plasmid dna  (Bio-Rad)

 
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    Bio-Rad plasmid dna
    Detection of polymorphisms in <t>Cm-eIF4E</t> . Gel images from the IRD700 ( A ) and IRD800 ( B ) channels of LI-COR analyzer. Each lane displays the 400 bp amplified product on Intron4-F/Full-cDNA3'-R primer combination digested with endonulcease ENDO-I . Heteroduplexes were produced after melting and annealing PCR products with the <t>DNA</t> of the reference genotype (cultivar Védrantais). A black arrow on the top left of each image indicates the position of homoduplex DNA. Arrows on the right of each panel indicate the molecular weight marker in bp. Cleaved products, indicated by boxes, correspond to sequence polymorphisms in exon 1. True polymorphisms should give rise to two complementary bands, one on each fluorescence channel.
    Plasmid Dna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "EcoTILLING for the identification of allelic variants of melon eIF4E, a factor that controls virus susceptibility"

    Article Title: EcoTILLING for the identification of allelic variants of melon eIF4E, a factor that controls virus susceptibility

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-7-34

    Detection of polymorphisms in Cm-eIF4E . Gel images from the IRD700 ( A ) and IRD800 ( B ) channels of LI-COR analyzer. Each lane displays the 400 bp amplified product on Intron4-F/Full-cDNA3'-R primer combination digested with endonulcease ENDO-I . Heteroduplexes were produced after melting and annealing PCR products with the DNA of the reference genotype (cultivar Védrantais). A black arrow on the top left of each image indicates the position of homoduplex DNA. Arrows on the right of each panel indicate the molecular weight marker in bp. Cleaved products, indicated by boxes, correspond to sequence polymorphisms in exon 1. True polymorphisms should give rise to two complementary bands, one on each fluorescence channel.
    Figure Legend Snippet: Detection of polymorphisms in Cm-eIF4E . Gel images from the IRD700 ( A ) and IRD800 ( B ) channels of LI-COR analyzer. Each lane displays the 400 bp amplified product on Intron4-F/Full-cDNA3'-R primer combination digested with endonulcease ENDO-I . Heteroduplexes were produced after melting and annealing PCR products with the DNA of the reference genotype (cultivar Védrantais). A black arrow on the top left of each image indicates the position of homoduplex DNA. Arrows on the right of each panel indicate the molecular weight marker in bp. Cleaved products, indicated by boxes, correspond to sequence polymorphisms in exon 1. True polymorphisms should give rise to two complementary bands, one on each fluorescence channel.

    Techniques Used: Amplification, Produced, Polymerase Chain Reaction, Molecular Weight, Marker, Sequencing, Fluorescence

    Related Articles

    Incubation:

    Article Title: The ATP-Binding Cassette Proteins of the Deep-Branching Protozoan Parasite Trichomonas vaginalis
    Article Snippet: Transfection of T. vaginalis Late stage cultures of T. vaginalis C1 were centrifuged (1500 g, 10 minutes, 4°C) and resuspended in supplemented Diamond's medium to a density of 108 cells/ml. .. Cells (3×107 ) were incubated with plasmid DNA (50 µg) for 15 minutes on ice and then electroporated 350 V, 960 µF (BioRad GenePulser) in chilled 0.4 cm spacing electroporation cuvettes (GeneFlow). .. Electroporated cells were immediately diluted into 50 ml of complete media, pre-warmed to 37°C and then incubated for a further 4 hours, prior to the addition of G418 to 50 µg/ml, and left overnight, before surviving cells (those in motile suspension) were transferred into fresh complete, selective media and incubated for 3–21 days until a density of ca.

    Plasmid Preparation:

    Article Title: The ATP-Binding Cassette Proteins of the Deep-Branching Protozoan Parasite Trichomonas vaginalis
    Article Snippet: Transfection of T. vaginalis Late stage cultures of T. vaginalis C1 were centrifuged (1500 g, 10 minutes, 4°C) and resuspended in supplemented Diamond's medium to a density of 108 cells/ml. .. Cells (3×107 ) were incubated with plasmid DNA (50 µg) for 15 minutes on ice and then electroporated 350 V, 960 µF (BioRad GenePulser) in chilled 0.4 cm spacing electroporation cuvettes (GeneFlow). .. Electroporated cells were immediately diluted into 50 ml of complete media, pre-warmed to 37°C and then incubated for a further 4 hours, prior to the addition of G418 to 50 µg/ml, and left overnight, before surviving cells (those in motile suspension) were transferred into fresh complete, selective media and incubated for 3–21 days until a density of ca.

