pcr mixture  (Thermo Fisher)


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    Structured Review

    Thermo Fisher pcr mixture
    Gene expression from in vivo rAAV vector forms. A circle <t>PCR</t> approach was used to clone the predicted in vivo monomeric circular vector form. PCR primers, overlapping the unique Pst I site at their 3′ ends, were used to amplify rAAV vectors from PS-DNase-treated muscle <t>DNA.</t> The resulting PCR products were cloned into a TA-cloning vector for further analysis. To determine whether the cloned vectors were transcriptionally active, they were excised from the TA vector with Pst I, self-ligated, and transfected into HeLa cells. Cell lysates were analyzed by Western blotting with an anti-ova-horseradish peroxidase antibody. Four independently rescued clones (lanes 1 to 4) that express chicken ovalbumin protein are shown. Lane U, untransfected cell lysate; lane C, lysate from cells transfected with a DNA expression plasmid encoding chicken ovalbumin.
    Pcr Mixture, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1489 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle"

    Article Title: Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.6.3495-3504.2003

    Gene expression from in vivo rAAV vector forms. A circle PCR approach was used to clone the predicted in vivo monomeric circular vector form. PCR primers, overlapping the unique Pst I site at their 3′ ends, were used to amplify rAAV vectors from PS-DNase-treated muscle DNA. The resulting PCR products were cloned into a TA-cloning vector for further analysis. To determine whether the cloned vectors were transcriptionally active, they were excised from the TA vector with Pst I, self-ligated, and transfected into HeLa cells. Cell lysates were analyzed by Western blotting with an anti-ova-horseradish peroxidase antibody. Four independently rescued clones (lanes 1 to 4) that express chicken ovalbumin protein are shown. Lane U, untransfected cell lysate; lane C, lysate from cells transfected with a DNA expression plasmid encoding chicken ovalbumin.
    Figure Legend Snippet: Gene expression from in vivo rAAV vector forms. A circle PCR approach was used to clone the predicted in vivo monomeric circular vector form. PCR primers, overlapping the unique Pst I site at their 3′ ends, were used to amplify rAAV vectors from PS-DNase-treated muscle DNA. The resulting PCR products were cloned into a TA-cloning vector for further analysis. To determine whether the cloned vectors were transcriptionally active, they were excised from the TA vector with Pst I, self-ligated, and transfected into HeLa cells. Cell lysates were analyzed by Western blotting with an anti-ova-horseradish peroxidase antibody. Four independently rescued clones (lanes 1 to 4) that express chicken ovalbumin protein are shown. Lane U, untransfected cell lysate; lane C, lysate from cells transfected with a DNA expression plasmid encoding chicken ovalbumin.

    Techniques Used: Expressing, In Vivo, Plasmid Preparation, Polymerase Chain Reaction, Clone Assay, TA Cloning, Transfection, Western Blot

    2) Product Images from "Phase Variation in Myxococcus xanthus Yields Cells Specialized for Iron Sequestration"

    Article Title: Phase Variation in Myxococcus xanthus Yields Cells Specialized for Iron Sequestration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095189

    Regulation of gene expression by iron. RNA harvested from WT-Y cells was used to prepare cDNA for qRT-PCR analysis of representative genes whose expression is affected during phase variation; MXAN_0228 (Xre228), MXAN_7370 (ST_kinase), MXAN_3641 (myxochelin), MXAN_6911 (FepA homolog), MXAN_3639 (myxovirescin) and MXAN_4305 (DKX). The results represent three biological and technical replicates; data are from cells grown in CTPM+FeCl 3 and CTPM+dipyridyl medium relative to levels from cells grown in CTPM alone (value = 1) are presented.
    Figure Legend Snippet: Regulation of gene expression by iron. RNA harvested from WT-Y cells was used to prepare cDNA for qRT-PCR analysis of representative genes whose expression is affected during phase variation; MXAN_0228 (Xre228), MXAN_7370 (ST_kinase), MXAN_3641 (myxochelin), MXAN_6911 (FepA homolog), MXAN_3639 (myxovirescin) and MXAN_4305 (DKX). The results represent three biological and technical replicates; data are from cells grown in CTPM+FeCl 3 and CTPM+dipyridyl medium relative to levels from cells grown in CTPM alone (value = 1) are presented.

    Techniques Used: Expressing, Quantitative RT-PCR

    3) Product Images from "Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism"

    Article Title: Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.13-13570

    GMS staining and the PCR products to demonstrate presence of Fusarium species in human TM samples. ( A , B ) Represent a GMS-stained positive control for Fusarium species in different magnifications (×10 and ×40 as indicated), respectively. ( C , D ) Representative human POAG TM stained with GMS (magnification power ×5 and ×20 as indicated). Arrowhead and star represent TM and Schlemm canal, respectively. ( E ) The PCR amplified product of Fusarium species electrophoresed on agarose gel (2%) and stained with ethidium bromide. Control, POAG-derived products (all Caucasian donors, age, and sex, and a positive Fusarium sp. DNA as positive control) used are as indicated. A negative control lacked any DNA template (−template). Molecular weight marker sizes are as indicated. M, male; F, female.
    Figure Legend Snippet: GMS staining and the PCR products to demonstrate presence of Fusarium species in human TM samples. ( A , B ) Represent a GMS-stained positive control for Fusarium species in different magnifications (×10 and ×40 as indicated), respectively. ( C , D ) Representative human POAG TM stained with GMS (magnification power ×5 and ×20 as indicated). Arrowhead and star represent TM and Schlemm canal, respectively. ( E ) The PCR amplified product of Fusarium species electrophoresed on agarose gel (2%) and stained with ethidium bromide. Control, POAG-derived products (all Caucasian donors, age, and sex, and a positive Fusarium sp. DNA as positive control) used are as indicated. A negative control lacked any DNA template (−template). Molecular weight marker sizes are as indicated. M, male; F, female.

    Techniques Used: Staining, Polymerase Chain Reaction, Positive Control, Amplification, Agarose Gel Electrophoresis, Derivative Assay, Negative Control, Molecular Weight, Marker

    4) Product Images from "Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus"

    Article Title: Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus

    Journal: Infection and Immunity

    doi: 10.1128/IAI.72.4.2177-2185.2004

    RFLP electrophoretic patterns of PCR-amplified coagulase gene digested with CfoI. The different patterns observed in the analysis of the bovine S. aureus strains are shown in individual lanes. M, molecular weight marker (gene ruler 100-bp DNA ladder plus; Fermentas).
    Figure Legend Snippet: RFLP electrophoretic patterns of PCR-amplified coagulase gene digested with CfoI. The different patterns observed in the analysis of the bovine S. aureus strains are shown in individual lanes. M, molecular weight marker (gene ruler 100-bp DNA ladder plus; Fermentas).

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    5) Product Images from "Non-Sulfate-Reducing, Syntrophic Bacteria Affiliated with Desulfotomaculum Cluster I Are Widely Distributed in Methanogenic Environments"

    Article Title: Non-Sulfate-Reducing, Syntrophic Bacteria Affiliated with Desulfotomaculum Cluster I Are Widely Distributed in Methanogenic Environments

    Journal:

    doi: 10.1128/AEM.72.3.2080-2091.2006

    RT-PCR amplification of dsrAB mRNA and 16S rRNA from strain MGP. Lane M, marker; lanes 1, 4, and 7, DNA of strain MGP (positive controls); lanes 2, 5, and 8, RNA extracted from strain MGP grown on only propionate (20 mM); lanes 3, 6, and 9, RNA extracted
    Figure Legend Snippet: RT-PCR amplification of dsrAB mRNA and 16S rRNA from strain MGP. Lane M, marker; lanes 1, 4, and 7, DNA of strain MGP (positive controls); lanes 2, 5, and 8, RNA extracted from strain MGP grown on only propionate (20 mM); lanes 3, 6, and 9, RNA extracted

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Marker

    6) Product Images from "Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay"

    Article Title: Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.01669-13

    Detection of drug resistance mutations using plasmid DNA templates. After PCR amplification of each template individually, a mixture of the wild-type template and the mutant template (Mut) at a ratio of 1:1 (wt/wt), amplicons of each individual template,
    Figure Legend Snippet: Detection of drug resistance mutations using plasmid DNA templates. After PCR amplification of each template individually, a mixture of the wild-type template and the mutant template (Mut) at a ratio of 1:1 (wt/wt), amplicons of each individual template,

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Mutagenesis

    7) Product Images from "Modeling of 5? Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica"

    Article Title: Modeling of 5? Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.40.1.52-60.2002

    Graphical appearance of the model used to estimate PCR performance. The experiment was performed three times with 10-fold dilutions of DNA, and the model (the line connecting the datum points) fits the experimental data (✻) that were obtained well. Salmonella DNA amplification took place in the presence of ddH 2 O and different media with two amplification mixtures: (A) Ampli Taq Gold with ddH 2 O; (B) r Tt h with ddH 2 O; (C) Ampli Taq Gold with BPW; (D) r Tt h with BPW; (E) Ampli Taq Gold with BHI; (F) r Tt h with BHI. The vertical dashed lines in all graphs are the log DNA concentration at a detection probability of 0.95 from the estimated model. The horizontal dashed lines at C T equal to 30 marks the proposed upper limit for sufficient PCR performance. From the model the slope was determined and all graphs (A to F) gave close to optimal amplification efficiencies.
    Figure Legend Snippet: Graphical appearance of the model used to estimate PCR performance. The experiment was performed three times with 10-fold dilutions of DNA, and the model (the line connecting the datum points) fits the experimental data (✻) that were obtained well. Salmonella DNA amplification took place in the presence of ddH 2 O and different media with two amplification mixtures: (A) Ampli Taq Gold with ddH 2 O; (B) r Tt h with ddH 2 O; (C) Ampli Taq Gold with BPW; (D) r Tt h with BPW; (E) Ampli Taq Gold with BHI; (F) r Tt h with BHI. The vertical dashed lines in all graphs are the log DNA concentration at a detection probability of 0.95 from the estimated model. The horizontal dashed lines at C T equal to 30 marks the proposed upper limit for sufficient PCR performance. From the model the slope was determined and all graphs (A to F) gave close to optimal amplification efficiencies.

    Techniques Used: Polymerase Chain Reaction, Amplification, Concentration Assay

    Enrichment PCR for S. enterica serovar Enteritidis. The graph illustrates the dynamic detection range, i.e., the time during which positive detection is possible, by plotting the C T value against the incubation time. BPW was inoculated with S. enterica serovar Enteritidis at a concentration of 1 CFU/ml, and the mixture was incubated at 37°C. Samples for PCR analysis were withdrawn every 4 h. •, numbers of CFU per milliliter; ▪, results for Ampli Taq Gold mixture; ▴, results for r Tth mixture.
    Figure Legend Snippet: Enrichment PCR for S. enterica serovar Enteritidis. The graph illustrates the dynamic detection range, i.e., the time during which positive detection is possible, by plotting the C T value against the incubation time. BPW was inoculated with S. enterica serovar Enteritidis at a concentration of 1 CFU/ml, and the mixture was incubated at 37°C. Samples for PCR analysis were withdrawn every 4 h. •, numbers of CFU per milliliter; ▪, results for Ampli Taq Gold mixture; ▴, results for r Tth mixture.

