolates of the species. The 490-bp band which was prevalent in biocontrol and environmental isolates, but was absent from clinical isolates and from strain C9-1 is indicated by the arrow.

    Journal: BMC Microbiology

    Article Title: Genotypic comparison of Pantoea agglomerans plant and clinical strains

    doi: 10.1186/1471-2180-9-204

    Figure Lengend Snippet: The fAFLP pattern generated with EcoRI-G and MseI-G primers from different biocontrol, environmental and clinical P. agglomerans isolates . ( A ) C9-1, ( B ) CIP 82.100, ( C ) P10c, ( D ) Eh325, ( E ) EM21cb, ( F ) EM22cb, ( G ) CIP A181. Biocontrol strain P. agglomerans C9-1 showed a completely divergent fAFLP pattern with respect to the other isolates of the species. The 490-bp band which was prevalent in biocontrol and environmental isolates, but was absent from clinical isolates and from strain C9-1 is indicated by the arrow.

    Article Snippet: Between 200-400 ng genomic DNA from each of the strains was used for each reaction in a mix containing 5 units EcoRI (Roche, Basel, Switzerland), 1 unit of MseI (Roche) and 1 unit of T4 DNA Ligase (Epicentre, Madison, U.S.A.), 5 mM 1,4-Dithio-DL-threitol (DTT) (Sigma-Aldrich, Buchs, Switzerland), 200 μM ATP (Fermentas, St. Leon-Rot, Germany), 50 μg/ml Bovine Serum Albumin (BSA), 0.25 μM of each EcoRI adaptor (EcoRI-F 5'-CTCGTAGACTGCGTACC-3', EcoRI-R 5'-AATTGGTACGCAGTCTAC-3') and 2.5 μM of each MseI adaptor (MseI-F 5'-GACGATGAGTCCTGAG-3', MseI-R 5'-TACTCAGGACTCAT-3') in a total volume of 11.1 μl of 1× One-Phor-All Buffer PLUS (GE Healthcare, Otelfingen, Switzerland).

    Techniques: Generated

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkh192

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

    Article Snippet: The purified MxA fragment was cloned into pVP16 that had been digested with EcoRI (TOYOBO) and blunted with Klenow fragment followed by BamHI digestion.

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

    Rescue of the inhibitory activity of MxA proteins by over- expression of influenza RNA polymerase subunits and NP. ( A ) Effect of over-expression of viral RNA polymerase subunits. Swiss3T3 cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP (0.1 µg), pVP16-MxA (0 or 0.1 µg indicated – or +, respectively), and three RNA polymerase subunits and NP (0.05 µg each). The additional amount (0.3 µg) of a plasmid encoding either PB1, PB2 or PA was added as indicated. The luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Effect of over-expression of NP. Swiss3T3 cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP (0.1 µg), pVP16-MxA (0, 0.1 or 0.5 µg), and three RNA polymerase subunits (0.05 µg each) and NP (0.15 µg). The additional amounts of the plasmid encoding NP were added as indicated. The luciferase activity was determined and shown as described in Materials and Methods. The result is shown as the average of two independent experiments. ( C ) Titration of plasmid encoding NP. Swiss3T3 cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP (0.1 µg), three RNA polymerase subunits (0.05 µg each) and NP (0.1, 0.3 or 0.5 µg) in the absence or presence of pVP16-MxA. The relative luciferase activity in the presence of MxA (0.1 µg, open circle; 0.3 µg, closed circle) was normalized with that in the absence of MxA, and plotted as a function of increasing amounts of NP. The result is shown as the average of two independent experiments. ( D ) Titration of VP16-MxA, and C-terminal (NPΔC) and N-terminal (NPΔN) deletion mutants of NP. The same procedure for (B) was carried out with mutant NPs. For expression of NP proteins lacking its C-terminal region (NPΔC) corresponding amino acid positions between 182 and 498 (where amino acid position 1 is set the first amino acid of the wild-type NP) and its N-terminal region (NPΔN) corresponding amino acid positions between 1 and 190, pCAGGS-NPΔC and pCAGGS-NPΔN plasmids were constructed as follows: portions of NP fragments were amplified by PCR with pCAGGS-NP as a template and a set of primers, 5′-TTGAATTCGCCACCATGGCGACCAAAGGCACC-3′ and 5′-TTGAATT CTTAAGCACCTGCGGCCCCAGACC-3′ for NPΔC and a set of primers, 5′-TTGAATTCGCCACCATGGAATTGATCAGAATG-3′ and 5′-GGGA ATTCTTAATTGTCGTACTCCTCTGCA-3′ for NPΔN. Synthesized NP fragments were digested with EcoRI and cloned into pCHA that had been digested with the same restriction enzyme. The additional amounts of the plasmid encoding deletion mutants of NP were transfected as indicated. The result is shown as the average of two independent experiments.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkh192