    Article Title: Plasmid DNA contaminant in molecular reagents
    Article Snippet: The HotStarTaq was still positive for Ori- and Ampicillin presence and the EvaGreen 2X qPCR Express Mix-ROX remained only positive for Ori presence, indicative for possible presence of artificial expression plasmids. .. All previous positive tested Taq enzymes from BioRad had been tested negative and, therefore, reconfirmed negative for plasmid presence (Table ). .. Finally, we analyzed previously published metagenomic data sets of human gut and plasma samples as well as a data set using different whole genome amplification kits – for the presence of plasmid residues.

    Article Title: Identification of a Novel and Unique Transcription Factor in the Intraerythrocytic Stage of Plasmodium falciparum
    Article Snippet: .. Ring-stage parasites (3–10×107 ) were transfected with 100 µg of plasmid DNA at 0.310 kV and 975 µF in a 0.2 cm gap cuvette with a Gene Pulser II (Bio-Rad). .. After electroporation, parasites were transferred into a culture dish containing 20 ml RPMI medium and red blood cells at a final hematocrit of 2%.

    Article Title: Elicitor-Induced Association of Isoflavone O-Methyltransferase with Endomembranes Prevents the Formation and 7-O-Methylation of Daidzein during Isoflavonoid Phytoalexin Biosynthesis
    Article Snippet: .. Plasmid DNAs (∼5 μg) harboring the IOMT8–EGFP, 2-HIS–EGFP, mGFP4–HDEL , RFP–HDEL, or free EGFP gene under the control of the double 35S promoter was mixed with 50 μL of an aqueous suspension containing 7.5 mg of 1.0-μm gold particles (Bio-Rad). .. The gold–DNA suspension was dispersed with moderate vortexing and sonication in the presence of 1.25 M CaCl2 and 17 mM spermidine and kept on ice for 5 to 30 min.

    Electroporation:

    Article Title: The ATP-Binding Cassette Proteins of the Deep-Branching Protozoan Parasite Trichomonas vaginalis
    Article Snippet: Transfection of T. vaginalis Late stage cultures of T. vaginalis C1 were centrifuged (1500 g, 10 minutes, 4°C) and resuspended in supplemented Diamond's medium to a density of 108 cells/ml. .. Cells (3×107 ) were incubated with plasmid DNA (50 µg) for 15 minutes on ice and then electroporated 350 V, 960 µF (BioRad GenePulser) in chilled 0.4 cm spacing electroporation cuvettes (GeneFlow). .. Electroporated cells were immediately diluted into 50 ml of complete media, pre-warmed to 37°C and then incubated for a further 4 hours, prior to the addition of G418 to 50 µg/ml, and left overnight, before surviving cells (those in motile suspension) were transferred into fresh complete, selective media and incubated for 3–21 days until a density of ca.

    Transfection:

    Article Title: Identification of a Novel and Unique Transcription Factor in the Intraerythrocytic Stage of Plasmodium falciparum
    Article Snippet: .. Ring-stage parasites (3–10×107 ) were transfected with 100 µg of plasmid DNA at 0.310 kV and 975 µF in a 0.2 cm gap cuvette with a Gene Pulser II (Bio-Rad). .. After electroporation, parasites were transferred into a culture dish containing 20 ml RPMI medium and red blood cells at a final hematocrit of 2%.

    Electrophoresis:

    Article Title: Plasmidic Extended-Spectrum ?-Lactamases in Vibrio cholerae O1 El Tor Isolates in Argentina †
    Article Snippet: Plasmid DNA was purified from V. cholerae or E. coli transconjugant cells as described previously ( ). .. Plasmidic DNAs (about 250 ng) were analyzed by electrophoresis in 0.7% agarose gels (Tris-acetate buffer), by using plasmids of known size as standards, and were then transferred onto and immobilized on a nylon transfer membrane (Zeta-Probe GT; Bio-Rad, Richmond, Calif.). ..