    Techniques Used: Polymerase Chain Reaction, Incubation, Concentration Assay

    DNA amplification by the Salmonella 5′ nuclease PCR assay in the presence of 5 μl of BHI and 5 μl of BPW. The graphs show the results of the C T response at a constant Salmonella DNA concentration (2 × 10 −9 g/microwell) while the concentrations of BHI and BPW in the media were changed. The experiment was performed with both the Ampli Taq Gold mixture and the r Tth mixture. The PCR results correspond to the following DNA amplification combinations: Ampli Taq Gold mixture and BPW medium (⧫), Ampli Taq Gold mixture and BHI medium (▴), r Tth mixture and BPW medium (⋄), and r Tth mixture and BHI medium (▵).
    Figure Legend Snippet: DNA amplification by the Salmonella 5′ nuclease PCR assay in the presence of 5 μl of BHI and 5 μl of BPW. The graphs show the results of the C T response at a constant Salmonella DNA concentration (2 × 10 −9 g/microwell) while the concentrations of BHI and BPW in the media were changed. The experiment was performed with both the Ampli Taq Gold mixture and the r Tth mixture. The PCR results correspond to the following DNA amplification combinations: Ampli Taq Gold mixture and BPW medium (⧫), Ampli Taq Gold mixture and BHI medium (▴), r Tth mixture and BPW medium (⋄), and r Tth mixture and BHI medium (▵).

    Techniques Used: Amplification, Polymerase Chain Reaction, Concentration Assay

    8) Product Images from "Considerations When Analyzing the Methylation Status of PTEN Tumor Suppressor Gene"

    Article Title: Considerations When Analyzing the Methylation Status of PTEN Tumor Suppressor Gene

    Journal: The American Journal of Pathology

    doi:

    Methylation-specific PCR using set III PTEN specific primers ( a ) and methylation-sensitive restriction analysis ( b, c, d ) using primer set IV-A ( PTEN ) respectively for cell lines. MSP analysis, as well as MSRA using primer set IV-A ( PTEN ), show lack of methylation in cell lines ( a, b ). Results for MSRA using set IV-B and -C primers ( c, d ) show methylation positive products in the same cell lines. NL treated with Sss I methyltransferase was used as a methylated positive control. Following Aci I digestion, equal amounts of cell line DNA were used for PCR analysis. U, unmethylated; M, methylated; N, undigested DNA; D, digested DNA; NL, normal lymphocyte DNA.
    Figure Legend Snippet: Methylation-specific PCR using set III PTEN specific primers ( a ) and methylation-sensitive restriction analysis ( b, c, d ) using primer set IV-A ( PTEN ) respectively for cell lines. MSP analysis, as well as MSRA using primer set IV-A ( PTEN ), show lack of methylation in cell lines ( a, b ). Results for MSRA using set IV-B and -C primers ( c, d ) show methylation positive products in the same cell lines. NL treated with Sss I methyltransferase was used as a methylated positive control. Following Aci I digestion, equal amounts of cell line DNA were used for PCR analysis. U, unmethylated; M, methylated; N, undigested DNA; D, digested DNA; NL, normal lymphocyte DNA.

    Techniques Used: Methylation, Polymerase Chain Reaction, Positive Control

    9) Product Images from "cytb as a New Genetic Marker for Differentiation of Prototheca Species"

    Article Title: cytb as a New Genetic Marker for Differentiation of Prototheca Species

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00584-18

    Evaluation of the cytb PCR restriction enzyme analysis profiling for selected Prototheca sp. strains. Lanes 1 to 3, P. zopfii genotype 1; lanes 4 to 6, P. zopfii genotype 2; lanes 7 to 9, P. blaschkeae ; lanes 10 to 12 – P. wickerhamii ; lanes M, size marker (Gene Ruler Low Range; Thermo Fisher Scientific, Waltham, MA, USA).
    Figure Legend Snippet: Evaluation of the cytb PCR restriction enzyme analysis profiling for selected Prototheca sp. strains. Lanes 1 to 3, P. zopfii genotype 1; lanes 4 to 6, P. zopfii genotype 2; lanes 7 to 9, P. blaschkeae ; lanes 10 to 12 – P. wickerhamii ; lanes M, size marker (Gene Ruler Low Range; Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques Used: Polymerase Chain Reaction, Marker

    10) Product Images from "Targeting miR-9 in gastric cancer cells using locked nucleic acid oligonucleotides"

    Article Title: Targeting miR-9 in gastric cancer cells using locked nucleic acid oligonucleotides

    Journal: BMC Molecular Biology

    doi: 10.1186/s12867-018-0107-6

    Silencing effect of the AMOs in MKN74 cells. Relative expression levels (measured by qRT-PCR) of a miR-9 and b CDH1 mRNA, after 4, 8, 12 and 24 h treatment with AM_LNA_PS, AM_LNA_2OMe_PS, and SC_LNA_PS. c Western blot analysis of the relative variation of E-cadherin expression in MKN74 cells after treatment with LNA-anti-miR oligonucleotides and the control, MKN74 cells without any treatment, at the same time-points used for qRT-PCR. All the qRT-PCR and western blot results were normalized against the MKN74 control and the levels of the respective endogenous control (RNU48 for miR-9 and GAPDH for CDH1 gene quantified by qRT-PCR and E-cadherin protein expression by western blot)
    Figure Legend Snippet: Silencing effect of the AMOs in MKN74 cells. Relative expression levels (measured by qRT-PCR) of a miR-9 and b CDH1 mRNA, after 4, 8, 12 and 24 h treatment with AM_LNA_PS, AM_LNA_2OMe_PS, and SC_LNA_PS. c Western blot analysis of the relative variation of E-cadherin expression in MKN74 cells after treatment with LNA-anti-miR oligonucleotides and the control, MKN74 cells without any treatment, at the same time-points used for qRT-PCR. All the qRT-PCR and western blot results were normalized against the MKN74 control and the levels of the respective endogenous control (RNU48 for miR-9 and GAPDH for CDH1 gene quantified by qRT-PCR and E-cadherin protein expression by western blot)

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

    11) Product Images from "Targeting miR-9 in gastric cancer cells using locked nucleic acid oligonucleotides"

    Article Title: Targeting miR-9 in gastric cancer cells using locked nucleic acid oligonucleotides

    Journal: BMC Molecular Biology

    doi: 10.1186/s12867-018-0107-6

    Silencing effect of the AMOs in MKN74 cells. Relative expression levels (measured by qRT-PCR) of a miR-9 and b CDH1 mRNA, after 4, 8, 12 and 24 h treatment with AM_LNA_PS, AM_LNA_2OMe_PS, and SC_LNA_PS. c Western blot analysis of the relative variation of E-cadherin expression in MKN74 cells after treatment with LNA-anti-miR oligonucleotides and the control, MKN74 cells without any treatment, at the same time-points used for qRT-PCR. All the qRT-PCR and western blot results were normalized against the MKN74 control and the levels of the respective endogenous control (RNU48 for miR-9 and GAPDH for CDH1 gene quantified by qRT-PCR and E-cadherin protein expression by western blot)
    Figure Legend Snippet: Silencing effect of the AMOs in MKN74 cells. Relative expression levels (measured by qRT-PCR) of a miR-9 and b CDH1 mRNA, after 4, 8, 12 and 24 h treatment with AM_LNA_PS, AM_LNA_2OMe_PS, and SC_LNA_PS. c Western blot analysis of the relative variation of E-cadherin expression in MKN74 cells after treatment with LNA-anti-miR oligonucleotides and the control, MKN74 cells without any treatment, at the same time-points used for qRT-PCR. All the qRT-PCR and western blot results were normalized against the MKN74 control and the levels of the respective endogenous control (RNU48 for miR-9 and GAPDH for CDH1 gene quantified by qRT-PCR and E-cadherin protein expression by western blot)

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

    12) Product Images from "The TT8 Gene Encodes a Basic Helix-Loop-Helix Domain Protein Required for Expression of DFR and BAN Genes in Arabidopsis Siliques"

    Article Title: The TT8 Gene Encodes a Basic Helix-Loop-Helix Domain Protein Required for Expression of DFR and BAN Genes in Arabidopsis Siliques

    Journal: The Plant Cell

    doi:

    Analysis of TT8 Expression during Plant Development. (A) TT8 transcripts were detected by quantitative RT-PCR in vegetative parts that included 4-day-old seedlings, rosette leaves, stems, and roots from 10-day-old plantlets and in reproductive organs that included buds, flowers, and developing seeds. Accumulation of the EF1 α A4 transcript was used as an internal control. The PCR products were detected by DNA gel blot analysis after 21 amplification cycles. The blots were hybridized with the respective probes. (B) Comparison of TT8 expression pattern in reproductive organs with those of C4H , CHS , and DFR genes. PCR and hybridization assays were conducted exactly as those in (A) .
    Figure Legend Snippet: Analysis of TT8 Expression during Plant Development. (A) TT8 transcripts were detected by quantitative RT-PCR in vegetative parts that included 4-day-old seedlings, rosette leaves, stems, and roots from 10-day-old plantlets and in reproductive organs that included buds, flowers, and developing seeds. Accumulation of the EF1 α A4 transcript was used as an internal control. The PCR products were detected by DNA gel blot analysis after 21 amplification cycles. The blots were hybridized with the respective probes. (B) Comparison of TT8 expression pattern in reproductive organs with those of C4H , CHS , and DFR genes. PCR and hybridization assays were conducted exactly as those in (A) .

    Techniques Used: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Western Blot, Amplification, Hybridization

    13) Product Images from "Rotavirus shedding following administration of RV3-BB human neonatal rotavirus vaccine"

    Article Title: Rotavirus shedding following administration of RV3-BB human neonatal rotavirus vaccine

    Journal: Human Vaccines & Immunotherapeutics

    doi: 10.1080/21645515.2017.1323591

    Scatter plot of maximum stool viral load on day 3–7 after any dose of RV3-BB against serum IgA response after 3 doses of RV3-BB (log scale) in neonatal (a) and infant (b) schedules. Participants with a viral load undetectable in the NSP3 qRT-PCR and were arbitrary assigned a value of 10 copies/g stool. Participants without an IgA serological response were assigned a titer of 10 U/ml. Gray circle identify participants without serological and/or stool viral load.
    Figure Legend Snippet: Scatter plot of maximum stool viral load on day 3–7 after any dose of RV3-BB against serum IgA response after 3 doses of RV3-BB (log scale) in neonatal (a) and infant (b) schedules. Participants with a viral load undetectable in the NSP3 qRT-PCR and were arbitrary assigned a value of 10 copies/g stool. Participants without an IgA serological response were assigned a titer of 10 U/ml. Gray circle identify participants without serological and/or stool viral load.