    Figure Lengend Snippet: Rescue of the inhibitory activity of MxA proteins by over- expression of influenza RNA polymerase subunits and NP. ( A ) Effect of over-expression of viral RNA polymerase subunits. Swiss3T3 cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP (0.1 µg), pVP16-MxA (0 or 0.1 µg indicated – or +, respectively), and three RNA polymerase subunits and NP (0.05 µg each). The additional amount (0.3 µg) of a plasmid encoding either PB1, PB2 or PA was added as indicated. The luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Effect of over-expression of NP. Swiss3T3 cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP (0.1 µg), pVP16-MxA (0, 0.1 or 0.5 µg), and three RNA polymerase subunits (0.05 µg each) and NP (0.15 µg). The additional amounts of the plasmid encoding NP were added as indicated. The luciferase activity was determined and shown as described in Materials and Methods. The result is shown as the average of two independent experiments. ( C ) Titration of plasmid encoding NP. Swiss3T3 cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP (0.1 µg), three RNA polymerase subunits (0.05 µg each) and NP (0.1, 0.3 or 0.5 µg) in the absence or presence of pVP16-MxA. The relative luciferase activity in the presence of MxA (0.1 µg, open circle; 0.3 µg, closed circle) was normalized with that in the absence of MxA, and plotted as a function of increasing amounts of NP. The result is shown as the average of two independent experiments. ( D ) Titration of VP16-MxA, and C-terminal (NPΔC) and N-terminal (NPΔN) deletion mutants of NP. The same procedure for (B) was carried out with mutant NPs. For expression of NP proteins lacking its C-terminal region (NPΔC) corresponding amino acid positions between 182 and 498 (where amino acid position 1 is set the first amino acid of the wild-type NP) and its N-terminal region (NPΔN) corresponding amino acid positions between 1 and 190, pCAGGS-NPΔC and pCAGGS-NPΔN plasmids were constructed as follows: portions of NP fragments were amplified by PCR with pCAGGS-NP as a template and a set of primers, 5′-TTGAATTCGCCACCATGGCGACCAAAGGCACC-3′ and 5′-TTGAATT CTTAAGCACCTGCGGCCCCAGACC-3′ for NPΔC and a set of primers, 5′-TTGAATTCGCCACCATGGAATTGATCAGAATG-3′ and 5′-GGGA ATTCTTAATTGTCGTACTCCTCTGCA-3′ for NPΔN. Synthesized NP fragments were digested with EcoRI and cloned into pCHA that had been digested with the same restriction enzyme. The additional amounts of the plasmid encoding deletion mutants of NP were transfected as indicated. The result is shown as the average of two independent experiments.

    Article Snippet: The purified MxA fragment was cloned into pVP16 that had been digested with EcoRI (TOYOBO) and blunted with Klenow fragment followed by BamHI digestion.