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    • plasmid dna  (Bio-Rad)
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    Bio-Rad plasmid dna
    Effects of EBNA2 on miR-155HG promoter and enhancer elements. (A) mIR-155HG luciferase reporter assays in the presence or absence of EBNA2. <t>DG75</t> cells were transfected with 2 μg of pGL3 firefly luciferase reporter constructs containing the miR-155HG promoter (pro) either alone or in the presence of E1, E2, or both E1 and E2. Assays were carried out in the absence or presence of 10 or 20 μg of the EBNA2-expressing plasmid pSG5-EBNA2 and 0.5 μg of the Renilla luciferase control plasmid (pRL-TK). Firefly (FFL) luciferase signals were normalized to Renilla (RL) luciferase signals and are expressed relative to the signal obtained for the miR-155HG promoter in the absence of EBNA2. Results show the means of data from three independent experiments ± standard deviations. Fold activation by EBNA2 relative to the signal obtained for each construct in the absence of EBNA2 is shown above each bar. Western blot analysis of EBNA2 and IRF4 expression is shown below each bar chart, with actin providing a loading control. All blots shown were probed at the same time with the same batch of antibody solution and for each protein show the same exposure. They are therefore directly comparable but have been cut and placed to align with the respective luciferase assay graphs. The asterisk shows the position of a nonspecific band visible upon longer exposures of EBNA2 blots. (B) EBNA2 activation of an EBV C promoter reporter construct was used as a positive control. (C) ChIP-QPCR analysis of RBPJ binding at the miR-155HG locus in GM12878 cells. Precipitated <t>DNA</t> was analyzed using primer sets located at the promoter, E1, and E2 and in a trough between E1 and E2 (T). EBNA2 binding at the transcription start site of PPIA and at the previously characterized CTBP2 binding site were used as negative and positive binding controls, respectively. Mean percent input signals, after subtraction of signals for the no-antibody controls, ± standard deviations are shown for three independent ChIP experiments. (D) Luciferase reporter assays carried out using the miR-155HG promoter or the miR-155HG E1 and E2 constructs in DG75 wt parental cells that lack IRF4 expression and the corresponding RBPJ knockout (KO) cell line. Results are displayed as described above for panel B. (E) Luciferase reporter assays carried out as described above for panel D, using the RBPJ-dependent C promoter reporter construct.
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    Image Search Results


    Effects of EBNA2 on miR-155HG promoter and enhancer elements. (A) mIR-155HG luciferase reporter assays in the presence or absence of EBNA2. DG75 cells were transfected with 2 μg of pGL3 firefly luciferase reporter constructs containing the miR-155HG promoter (pro) either alone or in the presence of E1, E2, or both E1 and E2. Assays were carried out in the absence or presence of 10 or 20 μg of the EBNA2-expressing plasmid pSG5-EBNA2 and 0.5 μg of the Renilla luciferase control plasmid (pRL-TK). Firefly (FFL) luciferase signals were normalized to Renilla (RL) luciferase signals and are expressed relative to the signal obtained for the miR-155HG promoter in the absence of EBNA2. Results show the means of data from three independent experiments ± standard deviations. Fold activation by EBNA2 relative to the signal obtained for each construct in the absence of EBNA2 is shown above each bar. Western blot analysis of EBNA2 and IRF4 expression is shown below each bar chart, with actin providing a loading control. All blots shown were probed at the same time with the same batch of antibody solution and for each protein show the same exposure. They are therefore directly comparable but have been cut and placed to align with the respective luciferase assay graphs. The asterisk shows the position of a nonspecific band visible upon longer exposures of EBNA2 blots. (B) EBNA2 activation of an EBV C promoter reporter construct was used as a positive control. (C) ChIP-QPCR analysis of RBPJ binding at the miR-155HG locus in GM12878 cells. Precipitated DNA was analyzed using primer sets located at the promoter, E1, and E2 and in a trough between E1 and E2 (T). EBNA2 binding at the transcription start site of PPIA and at the previously characterized CTBP2 binding site were used as negative and positive binding controls, respectively. Mean percent input signals, after subtraction of signals for the no-antibody controls, ± standard deviations are shown for three independent ChIP experiments. (D) Luciferase reporter assays carried out using the miR-155HG promoter or the miR-155HG E1 and E2 constructs in DG75 wt parental cells that lack IRF4 expression and the corresponding RBPJ knockout (KO) cell line. Results are displayed as described above for panel B. (E) Luciferase reporter assays carried out as described above for panel D, using the RBPJ-dependent C promoter reporter construct.