    Techniques Used: Quantitative RT-PCR

    Viral load in stool of participants who shed rotavirus on day 3–7 following each dose of RV3-BB in neonatal (a) and infant (b) schedules. Data points represent the mean of assay duplicates, horizontal line represents the median value. Participants who shed rotavirus (VP6 RT-PCR positive) which had a viral load undetectable in the NSP3 qRT-PCR and were arbitrary assigned a value of 10 copies/g stool.
    Figure Legend Snippet: Viral load in stool of participants who shed rotavirus on day 3–7 following each dose of RV3-BB in neonatal (a) and infant (b) schedules. Data points represent the mean of assay duplicates, horizontal line represents the median value. Participants who shed rotavirus (VP6 RT-PCR positive) which had a viral load undetectable in the NSP3 qRT-PCR and were arbitrary assigned a value of 10 copies/g stool.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    14) Product Images from "Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars"

    Article Title: Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars

    Journal: Journal of Industrial Microbiology & Biotechnology

    doi: 10.1007/s10295-011-1012-x

    Variable nucleotide tandem repeat (VNTR) fingerprint of the changes to the genomic DNA of mutagenized S. ( Pichia ) stipitis strains WT-2-1, WT-1-11, 14-2-6, 22-1-12, and 22-1-1 compared to Saccharomyces cerevisiae NRRL Y-2034 and the wild-type (WT) strain, S. stipitis NRRL Y-7124. Resulting PCR products were obtained using the VNTR primer-generated fragments and are indicative of changes to the genomes of the mutagenized strains and analyzed on a 1% (w/v) agarose gel. Bionexus DNA base-pair markers ( Lane M ) are identified by length on the left of the figure. Bands present in mutagenized strains but not present in WT S. stipitis NRRL Y-7124 are indicated by arrows . Gel was run at 80 V on a Bio-Rad Power Pac 3000 system, and a high-resolution digital image file was generated with an AlphaImager 3400 gel imaging system using a trans-UV light (Alpha Innotech Corp, San Leandro, CA)
    Figure Legend Snippet: Variable nucleotide tandem repeat (VNTR) fingerprint of the changes to the genomic DNA of mutagenized S. ( Pichia ) stipitis strains WT-2-1, WT-1-11, 14-2-6, 22-1-12, and 22-1-1 compared to Saccharomyces cerevisiae NRRL Y-2034 and the wild-type (WT) strain, S. stipitis NRRL Y-7124. Resulting PCR products were obtained using the VNTR primer-generated fragments and are indicative of changes to the genomes of the mutagenized strains and analyzed on a 1% (w/v) agarose gel. Bionexus DNA base-pair markers ( Lane M ) are identified by length on the left of the figure. Bands present in mutagenized strains but not present in WT S. stipitis NRRL Y-7124 are indicated by arrows . Gel was run at 80 V on a Bio-Rad Power Pac 3000 system, and a high-resolution digital image file was generated with an AlphaImager 3400 gel imaging system using a trans-UV light (Alpha Innotech Corp, San Leandro, CA)

    Techniques Used: Polymerase Chain Reaction, Generated, Agarose Gel Electrophoresis, Imaging

    15) Product Images from "Phylogenetic Analysis of Kindlins Suggests Subfunctionalization of an Ancestral Unduplicated Kindlin into Three Paralogs in Vertebrates"

    Article Title: Phylogenetic Analysis of Kindlins Suggests Subfunctionalization of an Ancestral Unduplicated Kindlin into Three Paralogs in Vertebrates

    Journal: Evolutionary Bioinformatics Online

    doi: 10.4137/EBO.S6179

    The expression profile of all three Kindlin paralogs was quantified by real-time RT-PCR using SYBR Green. Data are presented as the relative expression of Kindlins (fold change) normalized by a housekeeping gene, β-actin. Note: The error bars indicate the standard deviation.
    Figure Legend Snippet: The expression profile of all three Kindlin paralogs was quantified by real-time RT-PCR using SYBR Green. Data are presented as the relative expression of Kindlins (fold change) normalized by a housekeeping gene, β-actin. Note: The error bars indicate the standard deviation.

    Techniques Used: Expressing, Quantitative RT-PCR, SYBR Green Assay, Standard Deviation

    16) Product Images from "Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates"

    Article Title: Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates

    Journal: Applied Microbiology and Biotechnology

    doi: 10.1007/s00253-015-6852-2

    Variable nucleotide tandem repeat (VNTR) and Coomassie SDS PAGE analyses of Yarrowia lipolytica wild-type and mutant strains A , B , B2 , C , D , E , and F . a Genomic DNA extracted from strains grown in LNG medium was amplified by PCR using the VNTR oligonucleotide indicated at upper right and the amplified DNA loaded onto 1 % ( w / v ) agarose gels stained with ethidium bromide. Marker in lanes labeled M is lambda DNA- Hin d III/phiX-174 RF DNA- Hae III (72 bp to 23 kb); Y. lipolytica mutant strains are in lanes labeled B2 , A , B , C , D , E , and F ; wild-type strain is in lane WT . b Cell protein profile of samples prepared from cultures grown in YPD medium. Samples were loaded onto a 4 to 20 % tris-glycine SDS-PAGE gel and gel was stained with Coomassie Brilliant Blue R-250. Lane M is SeeBlue® Plus2 marker; Y. lipolytica mutant strains are in lanes labeled B2 , A , B , C , D , E , and F ; wild-type strain is in lane WT
    Figure Legend Snippet: Variable nucleotide tandem repeat (VNTR) and Coomassie SDS PAGE analyses of Yarrowia lipolytica wild-type and mutant strains A , B , B2 , C , D , E , and F . a Genomic DNA extracted from strains grown in LNG medium was amplified by PCR using the VNTR oligonucleotide indicated at upper right and the amplified DNA loaded onto 1 % ( w / v ) agarose gels stained with ethidium bromide. Marker in lanes labeled M is lambda DNA- Hin d III/phiX-174 RF DNA- Hae III (72 bp to 23 kb); Y. lipolytica mutant strains are in lanes labeled B2 , A , B , C , D , E , and F ; wild-type strain is in lane WT . b Cell protein profile of samples prepared from cultures grown in YPD medium. Samples were loaded onto a 4 to 20 % tris-glycine SDS-PAGE gel and gel was stained with Coomassie Brilliant Blue R-250. Lane M is SeeBlue® Plus2 marker; Y. lipolytica mutant strains are in lanes labeled B2 , A , B , C , D , E , and F ; wild-type strain is in lane WT

    Techniques Used: SDS Page, Mutagenesis, Amplification, Polymerase Chain Reaction, Staining, Marker, Labeling, Lambda DNA Preparation

    17) Product Images from "Dual transcripts of BCR-ABL different polymorphisms in chronic myeloid leukaemia patients"

    Article Title: Dual transcripts of BCR-ABL different polymorphisms in chronic myeloid leukaemia patients

    Journal: The Indian Journal of Medical Research

    doi: 10.4103/0971-5916.191816

    Agarose gel electrophoresis of dual transcript samples. Lane 1- 100 bp ladder, lane 2- ABL control (288 bp), lane 3- breakpoint region ( BCR-ABL ) (e13a2-308 bp and e14a2-383 bp), lanes 4 and 5- e13a2 (123 bp and 198 bp) and e14a2 (129 bp) transcript specific PCR, respectively.
    Figure Legend Snippet: Agarose gel electrophoresis of dual transcript samples. Lane 1- 100 bp ladder, lane 2- ABL control (288 bp), lane 3- breakpoint region ( BCR-ABL ) (e13a2-308 bp and e14a2-383 bp), lanes 4 and 5- e13a2 (123 bp and 198 bp) and e14a2 (129 bp) transcript specific PCR, respectively.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction

    18) Product Images from "Transcriptional Silencing of the Wnt-Antagonist DKK1 by Promoter Methylation Is Associated with Enhanced Wnt Signaling in Advanced Multiple Myeloma"

    Article Title: Transcriptional Silencing of the Wnt-Antagonist DKK1 by Promoter Methylation Is Associated with Enhanced Wnt Signaling in Advanced Multiple Myeloma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030359

    DKK1 promoter methylation in MM cell lines. (A) Total RNA was isolated and RT–PCR analyses were performed with the specific primers indicated. Complementary DNA from prostate cancer cell line (PC-3) was used as positive control (PC) for DKK1 expression. The β-actin expression was used as a loading control. (B) Schematic representation of the promoter area analyzed for DKK1 , containing a CpG island. White arrows indicate the positions of primers used for bisulfite sequencing, and black arrows indicate the positions of primers used for methylation specific PCR. Each of the CpG dinucleotides is presented as open circle. (C) Upper panel . Representation of bisulfite genomic sequencing results of 5 clones of the DKK1 promoter region in HT-29 and DLD-1 colon cell lines used as unmethylated (U_DNA) and methylated (M_DNA) control, respectively. The amplified 326 bp product corresponds to the DKK1 promoter region from −193 to +122. In total, 18 CpG dinucleotides (CpGs) within the CpG island were analyzed and are represented as open and closed circles, which indicate unmethylated and methylated CpG sites, respectively. Lower panel . Electropherograms of bisulfite modified DNA from DKK1 CpG island in HT-29 (U_DNA) and DLD-1 (M_DNA) cells. (D) Methylation specific PCR of the CpG island of the DKK1 promoter region in MM cell lines. DNA bands in lanes labeled with U indicate PCR products amplified with primers recognizing unmethylated promoter sequences, whereas DNA bands in lanes labeled with M represent amplified products with primers designed for the methylated template. (E) Bisulfite sequencing analysis of the the DKK1 promoter region in multiple myeloma cell lines, open circles indicating unmethylated CpG sites, and closed circles representing methylated CpG sites. Percentages indicate the fraction of methylated CpG dinucleotides of the total CpG sites analyzed.
    Figure Legend Snippet: DKK1 promoter methylation in MM cell lines. (A) Total RNA was isolated and RT–PCR analyses were performed with the specific primers indicated. Complementary DNA from prostate cancer cell line (PC-3) was used as positive control (PC) for DKK1 expression. The β-actin expression was used as a loading control. (B) Schematic representation of the promoter area analyzed for DKK1 , containing a CpG island. White arrows indicate the positions of primers used for bisulfite sequencing, and black arrows indicate the positions of primers used for methylation specific PCR. Each of the CpG dinucleotides is presented as open circle. (C) Upper panel . Representation of bisulfite genomic sequencing results of 5 clones of the DKK1 promoter region in HT-29 and DLD-1 colon cell lines used as unmethylated (U_DNA) and methylated (M_DNA) control, respectively. The amplified 326 bp product corresponds to the DKK1 promoter region from −193 to +122. In total, 18 CpG dinucleotides (CpGs) within the CpG island were analyzed and are represented as open and closed circles, which indicate unmethylated and methylated CpG sites, respectively. Lower panel . Electropherograms of bisulfite modified DNA from DKK1 CpG island in HT-29 (U_DNA) and DLD-1 (M_DNA) cells. (D) Methylation specific PCR of the CpG island of the DKK1 promoter region in MM cell lines. DNA bands in lanes labeled with U indicate PCR products amplified with primers recognizing unmethylated promoter sequences, whereas DNA bands in lanes labeled with M represent amplified products with primers designed for the methylated template. (E) Bisulfite sequencing analysis of the the DKK1 promoter region in multiple myeloma cell lines, open circles indicating unmethylated CpG sites, and closed circles representing methylated CpG sites. Percentages indicate the fraction of methylated CpG dinucleotides of the total CpG sites analyzed.