    Techniques: Activity Assay, Over Expression, Transfection, Plasmid Preparation, Luciferase, Standard Deviation, Titration, Mutagenesis, Expressing, Construct, Amplification, Polymerase Chain Reaction, Synthesized, Clone Assay

    Structure and targeting strategy for the mouse Ereg gene. Four genes for the EGF family of ligands, Epgn , Ereg , Areg , and Btc , are located in chromosome 5 from Mb 90.7 to 91.0. (a) The exons of Ereg are labeled 1 to 5. The sizes of exons are given under each exon, and the sizes of introns (introns 1 to 4) are given in parentheses. The empty square at exons 1 and 5 indicates 5′ and 3′ nontranslated region, respectively. The Ereg wild-type allele, targeting vector, and targeted Ereg tm1Dwt allele are shown in b. RI, RV, and N denote EcoRI, EcoRV, and NdeI sites, respectively. The vector region flanking the 5′ homology arm of the targeting construct is not included in this schematic, and nls, SV40pA, and neo represent the nuclear localization signal-tagged lacZ reporter gene, a simian virus 40 polyadenylation signal, and a neomycin transphosphorylase expression cassette, respectively. The genomic DNA fragment between NsiI and EcoRI, represented by a solid line, was used as a probe for Southern blot hybridizations. Arrows indicate primer sets used for genotyping wild-type and targeted alleles.

    Journal: Molecular and Cellular Biology

    Article Title: Epiregulin Is Not Essential for Development of Intestinal Tumors but Is Required for Protection from Intestinal Damage

    doi: 10.1128/MCB.24.20.8907-8916.2004

    Figure Lengend Snippet: Structure and targeting strategy for the mouse Ereg gene. Four genes for the EGF family of ligands, Epgn , Ereg , Areg , and Btc , are located in chromosome 5 from Mb 90.7 to 91.0. (a) The exons of Ereg are labeled 1 to 5. The sizes of exons are given under each exon, and the sizes of introns (introns 1 to 4) are given in parentheses. The empty square at exons 1 and 5 indicates 5′ and 3′ nontranslated region, respectively. The Ereg wild-type allele, targeting vector, and targeted Ereg tm1Dwt allele are shown in b. RI, RV, and N denote EcoRI, EcoRV, and NdeI sites, respectively. The vector region flanking the 5′ homology arm of the targeting construct is not included in this schematic, and nls, SV40pA, and neo represent the nuclear localization signal-tagged lacZ reporter gene, a simian virus 40 polyadenylation signal, and a neomycin transphosphorylase expression cassette, respectively. The genomic DNA fragment between NsiI and EcoRI, represented by a solid line, was used as a probe for Southern blot hybridizations. Arrows indicate primer sets used for genotyping wild-type and targeted alleles.

    Article Snippet: Genomic DNA from BAC clone 96A12 was digested with EcoRI, and an approximately 9-kb fragment containing exon 1 of Ereg was subcloned into the pBSII vector (Stratagene) as follows.

    Techniques: Labeling, Plasmid Preparation, Construct, Expressing, Southern Blot

    Restriction endonuclease EcoRI cleavage protection by 23-nt chimeric TFOs in models A , B and C . Incubation of labelled DNA duplexes in restriction buffer containing 30 mM MgCl 2 for 1 h at 25°C alone (lane 1), with EcoRI (lane 2) and with EcoRI and a 20-fold excess of chimeric TFOs (lane 3). Electrophoresis was done with 20% polyacrylamide gel in 7 M urea and 0.1 M TBE at 25°C. Alpha nucleotides are shown in lower case italics.

    Journal: Nucleic Acids Research

    Article Title: Targeting duplex DNA with chimeric ?,?-triplex-forming oligonucleotides

    doi: 10.1093/nar/gks410

    Figure Lengend Snippet: Restriction endonuclease EcoRI cleavage protection by 23-nt chimeric TFOs in models A , B and C . Incubation of labelled DNA duplexes in restriction buffer containing 30 mM MgCl 2 for 1 h at 25°C alone (lane 1), with EcoRI (lane 2) and with EcoRI and a 20-fold excess of chimeric TFOs (lane 3). Electrophoresis was done with 20% polyacrylamide gel in 7 M urea and 0.1 M TBE at 25°C. Alpha nucleotides are shown in lower case italics.