    Journal: Journal of Virology

    Article Title: Enhancer Control of MicroRNA miR-155 Expression in Epstein-Barr Virus-Infected B Cells

    doi: 10.1128/JVI.00716-18

    Figure Lengend Snippet: Effects of EBNA2 on miR-155HG promoter and enhancer elements. (A) mIR-155HG luciferase reporter assays in the presence or absence of EBNA2. DG75 cells were transfected with 2 μg of pGL3 firefly luciferase reporter constructs containing the miR-155HG promoter (pro) either alone or in the presence of E1, E2, or both E1 and E2. Assays were carried out in the absence or presence of 10 or 20 μg of the EBNA2-expressing plasmid pSG5-EBNA2 and 0.5 μg of the Renilla luciferase control plasmid (pRL-TK). Firefly (FFL) luciferase signals were normalized to Renilla (RL) luciferase signals and are expressed relative to the signal obtained for the miR-155HG promoter in the absence of EBNA2. Results show the means of data from three independent experiments ± standard deviations. Fold activation by EBNA2 relative to the signal obtained for each construct in the absence of EBNA2 is shown above each bar. Western blot analysis of EBNA2 and IRF4 expression is shown below each bar chart, with actin providing a loading control. All blots shown were probed at the same time with the same batch of antibody solution and for each protein show the same exposure. They are therefore directly comparable but have been cut and placed to align with the respective luciferase assay graphs. The asterisk shows the position of a nonspecific band visible upon longer exposures of EBNA2 blots. (B) EBNA2 activation of an EBV C promoter reporter construct was used as a positive control. (C) ChIP-QPCR analysis of RBPJ binding at the miR-155HG locus in GM12878 cells. Precipitated DNA was analyzed using primer sets located at the promoter, E1, and E2 and in a trough between E1 and E2 (T). EBNA2 binding at the transcription start site of PPIA and at the previously characterized CTBP2 binding site were used as negative and positive binding controls, respectively. Mean percent input signals, after subtraction of signals for the no-antibody controls, ± standard deviations are shown for three independent ChIP experiments. (D) Luciferase reporter assays carried out using the miR-155HG promoter or the miR-155HG E1 and E2 constructs in DG75 wt parental cells that lack IRF4 expression and the corresponding RBPJ knockout (KO) cell line. Results are displayed as described above for panel B. (E) Luciferase reporter assays carried out as described above for panel D, using the RBPJ-dependent C promoter reporter construct.

    Article Snippet: DG75 cell lines were electroporated with plasmid DNA at 260 V and 950 μF (Gene Pulser II; Bio-Rad) using 0.4-cm cuvettes, and luciferase assays were carried out as described previously, with some modifications ( ).

    Techniques: Luciferase, Transfection, Construct, Expressing, Plasmid Preparation, Activation Assay, Western Blot, Positive Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Knock-Out

    Epi-fluorescent microscopy of electroporated SW13 cells. ( a ) SW13 cells electroporated with a mixture of K14 ncIBs and a plasmid containing EYFP labeled K5 cDNA. ( b ) The positive control experiment, where cells were electroporated with plasmid DNAs encoding for EYFP-K5 and EYFP-K14. (C) The negative control experiment, where cells were electroporated exclusively with EYFP-K5 plasmid DNA. Experiments in ( a ) and ( b ) resulted in a multitude of speckles and short filamentous structures (see arrows). The scale bar is 20 μm.

    Journal: Microbial Cell Factories

    Article Title: Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells

    doi: 10.1186/1475-2859-11-67

    Figure Lengend Snippet: Epi-fluorescent microscopy of electroporated SW13 cells. ( a ) SW13 cells electroporated with a mixture of K14 ncIBs and a plasmid containing EYFP labeled K5 cDNA. ( b ) The positive control experiment, where cells were electroporated with plasmid DNAs encoding for EYFP-K5 and EYFP-K14. (C) The negative control experiment, where cells were electroporated exclusively with EYFP-K5 plasmid DNA. Experiments in ( a ) and ( b ) resulted in a multitude of speckles and short filamentous structures (see arrows). The scale bar is 20 μm.

    Article Snippet: Approximately 150 μg of K14 IBs and 10 μg of plasmid DNA (with EYFP-K5) was mixed and electroporated into SW13 cells using a Gene Pulser (Bio-Rad) electroporator set at 125 μF capacitance extender, 200 Ω pulse controler, 25 μF gene pulser capacitance and 1.5 kV voltage.