    Techniques Used: Methylation, Isolation, Reverse Transcription Polymerase Chain Reaction, Positive Control, Expressing, Methylation Sequencing, Polymerase Chain Reaction, Genomic Sequencing, Clone Assay, Amplification, Modification, Labeling

    19) Product Images from "Internal Transcribed Spacer Region Sequence Heterogeneity in Rhizopus microsporus: Implications for Molecular Diagnosis in Clinical Microbiology Laboratories ▿"

    Article Title: Internal Transcribed Spacer Region Sequence Heterogeneity in Rhizopus microsporus: Implications for Molecular Diagnosis in Clinical Microbiology Laboratories ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.01750-09

    DNA products from PCR of ITS in R. microsporus . Lane M, molecular marker Lambda AvaII digest; lane 1, strain P11; lane 2, strain P12; lane 3; strain D3-1; lane 4, strain D4-1; lane 5, strain P2 (positive control); lane 6, negative control containing DNase
    Figure Legend Snippet: DNA products from PCR of ITS in R. microsporus . Lane M, molecular marker Lambda AvaII digest; lane 1, strain P11; lane 2, strain P12; lane 3; strain D3-1; lane 4, strain D4-1; lane 5, strain P2 (positive control); lane 6, negative control containing DNase

    Techniques Used: Polymerase Chain Reaction, Marker, Positive Control, Negative Control

    20) Product Images from "CagL polymorphisms D58/K59 are predominant in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis"

    Article Title: CagL polymorphisms D58/K59 are predominant in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis

    Journal: Gut Pathogens

    doi: 10.1186/s13099-019-0286-9

    Representative gels of PCR amplified fragments. a 16S RNA fragment. Lanes: MW—molecular weight marker (bp), NC—negative control, PC—positive control (DNA from strain ATCC 43504), 4 to 7—clinical isolates UEGE-640, UEGE-697, UEGE-748, and UEGE-752. b cagL fragment amplified with the first set of primers (651 bp). Lanes: MW—molecular weight marker (bp), NC—negative control, PC—positive control (DNA from strain ATCC 43504), 4 to 7—clinical isolates UEGE-640, UEGE-696, UEGE-847, and UEGE-752. c cagL fragment amplified with the second set of primers (165 bp). Lanes: MW—molecular weight marker (bp), NC—negative control, PC—positive control (DNA from strain HP26695), 4 to 7—clinical isolates: UEGE-845, UEGE-652, UEGE-748, and UEGE-752. d cagL fragment amplified with the third set of primers (611 bp). Lanes: MW—molecular weight marker (bp), NC—negative control, PC—positive control (DNA from strain HP26695), 4 to 7—clinical isolates: HG-65, HG-155, HG-177, and UEGE-748
    Figure Legend Snippet: Representative gels of PCR amplified fragments. a 16S RNA fragment. Lanes: MW—molecular weight marker (bp), NC—negative control, PC—positive control (DNA from strain ATCC 43504), 4 to 7—clinical isolates UEGE-640, UEGE-697, UEGE-748, and UEGE-752. b cagL fragment amplified with the first set of primers (651 bp). Lanes: MW—molecular weight marker (bp), NC—negative control, PC—positive control (DNA from strain ATCC 43504), 4 to 7—clinical isolates UEGE-640, UEGE-696, UEGE-847, and UEGE-752. c cagL fragment amplified with the second set of primers (165 bp). Lanes: MW—molecular weight marker (bp), NC—negative control, PC—positive control (DNA from strain HP26695), 4 to 7—clinical isolates: UEGE-845, UEGE-652, UEGE-748, and UEGE-752. d cagL fragment amplified with the third set of primers (611 bp). Lanes: MW—molecular weight marker (bp), NC—negative control, PC—positive control (DNA from strain HP26695), 4 to 7—clinical isolates: HG-65, HG-155, HG-177, and UEGE-748

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Negative Control, Positive Control

    21) Product Images from "Development of a Novel Real-Time PCR Assay with High-Resolution Melt Analysis To Detect and Differentiate OXA-48-Like β-Lactamases in Carbapenem-Resistant Enterobacteriaceae"

    Article Title: Development of a Novel Real-Time PCR Assay with High-Resolution Melt Analysis To Detect and Differentiate OXA-48-Like β-Lactamases in Carbapenem-Resistant Enterobacteriaceae

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00425-15

    Normalized temperature-shifted difference plots, demonstrating different PCR product and ssDNA-LunaProbe duplex melting profiles for replicates of bla OXA-48-like genes. (A) Differentiation of bla OXA-162 and bla OXA-244 based on PCR product melting profiles.
    Figure Legend Snippet: Normalized temperature-shifted difference plots, demonstrating different PCR product and ssDNA-LunaProbe duplex melting profiles for replicates of bla OXA-48-like genes. (A) Differentiation of bla OXA-162 and bla OXA-244 based on PCR product melting profiles.

    Techniques Used: Polymerase Chain Reaction

    Nucleic acid alignment of bla OXA-48-like genes with PCR primer and LunaProbe binding regions.
    Figure Legend Snippet: Nucleic acid alignment of bla OXA-48-like genes with PCR primer and LunaProbe binding regions.

    Techniques Used: Polymerase Chain Reaction, Binding Assay

    Clinical isolates used for bla OXA-48-like PCR assay validation.
    Figure Legend Snippet: Clinical isolates used for bla OXA-48-like PCR assay validation.

    Techniques Used: Polymerase Chain Reaction

    22) Product Images from "Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes"

    Article Title: Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes

    Journal: mBio

    doi: 10.1128/mBio.02273-14

    TAC-HRM of rrs (amplicon 3) and gyrB (amplicon 2) gene segments. Singleplex PCR mixtures with primers and probes were loaded into empty microfluidic cards (1 sample per port, yielding 48 reaction mixtures per sample). Results are shown for probe-based detection (A and E), for amplification with SYTO9 (B and F), and for melt curve and difference curve analysis (C and D, G and H). The aligned melt curves show melt temperature shifts versus those for the M. tuberculosis H37Rv wild-type control based on nucleotide changes (leftward variation indicates a lower melt temperature and rightward variation a higher melt temperature). The difference plot uses the same data but plots the negative first derivative (− dF / dt ) on the y axis. The HRM software automatically labels samples that are variants from the wild-type control with a different color. Underlining indicates nucleotides that changed. The rrs (amplicon 3) primer/probe results show the amplification curve obtained with the rrs -A(1401)G-specific probe for a mutant isolate, while the results for M. tuberculosis H37Rv were negative. Both the mutant isolate and H37Rv amplified with the primers using SYTO9, and the HRM result reinforced the probe result as being a mutant/variant sample. The gyrB (amplicon 2) primer/probe results demonstrated the benefit of combining the probe with HRM, since the specific probe (E) detected the Asp461His (GAC→CAC) transversion, while HRM would not (F, G, and H); however, HRM detected the rare mutations Asp461Ala and Asp461Asn.
    Figure Legend Snippet: TAC-HRM of rrs (amplicon 3) and gyrB (amplicon 2) gene segments. Singleplex PCR mixtures with primers and probes were loaded into empty microfluidic cards (1 sample per port, yielding 48 reaction mixtures per sample). Results are shown for probe-based detection (A and E), for amplification with SYTO9 (B and F), and for melt curve and difference curve analysis (C and D, G and H). The aligned melt curves show melt temperature shifts versus those for the M. tuberculosis H37Rv wild-type control based on nucleotide changes (leftward variation indicates a lower melt temperature and rightward variation a higher melt temperature). The difference plot uses the same data but plots the negative first derivative (− dF / dt ) on the y axis. The HRM software automatically labels samples that are variants from the wild-type control with a different color. Underlining indicates nucleotides that changed. The rrs (amplicon 3) primer/probe results show the amplification curve obtained with the rrs -A(1401)G-specific probe for a mutant isolate, while the results for M. tuberculosis H37Rv were negative. Both the mutant isolate and H37Rv amplified with the primers using SYTO9, and the HRM result reinforced the probe result as being a mutant/variant sample. The gyrB (amplicon 2) primer/probe results demonstrated the benefit of combining the probe with HRM, since the specific probe (E) detected the Asp461His (GAC→CAC) transversion, while HRM would not (F, G, and H); however, HRM detected the rare mutations Asp461Ala and Asp461Asn.

    Techniques Used: Amplification, Polymerase Chain Reaction, Software, Mutagenesis, Variant Assay

    23) Product Images from "Performance of Nanoliter-Sized Droplet-based Microfluidic PCR"

    Article Title: Performance of Nanoliter-Sized Droplet-based Microfluidic PCR

    Journal: Biomedical microdevices

    doi: 10.1007/s10544-009-9324-6

    Fluorescence images of the reaction droplet (a) before PCR and (b) after 40 PCR cycles. The template λDNA concentration is 3.5ng/µL (~6.69×10 7 copies/µL). The droplet size is ~225nL. The scale bar represents 250µm.
    Figure Legend Snippet: Fluorescence images of the reaction droplet (a) before PCR and (b) after 40 PCR cycles. The template λDNA concentration is 3.5ng/µL (~6.69×10 7 copies/µL). The droplet size is ~225nL. The scale bar represents 250µm.

    Techniques Used: Fluorescence, Polymerase Chain Reaction, Concentration Assay

    Fluorescence intensity of reaction droplet containing different concentrations of template λDNA after 16 PCR cycles. The droplet size is ~150nL. The error bar represents the standard deviation.
    Figure Legend Snippet: Fluorescence intensity of reaction droplet containing different concentrations of template λDNA after 16 PCR cycles. The droplet size is ~150nL. The error bar represents the standard deviation.

    Techniques Used: Fluorescence, Polymerase Chain Reaction, Standard Deviation

    24) Product Images from "Characterization of Chlamydia trachomatis omp1 Genotypes Detected in Eye Swab Samples from Remote Australian Communities"

    Article Title: Characterization of Chlamydia trachomatis omp1 Genotypes Detected in Eye Swab Samples from Remote Australian Communities

    Journal:

    doi: 10.1128/JCM.42.6.2501-2507.2004

    Schematic representation of the C . trachomatis genome. The omp1 gene, approximately 1.2 kb in length, was PCR amplified with various combinations of primers. Primers P1 and OMP2 were used in the primary PCR. DNA which failed to generate primary PCR products
    Figure Legend Snippet: Schematic representation of the C . trachomatis genome. The omp1 gene, approximately 1.2 kb in length, was PCR amplified with various combinations of primers. Primers P1 and OMP2 were used in the primary PCR. DNA which failed to generate primary PCR products

    Techniques Used: Polymerase Chain Reaction, Amplification

    25) Product Images from "In Vitro and in Vivo Selection of Potentially Probiotic Lactobacilli From Nocellara del Belice Table Olives"

    Article Title: In Vitro and in Vivo Selection of Potentially Probiotic Lactobacilli From Nocellara del Belice Table Olives

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00595

    Strain typing of the selected L. pentosus and L. coryniformis isolates by rep-PCR. Agarose gel electrophoresis of GTG 5 rep-PCR fingerprinting profiles of L. coryniformis and L. pentosus strains. M: 1 kb DNA ladder (Microzone, UK).
    Figure Legend Snippet: Strain typing of the selected L. pentosus and L. coryniformis isolates by rep-PCR. Agarose gel electrophoresis of GTG 5 rep-PCR fingerprinting profiles of L. coryniformis and L. pentosus strains. M: 1 kb DNA ladder (Microzone, UK).