    Article Snippet: EcoRI cleavage protection assay The inhibition of EcoRI duplex cleavage by chimeric TFOs was performed by incubation of labelled DNA duplexes in restriction buffer containing 30 mM MgCl2 for 1 h at 25°C with 30 units of EcoRI (SibEnzyme) in the presence of a 20-fold excess of chimeric TFOs.

    Techniques: Incubation, Electrophoresis

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Journal: PLoS ONE

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    doi: 10.1371/journal.pone.0189892

    Figure Lengend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Article Snippet: Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel).

    Techniques: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    Continuous monitoring of methyltransferases. To perform the methyltransferase assay, a 100 μl reaction typically contained the following assay components: 3.5 μg purified recombinant Mtn, 1 μg XOD (10 units/mg), 0.2 μg HRP (300 units/mg), 0.1 mM Amplex Red, 5 mM MgCl 2 , 0.5 mM SAM (32 mM stock), 1 mM salicylic acid or ~14 nM lambda DNA (0.5 mg/ml), 1-3 μg SA MTase or 80 units Eco RI MTase, and 50 mM Tris-HCl (pH 7.5) or 50 mM potassium phosphate buffer (pH 7.5). For SA MTase, the mixture was additionally supplemented with 1 mM KCl to stimulate activity. Reactions were initiated either with addition of the substrate or enzyme. Fluorescence output was monitored in 96-well microplates at 590 nm with excitation set at 530 nm. The reactions were incubated at 30°C without shaking for up to 60 mins. (A) Methyltransferase activity of Eco RI MTase in the presence of lambda DNA and SAM. (B) Confirmation of lambda DNA methylation by agarose gel electrophoresis. (C) Methyltransferase activity of SA MTase in the presence of salicylic acid (SA) and SAM. (D) Confirmation of the synthesis of methyl salicylate by HPLC.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Hydrogen Peroxide-Based Fluorometric Assay for Real-Time Monitoring of SAM-Dependent Methyltransferases

    doi: 10.3389/fbioe.2018.00146

    Figure Lengend Snippet: Continuous monitoring of methyltransferases. To perform the methyltransferase assay, a 100 μl reaction typically contained the following assay components: 3.5 μg purified recombinant Mtn, 1 μg XOD (10 units/mg), 0.2 μg HRP (300 units/mg), 0.1 mM Amplex Red, 5 mM MgCl 2 , 0.5 mM SAM (32 mM stock), 1 mM salicylic acid or ~14 nM lambda DNA (0.5 mg/ml), 1-3 μg SA MTase or 80 units Eco RI MTase, and 50 mM Tris-HCl (pH 7.5) or 50 mM potassium phosphate buffer (pH 7.5). For SA MTase, the mixture was additionally supplemented with 1 mM KCl to stimulate activity. Reactions were initiated either with addition of the substrate or enzyme. Fluorescence output was monitored in 96-well microplates at 590 nm with excitation set at 530 nm. The reactions were incubated at 30°C without shaking for up to 60 mins. (A) Methyltransferase activity of Eco RI MTase in the presence of lambda DNA and SAM. (B) Confirmation of lambda DNA methylation by agarose gel electrophoresis. (C) Methyltransferase activity of SA MTase in the presence of salicylic acid (SA) and SAM. (D) Confirmation of the synthesis of methyl salicylate by HPLC.

    Article Snippet: During the assay, a linear increase in fluorescence intensity was observed upon addition of Eco RI MTAse with lambda DNA as substrate at a concentration of 96 nM (Figure ).

    Techniques: Purification, Recombinant, Lambda DNA Preparation, Activity Assay, Fluorescence, Incubation, Methylation, Agarose Gel Electrophoresis, High Performance Liquid Chromatography