    Techniques: Microscopy, Plasmid Preparation, Labeling, Positive Control, Negative Control

    Virus titer in wild-type and FEC-derived TME 7 (Oko-iyawo) plants challenged with GFP-VIGS and MeSPY1-VIGS at 9 DPI. a Virus titer determination by Southern blot and b qPCR performed on total DNA extracted from leaves of wild-type (resistant to CMD) and FEC-derived (susceptible to CMD) plants. c RT-qPCR expression analysis of MeSPY. Higher virus titer is detected in both GFP-VIGS and MeSPY1-VIGS-challenged FEC-TME7 (susceptible plants) compared to the wild-type TME 7. Primers used for labelling probes for Southern blotting and for qPCR and RT-qPCR assays are shown in Table 1 . Bars show SE ( n = 4). P, positive control, N genomic DNA from unchallenged plants. Samples from three infected plants were pooled to make one sample, and a total of 4 samples (from 12 plants) were used per treatment combination

    Journal: Virology Journal

    Article Title: A rapid virus-induced gene silencing (VIGS) method for assessing resistance and susceptibility to cassava mosaic disease

    doi: 10.1186/s12985-017-0716-6

    Figure Lengend Snippet: Virus titer in wild-type and FEC-derived TME 7 (Oko-iyawo) plants challenged with GFP-VIGS and MeSPY1-VIGS at 9 DPI. a Virus titer determination by Southern blot and b qPCR performed on total DNA extracted from leaves of wild-type (resistant to CMD) and FEC-derived (susceptible to CMD) plants. c RT-qPCR expression analysis of MeSPY. Higher virus titer is detected in both GFP-VIGS and MeSPY1-VIGS-challenged FEC-TME7 (susceptible plants) compared to the wild-type TME 7. Primers used for labelling probes for Southern blotting and for qPCR and RT-qPCR assays are shown in Table 1 . Bars show SE ( n = 4). P, positive control, N genomic DNA from unchallenged plants. Samples from three infected plants were pooled to make one sample, and a total of 4 samples (from 12 plants) were used per treatment combination

    Article Snippet: Inoculation of VIGS clones and assessment of phenotype in the greenhouse Four- to 6-week-old greenhouse-grown plants were inoculated with plasmid DNA of MeSPY1-VIGS, GFP-VIGS or MePSY2-VIGS vectors plus the DNA-B component of EACMV-K201 using a Helios® Gene Gun (BioRad, Hercules, California), following Beyene, et al. [ ].

    Techniques: Derivative Assay, Southern Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Positive Control, Infection

    East African Cassava mosaic virus (EACMV-K201) infectious clone-based virus-induced gene silencing (VIGS) vector. a Coat protein (CP) nucleotide sequence of EACMV-K201 DNA-A showing position and mutated nucleotides that introduced Avr II and Nhe I near 5′-end and Sbf I near 3′-end. Nucleotide positions were counted from the start codon “ATG” of the CP. b Infectivity of the GFP-VIGS and MePSY2-VIGS targeting GFP and cassava phytoene synthase coding sequences, respectively, in wild-type cassava cultivar TME 7S. GFP-VIGS has no known target in the challenged cassava plant, and these plants display typical mosaic symptoms characteristic of CMD on leaves. MePSY2-VIGS-challenged plants show chlorosis on leaves. Pictures were collected at 2 and 12 weeks post inoculation (WPI)

    Journal: Virology Journal

    Article Title: A rapid virus-induced gene silencing (VIGS) method for assessing resistance and susceptibility to cassava mosaic disease

    doi: 10.1186/s12985-017-0716-6

    Figure Lengend Snippet: East African Cassava mosaic virus (EACMV-K201) infectious clone-based virus-induced gene silencing (VIGS) vector. a Coat protein (CP) nucleotide sequence of EACMV-K201 DNA-A showing position and mutated nucleotides that introduced Avr II and Nhe I near 5′-end and Sbf I near 3′-end. Nucleotide positions were counted from the start codon “ATG” of the CP. b Infectivity of the GFP-VIGS and MePSY2-VIGS targeting GFP and cassava phytoene synthase coding sequences, respectively, in wild-type cassava cultivar TME 7S. GFP-VIGS has no known target in the challenged cassava plant, and these plants display typical mosaic symptoms characteristic of CMD on leaves. MePSY2-VIGS-challenged plants show chlorosis on leaves. Pictures were collected at 2 and 12 weeks post inoculation (WPI)

    Article Snippet: Inoculation of VIGS clones and assessment of phenotype in the greenhouse Four- to 6-week-old greenhouse-grown plants were inoculated with plasmid DNA of MeSPY1-VIGS, GFP-VIGS or MePSY2-VIGS vectors plus the DNA-B component of EACMV-K201 using a Helios® Gene Gun (BioRad, Hercules, California), following Beyene, et al. [ ].