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis

    26) Product Images from "Morphological and molecular identification of the brown dog tick Rhipicephalus sanguineus and the camel tick Hyalomma dromedarii (Acari: Ixodidae) vectors of Rickettsioses in Egypt"

    Article Title: Morphological and molecular identification of the brown dog tick Rhipicephalus sanguineus and the camel tick Hyalomma dromedarii (Acari: Ixodidae) vectors of Rickettsioses in Egypt

    Journal: Veterinary World

    doi: 10.14202/vetworld.2016.1087-1101

    Molecular identification of tick species ( Rhipicephalus sanguineus and Hyalomma dromedarii ) by polymerase chain reaction products of the five DNA markers detected in 1.5% agarose gels stained with ethiduim bromide. In all figures Lane M: 100 bp DNA ladder, Lane 1 or 3: Control negative. (a) Lane 1 present 780 bp amplicon of 18S rRNA gene of R. sanguineus , while Lane 2 present 780 bp amplicon of 18S ribosomal ribonucleic acid (rRNA) gene of H. dromedarii , (b) 1200 bp amplicons of Second Internal transcribed spacer (ITS2) of R. sanguineus , (c) 1500 bp amplicons of ITS2 of H. dromedarii , (d and e) 380 bp amplicon of 12S rRNA gene of both R. sanguineus and H. dromedarii , (f) Lane 2 present 850 bp amplicon of cytochrome c oxidase subunit-1 (CO1) gene of R. sanguineus , while Lane 3 present 850 bp amplicon of CO1 gene of H. dromedarii , (g) Lane 1 present 455 bp amplicon of 16S rRNA gene of R. sanguineus , while Lane 2 present 455 bp amplicon of 16S rRNA gene of H. dromedarii .
    Figure Legend Snippet: Molecular identification of tick species ( Rhipicephalus sanguineus and Hyalomma dromedarii ) by polymerase chain reaction products of the five DNA markers detected in 1.5% agarose gels stained with ethiduim bromide. In all figures Lane M: 100 bp DNA ladder, Lane 1 or 3: Control negative. (a) Lane 1 present 780 bp amplicon of 18S rRNA gene of R. sanguineus , while Lane 2 present 780 bp amplicon of 18S ribosomal ribonucleic acid (rRNA) gene of H. dromedarii , (b) 1200 bp amplicons of Second Internal transcribed spacer (ITS2) of R. sanguineus , (c) 1500 bp amplicons of ITS2 of H. dromedarii , (d and e) 380 bp amplicon of 12S rRNA gene of both R. sanguineus and H. dromedarii , (f) Lane 2 present 850 bp amplicon of cytochrome c oxidase subunit-1 (CO1) gene of R. sanguineus , while Lane 3 present 850 bp amplicon of CO1 gene of H. dromedarii , (g) Lane 1 present 455 bp amplicon of 16S rRNA gene of R. sanguineus , while Lane 2 present 455 bp amplicon of 16S rRNA gene of H. dromedarii .

    Techniques Used: Polymerase Chain Reaction, Staining, Amplification

    Molecular identification of tick-borne Rickettsiae by polymerase chain reaction products of the OmpA (a) and gltA (b) genes detected in Rhipicephalus sanguineus and Hyalomma dromedarii species in 1.5% agarose gels stained with ethiduim bromide. Lane M: 100 bp DNA ladder, Lane N: Control negative, Lane P: Control positive and Lane 1 and 6 are negative tick samples, while tick samples on Lanes 2 to 5 are OmpA positive (a) with molecular sized ranged from 600 bp. Whereas, Lane 1 (b) are gltA positive with molecular size 1200 bp.
    Figure Legend Snippet: Molecular identification of tick-borne Rickettsiae by polymerase chain reaction products of the OmpA (a) and gltA (b) genes detected in Rhipicephalus sanguineus and Hyalomma dromedarii species in 1.5% agarose gels stained with ethiduim bromide. Lane M: 100 bp DNA ladder, Lane N: Control negative, Lane P: Control positive and Lane 1 and 6 are negative tick samples, while tick samples on Lanes 2 to 5 are OmpA positive (a) with molecular sized ranged from 600 bp. Whereas, Lane 1 (b) are gltA positive with molecular size 1200 bp.

    Techniques Used: Polymerase Chain Reaction, Staining

    27) Product Images from "Superinfection with Woodchuck Hepatitis Virus Strain WHVNY of Livers Chronically Infected with Strain WHV7"

    Article Title: Superinfection with Woodchuck Hepatitis Virus Strain WHVNY of Livers Chronically Infected with Strain WHV7

    Journal: Journal of Virology

    doi: 10.1128/JVI.02361-14

    Detection of rcDNA of WHVNY and WHV7 in serum samples harvested from WHV7 chronic carrier woodchucks superinfected with strain WHVNY. The isolation of rcDNA and conventional nested WHV strain-specific PCR assays are described in Materials and Methods.
    Figure Legend Snippet: Detection of rcDNA of WHVNY and WHV7 in serum samples harvested from WHV7 chronic carrier woodchucks superinfected with strain WHVNY. The isolation of rcDNA and conventional nested WHV strain-specific PCR assays are described in Materials and Methods.

    Techniques Used: Isolation, Polymerase Chain Reaction

    Quantification of rcDNA of WHVNY and WHV7 in sera of superinfected woodchucks F7808, F7806, and M7761. The isolation of WHV rcDNA from serum samples and WHV strain-specific real-time PCR assays that were used to quantify the rcDNA levels of WHVNY and
    Figure Legend Snippet: Quantification of rcDNA of WHVNY and WHV7 in sera of superinfected woodchucks F7808, F7806, and M7761. The isolation of WHV rcDNA from serum samples and WHV strain-specific real-time PCR assays that were used to quantify the rcDNA levels of WHVNY and

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction

    28) Product Images from "Ginsenosides Rb1 and Rg1 Protect Primary Cultured Astrocytes against Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury via Improving Mitochondrial Function"

    Article Title: Ginsenosides Rb1 and Rg1 Protect Primary Cultured Astrocytes against Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury via Improving Mitochondrial Function

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20236086

    Rb1 and Rg1 attenuated the mitochondrial membrane potential (MMP) depolarization and increased the mitochondrial DNA (mtDNA) content in oxygen-glucose deprivation/reoxygenation (OGD/R)-treated astrocytes. After 6 h of OGD, astrocytes were reoxygenated for 0, 4, 12, or 24 h. ( a , b ) Cells were collected to detect the MMP. After 6 h of OGD, astrocytes were reoxygenated for 4 or 24 h. ( c ) Cells were collected to examine the mtDNA copy number by real-time quantitative PCR. Rb1 (5 µM) and Rg1 (10 µM) administration attenuated the MMP depolarization and increased the mtDNA content. The values are expressed as the mean ± SD ( n = 3). ## p
    Figure Legend Snippet: Rb1 and Rg1 attenuated the mitochondrial membrane potential (MMP) depolarization and increased the mitochondrial DNA (mtDNA) content in oxygen-glucose deprivation/reoxygenation (OGD/R)-treated astrocytes. After 6 h of OGD, astrocytes were reoxygenated for 0, 4, 12, or 24 h. ( a , b ) Cells were collected to detect the MMP. After 6 h of OGD, astrocytes were reoxygenated for 4 or 24 h. ( c ) Cells were collected to examine the mtDNA copy number by real-time quantitative PCR. Rb1 (5 µM) and Rg1 (10 µM) administration attenuated the MMP depolarization and increased the mtDNA content. The values are expressed as the mean ± SD ( n = 3). ## p

    Techniques Used: Real-time Polymerase Chain Reaction

    29) Product Images from "Regulation of human glioma cell apoptosis and invasion by miR-152-3p through targeting DNMT1 and regulating NF2"

    Article Title: Regulation of human glioma cell apoptosis and invasion by miR-152-3p through targeting DNMT1 and regulating NF2

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0567-4

    MiR-152-3p was involved in the regulation of NF2expression.mRNA expression of DNMT1, NF2, and miR-152-3p after transfection with miR-152-3p mimics, detected by RT-PCR in U251 cells ( a ). Protein expression of DNMT1 and NF2 was detected by Western blot after transfection with miR-152-3p mimics ( b ). The expression of DNMT1 and NF2in U251 cells was detected by immunofluorescence ( c ). After miR-152-3p transfection, the methylation status of NF2 in U251 cells was determined with MSP ( d ) and bisulfite Sanger sequencing ( e ). **, P
    Figure Legend Snippet: MiR-152-3p was involved in the regulation of NF2expression.mRNA expression of DNMT1, NF2, and miR-152-3p after transfection with miR-152-3p mimics, detected by RT-PCR in U251 cells ( a ). Protein expression of DNMT1 and NF2 was detected by Western blot after transfection with miR-152-3p mimics ( b ). The expression of DNMT1 and NF2in U251 cells was detected by immunofluorescence ( c ). After miR-152-3p transfection, the methylation status of NF2 in U251 cells was determined with MSP ( d ) and bisulfite Sanger sequencing ( e ). **, P

    Techniques Used: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Methylation, Sequencing

    DNMT1 negatively regulates the expression of NF2. Levels of mRNA expression of DNMT1, NF2, and miR-152-3p after transfection with DNMT1 siRNA and pcDNA-DNMT1 detected by RT-PCR in U251 cells ( a ). Protein expression of DNMT1 and NF2wasdetected by Western blot ( b ) and immunofluorescence ( c ). After DNMT1 knockdown, the methylation status of NF2 in U251 cells was determined with MSP ( d ) and bisulfite Sanger sequencing ( e ). *, P
    Figure Legend Snippet: DNMT1 negatively regulates the expression of NF2. Levels of mRNA expression of DNMT1, NF2, and miR-152-3p after transfection with DNMT1 siRNA and pcDNA-DNMT1 detected by RT-PCR in U251 cells ( a ). Protein expression of DNMT1 and NF2wasdetected by Western blot ( b ) and immunofluorescence ( c ). After DNMT1 knockdown, the methylation status of NF2 in U251 cells was determined with MSP ( d ) and bisulfite Sanger sequencing ( e ). *, P

    Techniques Used: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Methylation, Sequencing

    30) Product Images from "Morphological and molecular identification of the brown dog tick Rhipicephalus sanguineus and the camel tick Hyalomma dromedarii (Acari: Ixodidae) vectors of Rickettsioses in Egypt"

    Article Title: Morphological and molecular identification of the brown dog tick Rhipicephalus sanguineus and the camel tick Hyalomma dromedarii (Acari: Ixodidae) vectors of Rickettsioses in Egypt

    Journal: Veterinary World

    doi: 10.14202/vetworld.2016.1087-1101

    Molecular identification of tick species ( Rhipicephalus sanguineus and Hyalomma dromedarii ) by polymerase chain reaction products of the five DNA markers detected in 1.5% agarose gels stained with ethiduim bromide. In all figures Lane M: 100 bp DNA ladder, Lane 1 or 3: Control negative. (a) Lane 1 present 780 bp amplicon of 18S rRNA gene of R. sanguineus , while Lane 2 present 780 bp amplicon of 18S ribosomal ribonucleic acid (rRNA) gene of H. dromedarii , (b) 1200 bp amplicons of Second Internal transcribed spacer (ITS2) of R. sanguineus , (c) 1500 bp amplicons of ITS2 of H. dromedarii , (d and e) 380 bp amplicon of 12S rRNA gene of both R. sanguineus and H. dromedarii , (f) Lane 2 present 850 bp amplicon of cytochrome c oxidase subunit-1 (CO1) gene of R. sanguineus , while Lane 3 present 850 bp amplicon of CO1 gene of H. dromedarii , (g) Lane 1 present 455 bp amplicon of 16S rRNA gene of R. sanguineus , while Lane 2 present 455 bp amplicon of 16S rRNA gene of H. dromedarii .
    Figure Legend Snippet: Molecular identification of tick species ( Rhipicephalus sanguineus and Hyalomma dromedarii ) by polymerase chain reaction products of the five DNA markers detected in 1.5% agarose gels stained with ethiduim bromide. In all figures Lane M: 100 bp DNA ladder, Lane 1 or 3: Control negative. (a) Lane 1 present 780 bp amplicon of 18S rRNA gene of R. sanguineus , while Lane 2 present 780 bp amplicon of 18S ribosomal ribonucleic acid (rRNA) gene of H. dromedarii , (b) 1200 bp amplicons of Second Internal transcribed spacer (ITS2) of R. sanguineus , (c) 1500 bp amplicons of ITS2 of H. dromedarii , (d and e) 380 bp amplicon of 12S rRNA gene of both R. sanguineus and H. dromedarii , (f) Lane 2 present 850 bp amplicon of cytochrome c oxidase subunit-1 (CO1) gene of R. sanguineus , while Lane 3 present 850 bp amplicon of CO1 gene of H. dromedarii , (g) Lane 1 present 455 bp amplicon of 16S rRNA gene of R. sanguineus , while Lane 2 present 455 bp amplicon of 16S rRNA gene of H. dromedarii .

    Techniques Used: Polymerase Chain Reaction, Staining, Amplification

    Molecular identification of tick-borne Rickettsiae by polymerase chain reaction products of the OmpA (a) and gltA (b) genes detected in Rhipicephalus sanguineus and Hyalomma dromedarii species in 1.5% agarose gels stained with ethiduim bromide. Lane M: 100 bp DNA ladder, Lane N: Control negative, Lane P: Control positive and Lane 1 and 6 are negative tick samples, while tick samples on Lanes 2 to 5 are OmpA positive (a) with molecular sized ranged from 600 bp. Whereas, Lane 1 (b) are gltA positive with molecular size 1200 bp.
    Figure Legend Snippet: Molecular identification of tick-borne Rickettsiae by polymerase chain reaction products of the OmpA (a) and gltA (b) genes detected in Rhipicephalus sanguineus and Hyalomma dromedarii species in 1.5% agarose gels stained with ethiduim bromide. Lane M: 100 bp DNA ladder, Lane N: Control negative, Lane P: Control positive and Lane 1 and 6 are negative tick samples, while tick samples on Lanes 2 to 5 are OmpA positive (a) with molecular sized ranged from 600 bp. Whereas, Lane 1 (b) are gltA positive with molecular size 1200 bp.

    Techniques Used: Polymerase Chain Reaction, Staining

    31) Product Images from "Cloning and Sequence Analysis of LipL32, a Surface-Exposed Lipoprotein of Pathogenic Leptospira Spp"

    Article Title: Cloning and Sequence Analysis of LipL32, a Surface-Exposed Lipoprotein of Pathogenic Leptospira Spp

    Journal: Iranian Red Crescent Medical Journal

    doi: 10.5812/ircmj.8793

    PCR amplification of the 835 bp LipL32 gene of L.interrogans serovars: 100 bp DNA ladder, Positive control: serovar Pomona, Lane1: serovar Grippotyphosa, Lane 2: serovar Sejroe Hardjo, Lane3: serovar Canicola, Lane 4: saprophytic serovar Biflexa, and negative control.
    Figure Legend Snippet: PCR amplification of the 835 bp LipL32 gene of L.interrogans serovars: 100 bp DNA ladder, Positive control: serovar Pomona, Lane1: serovar Grippotyphosa, Lane 2: serovar Sejroe Hardjo, Lane3: serovar Canicola, Lane 4: saprophytic serovar Biflexa, and negative control.

    Techniques Used: Polymerase Chain Reaction, Amplification, Positive Control, Negative Control

    32) Product Images from "Episodes of horizontal gene-transfer and gene-fusion led to co-existence of different metal-ion specific glyoxalase I"

    Article Title: Episodes of horizontal gene-transfer and gene-fusion led to co-existence of different metal-ion specific glyoxalase I

    Journal: Scientific Reports

    doi: 10.1038/srep03076

    Transcript profiling of rice GlxI genes in response to various abiotic stress conditions. (A) Histogram depicting logarithmic expression change of rice GlxI genes based on qRT-PCR analysis. Real time PCR analysis was done with cDNA template generated from shoot tissue of 14 day old stressed or control seedlings. As OsGLYI7.2 and OsGLYI11.1 could not be amplified they are not included in the real-time PCR analysis. (B) Heat map and hierarchical cluster display of expression profile for GlxI genes showing different levels of expression in response to stress at 6 h and 24 h. Colour bar at the bottom represent expression values in terms of logarithmic fold, green (lowest), black (medium) and red (highest) expression levels.
    Figure Legend Snippet: Transcript profiling of rice GlxI genes in response to various abiotic stress conditions. (A) Histogram depicting logarithmic expression change of rice GlxI genes based on qRT-PCR analysis. Real time PCR analysis was done with cDNA template generated from shoot tissue of 14 day old stressed or control seedlings. As OsGLYI7.2 and OsGLYI11.1 could not be amplified they are not included in the real-time PCR analysis. (B) Heat map and hierarchical cluster display of expression profile for GlxI genes showing different levels of expression in response to stress at 6 h and 24 h. Colour bar at the bottom represent expression values in terms of logarithmic fold, green (lowest), black (medium) and red (highest) expression levels.

    Techniques Used: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Generated, Amplification

    33) Product Images from "Relationship of four vitamin D receptor gene polymorphisms with type 1 diabetes mellitus susceptibility in Kuwaiti children"

    Article Title: Relationship of four vitamin D receptor gene polymorphisms with type 1 diabetes mellitus susceptibility in Kuwaiti children

    Journal: BMC Pediatrics

    doi: 10.1186/s12887-019-1448-0

    Detection of VDR gene Taq I polymorphism. PCR amplification of the genomic DNA was carried out and the products of amplification were cleaved with restriction enzyme Taq I (details given in Methods ). Lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product, lanes 3,8 Taq I cleavage pattern from subjects with heterozygous TC genotype; lanes 4,7 Taq I cleaved PCR product from subjects with CC genotype; lanes 5,9–10 Taq I cleaved PCR products from a subject with TT genotype, lane 6, no sample. The products were analyzed on 2% agarose gel and visualized under UV light after staining with ethidium bromide. The number on the right side indicate the product sizes (bp)
    Figure Legend Snippet: Detection of VDR gene Taq I polymorphism. PCR amplification of the genomic DNA was carried out and the products of amplification were cleaved with restriction enzyme Taq I (details given in Methods ). Lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product, lanes 3,8 Taq I cleavage pattern from subjects with heterozygous TC genotype; lanes 4,7 Taq I cleaved PCR product from subjects with CC genotype; lanes 5,9–10 Taq I cleaved PCR products from a subject with TT genotype, lane 6, no sample. The products were analyzed on 2% agarose gel and visualized under UV light after staining with ethidium bromide. The number on the right side indicate the product sizes (bp)

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining

    Detection of VDR gene Bsm I polymorphism. PCR amplification of genomic DNA was carried out and the products of amplification were cleaved with restriction enzyme Mva I (details given in Methods ). A: lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lanes 3–7, cleavage products from subjects with BB genotype. B: lane 1, ϕX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lane 3,5, cleavage products from subjects with bb genotype; lanes 4, cleavage products from subjects having Bb genotype. The numbers on the side are sizes (bp) of the characteristic bands resulting from PCR-RFLP analysis of the subjects having different genotypes. The restriction enzyme cleavage products were analyzed on 2% agarose gel and visualized under UV light after staining with Ethidium bromide
    Figure Legend Snippet: Detection of VDR gene Bsm I polymorphism. PCR amplification of genomic DNA was carried out and the products of amplification were cleaved with restriction enzyme Mva I (details given in Methods ). A: lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lanes 3–7, cleavage products from subjects with BB genotype. B: lane 1, ϕX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lane 3,5, cleavage products from subjects with bb genotype; lanes 4, cleavage products from subjects having Bb genotype. The numbers on the side are sizes (bp) of the characteristic bands resulting from PCR-RFLP analysis of the subjects having different genotypes. The restriction enzyme cleavage products were analyzed on 2% agarose gel and visualized under UV light after staining with Ethidium bromide

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining

    Detection of VDR gene Apa I polymorphism. PCR amplification of the genomic DNA was carried out and products of amplification were cleaved with restriction enzyme Apa I (details given in Methods ). Lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lanes 3,4,8,9, products from subjects with heterozygous GT genotype; lanes 5–6, products from subjects with homozygous TT genotype; lane 7, products from homozygous GG genotype. The numbers on the side are sizes (bp) of the characteristic bands resulting from PCR-RFLP analysis. The cleavage products were analyzed on 2% agarose gel and visualized under UV light after staining with Ethidium bromide
    Figure Legend Snippet: Detection of VDR gene Apa I polymorphism. PCR amplification of the genomic DNA was carried out and products of amplification were cleaved with restriction enzyme Apa I (details given in Methods ). Lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lanes 3,4,8,9, products from subjects with heterozygous GT genotype; lanes 5–6, products from subjects with homozygous TT genotype; lane 7, products from homozygous GG genotype. The numbers on the side are sizes (bp) of the characteristic bands resulting from PCR-RFLP analysis. The cleavage products were analyzed on 2% agarose gel and visualized under UV light after staining with Ethidium bromide

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining

    Detection of VDR gene Fok I polymorphism. PCR amplification of the genomic DNA was carried out and the products of amplification were cleaved with restriction enzyme Fok I (details given in Methods ). Lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lane 3–5,7–9 13, PCR products from subjects having ff genotype; lane 6, pattern from a subject with homozygous FF genotype; lanes 10–12, pattern from subjects with heterozygous Ff genotype. The numbers on the side are sizes (bp) of the characteristic bands resulting from PCR-RFLP analysis. The restriction enzyme cleavage products were analyzed on 2% agarose gel and visualized under UV light after staining with Ethidium bromide
    Figure Legend Snippet: Detection of VDR gene Fok I polymorphism. PCR amplification of the genomic DNA was carried out and the products of amplification were cleaved with restriction enzyme Fok I (details given in Methods ). Lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lane 3–5,7–9 13, PCR products from subjects having ff genotype; lane 6, pattern from a subject with homozygous FF genotype; lanes 10–12, pattern from subjects with heterozygous Ff genotype. The numbers on the side are sizes (bp) of the characteristic bands resulting from PCR-RFLP analysis. The restriction enzyme cleavage products were analyzed on 2% agarose gel and visualized under UV light after staining with Ethidium bromide

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining

    34) Product Images from "Relationship of four vitamin D receptor gene polymorphisms with type 1 diabetes mellitus susceptibility in Kuwaiti children"

    Article Title: Relationship of four vitamin D receptor gene polymorphisms with type 1 diabetes mellitus susceptibility in Kuwaiti children

    Journal: BMC Pediatrics

    doi: 10.1186/s12887-019-1448-0

    Detection of VDR gene Taq I polymorphism. PCR amplification of the genomic DNA was carried out and the products of amplification were cleaved with restriction enzyme Taq I (details given in Methods ). Lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product, lanes 3,8 Taq I cleavage pattern from subjects with heterozygous TC genotype; lanes 4,7 Taq I cleaved PCR product from subjects with CC genotype; lanes 5,9–10 Taq I cleaved PCR products from a subject with TT genotype, lane 6, no sample. The products were analyzed on 2% agarose gel and visualized under UV light after staining with ethidium bromide. The number on the right side indicate the product sizes (bp)
    Figure Legend Snippet: Detection of VDR gene Taq I polymorphism. PCR amplification of the genomic DNA was carried out and the products of amplification were cleaved with restriction enzyme Taq I (details given in Methods ). Lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product, lanes 3,8 Taq I cleavage pattern from subjects with heterozygous TC genotype; lanes 4,7 Taq I cleaved PCR product from subjects with CC genotype; lanes 5,9–10 Taq I cleaved PCR products from a subject with TT genotype, lane 6, no sample. The products were analyzed on 2% agarose gel and visualized under UV light after staining with ethidium bromide. The number on the right side indicate the product sizes (bp)