    Techniques: Plasmid Preparation, Sequencing, Infection

    GR transcriptional activity is repressed by cytochalasin B, nocodazole, or c-Jun. Retinal tissue was precultured for 30 min in the presence of cytochalasin B (50 μg/ml) (bars 3 and 4) or nocodazole (40 μg/ml) (bars 5 and 6) or in their absence (bars 1 and 2) or transfected with the c-Jun expression vector RSVc-Jun (1 μg of DNA/8 × 10 6 cells) (bars 7 and 8). The tissue was transfected (bars 1 to 6) or cotransfected (bars 7 and 8) with the glucocorticoid-inducible CAT construct pΔG46TCO (1 μg of DNA/8 × 10 6 cells) (A) or the noninducible construct RSVCAT (1 μg of DNA/8 × 10 6 cells) (B) or cotransfected with pΔG46TCO (1 μg of DNA/8 × 10 6 cells) and the GR expression vector p6RGR (1 μg of DNA/8 × 10 6 cells) (C). In all cases, the luciferase reporter construct RSVL(SEL) (1 μg of DNA/8 × 10 6 cells) was cotransfected as a control. The transfected cultures were maintained for 24 h in the presence (solid bars) or absence (open bars) of cortisol. The CAT and luciferase activities were then examined. The CAT assays were adjusted to include an equal amount of luciferase activity. The percentage of CAT conversion was calculated by scanning the thin-layer chromatographic plates with a phosphorimager instrument. In each experiment the value of CAT conversion in the cortisol-treated control (bar 1) was used to normalize all other results. The data shown in A and B are the means plus standard errors of the mean of three separate experiments.

    Journal: Molecular and Cellular Biology

    Article Title: The Cytoskeletal Network Controls c-Jun Expression and Glucocorticoid Receptor Transcriptional Activity in an Antagonistic and Cell-Type-Specific Manner

    doi:

    Figure Lengend Snippet: GR transcriptional activity is repressed by cytochalasin B, nocodazole, or c-Jun. Retinal tissue was precultured for 30 min in the presence of cytochalasin B (50 μg/ml) (bars 3 and 4) or nocodazole (40 μg/ml) (bars 5 and 6) or in their absence (bars 1 and 2) or transfected with the c-Jun expression vector RSVc-Jun (1 μg of DNA/8 × 10 6 cells) (bars 7 and 8). The tissue was transfected (bars 1 to 6) or cotransfected (bars 7 and 8) with the glucocorticoid-inducible CAT construct pΔG46TCO (1 μg of DNA/8 × 10 6 cells) (A) or the noninducible construct RSVCAT (1 μg of DNA/8 × 10 6 cells) (B) or cotransfected with pΔG46TCO (1 μg of DNA/8 × 10 6 cells) and the GR expression vector p6RGR (1 μg of DNA/8 × 10 6 cells) (C). In all cases, the luciferase reporter construct RSVL(SEL) (1 μg of DNA/8 × 10 6 cells) was cotransfected as a control. The transfected cultures were maintained for 24 h in the presence (solid bars) or absence (open bars) of cortisol. The CAT and luciferase activities were then examined. The CAT assays were adjusted to include an equal amount of luciferase activity. The percentage of CAT conversion was calculated by scanning the thin-layer chromatographic plates with a phosphorimager instrument. In each experiment the value of CAT conversion in the cortisol-treated control (bar 1) was used to normalize all other results. The data shown in A and B are the means plus standard errors of the mean of three separate experiments.

    Article Snippet: Plasmid DNA was transfected into pieces of intact retinal tissue by electroporation with a gene pulser (Bio-Rad, Richmond, Calif.) with voltage and capacitance settings of 400 V and 960 μF, as described previously ( ).

    Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Construct, Luciferase