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining

    Detection of VDR gene Bsm I polymorphism. PCR amplification of genomic DNA was carried out and the products of amplification were cleaved with restriction enzyme Mva I (details given in Methods ). A: lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lanes 3–7, cleavage products from subjects with BB genotype. B: lane 1, ϕX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lane 3,5, cleavage products from subjects with bb genotype; lanes 4, cleavage products from subjects having Bb genotype. The numbers on the side are sizes (bp) of the characteristic bands resulting from PCR-RFLP analysis of the subjects having different genotypes. The restriction enzyme cleavage products were analyzed on 2% agarose gel and visualized under UV light after staining with Ethidium bromide
    Figure Legend Snippet: Detection of VDR gene Bsm I polymorphism. PCR amplification of genomic DNA was carried out and the products of amplification were cleaved with restriction enzyme Mva I (details given in Methods ). A: lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lanes 3–7, cleavage products from subjects with BB genotype. B: lane 1, ϕX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lane 3,5, cleavage products from subjects with bb genotype; lanes 4, cleavage products from subjects having Bb genotype. The numbers on the side are sizes (bp) of the characteristic bands resulting from PCR-RFLP analysis of the subjects having different genotypes. The restriction enzyme cleavage products were analyzed on 2% agarose gel and visualized under UV light after staining with Ethidium bromide

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining

    Detection of VDR gene Apa I polymorphism. PCR amplification of the genomic DNA was carried out and products of amplification were cleaved with restriction enzyme Apa I (details given in Methods ). Lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lanes 3,4,8,9, products from subjects with heterozygous GT genotype; lanes 5–6, products from subjects with homozygous TT genotype; lane 7, products from homozygous GG genotype. The numbers on the side are sizes (bp) of the characteristic bands resulting from PCR-RFLP analysis. The cleavage products were analyzed on 2% agarose gel and visualized under UV light after staining with Ethidium bromide
    Figure Legend Snippet: Detection of VDR gene Apa I polymorphism. PCR amplification of the genomic DNA was carried out and products of amplification were cleaved with restriction enzyme Apa I (details given in Methods ). Lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lanes 3,4,8,9, products from subjects with heterozygous GT genotype; lanes 5–6, products from subjects with homozygous TT genotype; lane 7, products from homozygous GG genotype. The numbers on the side are sizes (bp) of the characteristic bands resulting from PCR-RFLP analysis. The cleavage products were analyzed on 2% agarose gel and visualized under UV light after staining with Ethidium bromide

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining

    Detection of VDR gene Fok I polymorphism. PCR amplification of the genomic DNA was carried out and the products of amplification were cleaved with restriction enzyme Fok I (details given in Methods ). Lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lane 3–5,7–9 13, PCR products from subjects having ff genotype; lane 6, pattern from a subject with homozygous FF genotype; lanes 10–12, pattern from subjects with heterozygous Ff genotype. The numbers on the side are sizes (bp) of the characteristic bands resulting from PCR-RFLP analysis. The restriction enzyme cleavage products were analyzed on 2% agarose gel and visualized under UV light after staining with Ethidium bromide
    Figure Legend Snippet: Detection of VDR gene Fok I polymorphism. PCR amplification of the genomic DNA was carried out and the products of amplification were cleaved with restriction enzyme Fok I (details given in Methods ). Lane 1, phiX174 Hae III cut M r markers; lane 2, uncleaved PCR product; lane 3–5,7–9 13, PCR products from subjects having ff genotype; lane 6, pattern from a subject with homozygous FF genotype; lanes 10–12, pattern from subjects with heterozygous Ff genotype. The numbers on the side are sizes (bp) of the characteristic bands resulting from PCR-RFLP analysis. The restriction enzyme cleavage products were analyzed on 2% agarose gel and visualized under UV light after staining with Ethidium bromide

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining

    35) Product Images from "Duplex PCR for Differential Identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in Formalin- Fixed Paraffin-Embedded Tissues from Cattle"

    Article Title: Duplex PCR for Differential Identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in Formalin- Fixed Paraffin-Embedded Tissues from Cattle

    Journal: Journal of Clinical Microbiology

    doi:

    PCR duplex analysis of DNA purified from formalin-fixed and paraffin-embedded paratuberculosis tissues. DNA samples (100 ng) isolated from mesenteric lymph node biopsy specimens and were used in a duplex PCR procedure. (A) Aliquots of PCR-amplified DNA (50 μl of each amplified sample) were analyzed by 2% agarose gel electrophoresis. (B) Hybridization analysis of the same amplified products with the DIG-labeled f57 and us-p34 probes. Lanes 1, Bgl I- and Hinf I-cleaved pBR328 weight marker; lanes 2 and 3, specimens from pluribacillary cows; lanes 4 and 5, specimens from paratuberculous cows (lanes of panel B are as labeled in panel A).
    Figure Legend Snippet: PCR duplex analysis of DNA purified from formalin-fixed and paraffin-embedded paratuberculosis tissues. DNA samples (100 ng) isolated from mesenteric lymph node biopsy specimens and were used in a duplex PCR procedure. (A) Aliquots of PCR-amplified DNA (50 μl of each amplified sample) were analyzed by 2% agarose gel electrophoresis. (B) Hybridization analysis of the same amplified products with the DIG-labeled f57 and us-p34 probes. Lanes 1, Bgl I- and Hinf I-cleaved pBR328 weight marker; lanes 2 and 3, specimens from pluribacillary cows; lanes 4 and 5, specimens from paratuberculous cows (lanes of panel B are as labeled in panel A).

    Techniques Used: Polymerase Chain Reaction, Purification, Isolation, Amplification, Agarose Gel Electrophoresis, Hybridization, Labeling, Marker

    36) Product Images from "An Outbreak of Keratitis Caused by Mycobacterium immunogenum"

    Article Title: An Outbreak of Keratitis Caused by Mycobacterium immunogenum

    Journal:

    doi: 10.1128/JCM.00656-06

    (A) PFGE pattern of DraI digests of chromosomal DNA. Lanes: 1 and 6, lambda DNA standards (New England Biolabs); 2, 3, 4, and 5, outbreak isolates F1111, F1112, F1113, and F1114, respectively. (B) Electrophoretic patterns obtained by ERIC-PCR. Lanes:
    Figure Legend Snippet: (A) PFGE pattern of DraI digests of chromosomal DNA. Lanes: 1 and 6, lambda DNA standards (New England Biolabs); 2, 3, 4, and 5, outbreak isolates F1111, F1112, F1113, and F1114, respectively. (B) Electrophoretic patterns obtained by ERIC-PCR. Lanes:

    Techniques Used: Lambda DNA Preparation, Polymerase Chain Reaction

    37) Product Images from "The CD3 versus CD7 Plot in Multicolor Flow Cytometry Reflects Progression of Disease Stage in Patients Infected with HTLV-I"

    Article Title: The CD3 versus CD7 Plot in Multicolor Flow Cytometry Reflects Progression of Disease Stage in Patients Infected with HTLV-I

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053728

    HTLV-I proviral load (VL) and clonality in each subpopulation, based on the CD3 versus CD7 plot. (A) The three subpopulations (H, D, L) based on the CD3 versus CD7 plot were subjected to fluorescence-activated cell sorting (FACS) and VL analysis. Three representative cases are shown. G1 or G2 in the dotted box indicates Group 1 or Group 2, categorized by the percentage of the D and L subpopulations, respectively. (B)–(D) Analysis of clonality in the three subpopulations based on the CD3 versus CD7 plot. Genomic DNA was extracted from FACS-sorted cells of each subpopulation and subjected to inverse long polymerase chain reaction (PCR). Representative data of two cases of AC (B), three cases of smoldering type, including one with skin manifestations (C), and cases of a chronic type and an acute type (D) are shown. PCR was performed in duplicate (black bars) in cases when a sufficient amount of DNA was obtained.
    Figure Legend Snippet: HTLV-I proviral load (VL) and clonality in each subpopulation, based on the CD3 versus CD7 plot. (A) The three subpopulations (H, D, L) based on the CD3 versus CD7 plot were subjected to fluorescence-activated cell sorting (FACS) and VL analysis. Three representative cases are shown. G1 or G2 in the dotted box indicates Group 1 or Group 2, categorized by the percentage of the D and L subpopulations, respectively. (B)–(D) Analysis of clonality in the three subpopulations based on the CD3 versus CD7 plot. Genomic DNA was extracted from FACS-sorted cells of each subpopulation and subjected to inverse long polymerase chain reaction (PCR). Representative data of two cases of AC (B), three cases of smoldering type, including one with skin manifestations (C), and cases of a chronic type and an acute type (D) are shown. PCR was performed in duplicate (black bars) in cases when a sufficient amount of DNA was obtained.

    Techniques Used: Fluorescence, FACS, Polymerase Chain Reaction

    38) Product Images from "Ecology of Uncultivated Oscillospira Species in the Rumen of Cattle, Sheep, and Reindeer as Assessed by Microscopy and Molecular Approaches"

    Article Title: Ecology of Uncultivated Oscillospira Species in the Rumen of Cattle, Sheep, and Reindeer as Assessed by Microscopy and Molecular Approaches

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.69.11.6808-6815.2003

    DGGE analysis PCR-amplified 16S rRNA fragment of Oscillospira with the OSCI primers with an attached GC-clamp. The figure shows the DGGE separation pattern of PCR fragments obtained from Norwegian reindeer (lanes 1 to 4), cattle (lanes 5 and 6), and sheep (lanes 7 to 12). The arrows indicate the representatives of DNA bands, which were excised and subjected to cloning and sequencing.
    Figure Legend Snippet: DGGE analysis PCR-amplified 16S rRNA fragment of Oscillospira with the OSCI primers with an attached GC-clamp. The figure shows the DGGE separation pattern of PCR fragments obtained from Norwegian reindeer (lanes 1 to 4), cattle (lanes 5 and 6), and sheep (lanes 7 to 12). The arrows indicate the representatives of DNA bands, which were excised and subjected to cloning and sequencing.

    Techniques Used: Denaturing Gradient Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Clone Assay, Sequencing

    39) Product Images from "Lymphatic Dissemination of Simian Immunodeficiency Virus after Penile Inoculation"

    Article Title: Lymphatic Dissemination of Simian Immunodeficiency Virus after Penile Inoculation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02947-15

    SIV RNA levels in plasma of rhesus macaques after penile SIVmac251 inoculation. Total vRNA levels in plasma were determined by RT-PCR. The last blood sample was collected at necropsy. The animal number associated with each symbol is indicated.
    Figure Legend Snippet: SIV RNA levels in plasma of rhesus macaques after penile SIVmac251 inoculation. Total vRNA levels in plasma were determined by RT-PCR. The last blood sample was collected at necropsy. The animal number associated with each symbol is indicated.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    40) Product Images from "dsRNA silencing of an R2R3-MYB transcription factor affects flower cell shape in a Dendrobium hybrid"

    Article Title: dsRNA silencing of an R2R3-MYB transcription factor affects flower cell shape in a Dendrobium hybrid

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-015-0577-3

    Relative expression levels of the DhMYB1 cDNA in different floral tissues of floral buds. a Representative semi-quantitative RT-PCR results for DhMYB1 and β-actin cDNA in sepals, petals and labella of stage 5 to 7 floral buds. Lane 1: stage 5 sepals; lane 2: stage 5 petals; lane 3: stage 5 labella; lane 4: stage 6 sepals; lane 5: stage 6 petals; lane 6: stage 6 labella; lane 7: stage 7 sepals; lane 8: stage 7 petals; lane 9: stage 7 labella; lane 10: negative control. b Relative intensity of PCR bands for DhMYB1 normalized to the constitutive β-actin gene. Each bar represents the mean for three biological replicates with error bars indicating the SE. Letters indicate significant differences using one way ANOVA followed by Tukey HSD multi-comparison analysis test (P
    Figure Legend Snippet: Relative expression levels of the DhMYB1 cDNA in different floral tissues of floral buds. a Representative semi-quantitative RT-PCR results for DhMYB1 and β-actin cDNA in sepals, petals and labella of stage 5 to 7 floral buds. Lane 1: stage 5 sepals; lane 2: stage 5 petals; lane 3: stage 5 labella; lane 4: stage 6 sepals; lane 5: stage 6 petals; lane 6: stage 6 labella; lane 7: stage 7 sepals; lane 8: stage 7 petals; lane 9: stage 7 labella; lane 10: negative control. b Relative intensity of PCR bands for DhMYB1 normalized to the constitutive β-actin gene. Each bar represents the mean for three biological replicates with error bars indicating the SE. Letters indicate significant differences using one way ANOVA followed by Tukey HSD multi-comparison analysis test (P

    Techniques Used: Expressing, Quantitative RT-PCR, Negative Control, Polymerase Chain Reaction

    41) Product Images from "Genetic Analysis of Babesia Isolates from Cattle with Clinical Babesiosis in Sri Lanka"

    Article Title: Genetic Analysis of Babesia Isolates from Cattle with Clinical Babesiosis in Sri Lanka

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00895-18

    A PCR assay specific to Babesia sp. Mymensingh was developed. (A) Specificity testing. The specificity of the newly developed PCR assay was tested using DNA samples from Babesia sp. Mymensingh, B. bigemina , B. bovis , B. ovata , B . divergens , Babesia sp. Hue-1, Theileria annulata , Theileria parva , Th. orientalis , Trypanosoma evansi , Trypanosoma theileri , Tr. brucei , Anaplasma marginale , Anaplamsa bovis , and uninfected cattle (lanes 1 to 15, respectively). Lane M, 100-bp DNA marker. Note that the amplicon with the expected size was observed only with Babesia sp. Mymensingh. (B) Screening of 13 clinical samples for Babesia sp. Mymensingh. The PCR assay specific to Babesia sp. Mymensingh was used to screen DNA samples from 13 clinical cases. Lanes M and NC contained the 100-bp DNA marker and nontemplate control, respectively. Note that Babesia sp. Mymensingh was also detected in cow V.
    Figure Legend Snippet: A PCR assay specific to Babesia sp. Mymensingh was developed. (A) Specificity testing. The specificity of the newly developed PCR assay was tested using DNA samples from Babesia sp. Mymensingh, B. bigemina , B. bovis , B. ovata , B . divergens , Babesia sp. Hue-1, Theileria annulata , Theileria parva , Th. orientalis , Trypanosoma evansi , Trypanosoma theileri , Tr. brucei , Anaplasma marginale , Anaplamsa bovis , and uninfected cattle (lanes 1 to 15, respectively). Lane M, 100-bp DNA marker. Note that the amplicon with the expected size was observed only with Babesia sp. Mymensingh. (B) Screening of 13 clinical samples for Babesia sp. Mymensingh. The PCR assay specific to Babesia sp. Mymensingh was used to screen DNA samples from 13 clinical cases. Lanes M and NC contained the 100-bp DNA marker and nontemplate control, respectively. Note that Babesia sp. Mymensingh was also detected in cow V.

    Techniques Used: Polymerase Chain Reaction, Marker, Amplification

    42) Product Images from "Involvement of methylation of MicroRNA-122, −125b and -106b in regulation of Cyclin G1, CAT-1 and STAT3 target genes in isoniazid-induced liver injury"

    Article Title: Involvement of methylation of MicroRNA-122, −125b and -106b in regulation of Cyclin G1, CAT-1 and STAT3 target genes in isoniazid-induced liver injury

    Journal: BMC Pharmacology & Toxicology

    doi: 10.1186/s40360-018-0201-x

    MiR-122, miR-125b and miR-106b were epigenetically silenced in INH-induced liver injury. ( a ), ( d ) and ( g ) represent the CpG islands of miR-122, miR-125b and miR-106b that were predicted by MethPrimer, respectively. Agarose gel electrophoresis of the PCR products of gene promoter methylation of ( b ), miR-122, ( e ) miR-125b and ( h ) miR-106b. Marker: DNA ladder 50 bp, M: Methylation, U: Unmethylation. DNA methylation at particular CG dinucleotides within the miR-122 gene promoter ( c ), miR-125b gene promoter ( f ) and miR-106b gene promoter ( i ) in liver tissues from INH-administered rats was determined by qMSP. Methylated levels of miR-122, miR-125b and miR-106b in INH-administered rat liver tissues were significantly higher than those in the control rats (* p
    Figure Legend Snippet: MiR-122, miR-125b and miR-106b were epigenetically silenced in INH-induced liver injury. ( a ), ( d ) and ( g ) represent the CpG islands of miR-122, miR-125b and miR-106b that were predicted by MethPrimer, respectively. Agarose gel electrophoresis of the PCR products of gene promoter methylation of ( b ), miR-122, ( e ) miR-125b and ( h ) miR-106b. Marker: DNA ladder 50 bp, M: Methylation, U: Unmethylation. DNA methylation at particular CG dinucleotides within the miR-122 gene promoter ( c ), miR-125b gene promoter ( f ) and miR-106b gene promoter ( i ) in liver tissues from INH-administered rats was determined by qMSP. Methylated levels of miR-122, miR-125b and miR-106b in INH-administered rat liver tissues were significantly higher than those in the control rats (* p

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Methylation, Marker, DNA Methylation Assay

    43) Product Images from "Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology ▿"

    Article Title: Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology ▿

    Journal:

    doi: 10.1128/AEM.02987-06

    DGGE profiles of amplified partial 16S rRNA genes of a water sediment sample, which was either not exposed to heat or heated at 55°C for 15 min. Samples were treated with PMA or not prior to DNA extraction and PCR amplification. Arrows indicate
    Figure Legend Snippet: DGGE profiles of amplified partial 16S rRNA genes of a water sediment sample, which was either not exposed to heat or heated at 55°C for 15 min. Samples were treated with PMA or not prior to DNA extraction and PCR amplification. Arrows indicate

    Techniques Used: Denaturing Gradient Gel Electrophoresis, Amplification, DNA Extraction, Polymerase Chain Reaction

    DGGE profiles of amplified partial 16S rRNA genes of a water sediment sample, which was either not exposed to heat or heated at the indicated temperatures for 15 min. All samples but one were treated with PMA prior to DNA extraction and PCR amplification.
    Figure Legend Snippet: DGGE profiles of amplified partial 16S rRNA genes of a water sediment sample, which was either not exposed to heat or heated at the indicated temperatures for 15 min. All samples but one were treated with PMA prior to DNA extraction and PCR amplification.

    Techniques Used: Denaturing Gradient Gel Electrophoresis, Amplification, DNA Extraction, Polymerase Chain Reaction

    44) Product Images from "Involvement of methylation of MicroRNA-122, −125b and -106b in regulation of Cyclin G1, CAT-1 and STAT3 target genes in isoniazid-induced liver injury"

    Article Title: Involvement of methylation of MicroRNA-122, −125b and -106b in regulation of Cyclin G1, CAT-1 and STAT3 target genes in isoniazid-induced liver injury

    Journal: BMC Pharmacology & Toxicology

    doi: 10.1186/s40360-018-0201-x

    MiR-122, miR-125b and miR-106b were epigenetically silenced in INH-induced liver injury. ( a ), ( d ) and ( g ) represent the CpG islands of miR-122, miR-125b and miR-106b that were predicted by MethPrimer, respectively. Agarose gel electrophoresis of the PCR products of gene promoter methylation of ( b ), miR-122, ( e ) miR-125b and ( h ) miR-106b. Marker: DNA ladder 50 bp, M: Methylation, U: Unmethylation. DNA methylation at particular CG dinucleotides within the miR-122 gene promoter ( c ), miR-125b gene promoter ( f ) and miR-106b gene promoter ( i ) in liver tissues from INH-administered rats was determined by qMSP. Methylated levels of miR-122, miR-125b and miR-106b in INH-administered rat liver tissues were significantly higher than those in the control rats (* p
    Figure Legend Snippet: MiR-122, miR-125b and miR-106b were epigenetically silenced in INH-induced liver injury. ( a ), ( d ) and ( g ) represent the CpG islands of miR-122, miR-125b and miR-106b that were predicted by MethPrimer, respectively. Agarose gel electrophoresis of the PCR products of gene promoter methylation of ( b ), miR-122, ( e ) miR-125b and ( h ) miR-106b. Marker: DNA ladder 50 bp, M: Methylation, U: Unmethylation. DNA methylation at particular CG dinucleotides within the miR-122 gene promoter ( c ), miR-125b gene promoter ( f ) and miR-106b gene promoter ( i ) in liver tissues from INH-administered rats was determined by qMSP. Methylated levels of miR-122, miR-125b and miR-106b in INH-administered rat liver tissues were significantly higher than those in the control rats (* p

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Methylation, Marker, DNA Methylation Assay

    45) Product Images from "A Salt Overly Sensitive Pathway Member from Brassica juncea BjSOS3 Can Functionally Complement ΔAtsos3 in Arabidopsis"

    Article Title: A Salt Overly Sensitive Pathway Member from Brassica juncea BjSOS3 Can Functionally Complement ΔAtsos3 in Arabidopsis

    Journal: Current Genomics

    doi: 10.2174/1389202918666170228133621

    Transcript abundance of SOS3 members in BjSOS3 complemented transgenic arabidopsis and their ABA stress response. Transgenic arabidopsis seedlings were grown on MS agar plate and samples were harvested for expression analysis. qRT-PCR was performed using the cDNA prepared from total RNA isolated from whole seedlings. Relative expression of ( a ) BjSOS3 , ( b ) AtSOS1 ( c ) AtSOS2 . Bar graphs were plotted between stress duration (X-axis) and log 2 -dCt value in number (Y-axis). Gene expression data was normalised with the plant reference gene ‘actin’ as an internal control. The values represented are the mean of two biological and three technical replicates, standard error is shown above the bar. For statistical significance, student t -test was performed and asterisk above the graph means significant differences from WT at P ≤ 0.05. Four days old seedlings were transferred to MS media supplemented with ( d ) 0 or ( e ) 25 µM of ABA. After 7 days of growth, pictures were taken and ( f ) root length was measured. Error bars represent the standard deviation (n = 15). For statistical significance, student’s t -test was performed and asterisk above the graph means significant differences from WT at P ≤ 0.05.
    Figure Legend Snippet: Transcript abundance of SOS3 members in BjSOS3 complemented transgenic arabidopsis and their ABA stress response. Transgenic arabidopsis seedlings were grown on MS agar plate and samples were harvested for expression analysis. qRT-PCR was performed using the cDNA prepared from total RNA isolated from whole seedlings. Relative expression of ( a ) BjSOS3 , ( b ) AtSOS1 ( c ) AtSOS2 . Bar graphs were plotted between stress duration (X-axis) and log 2 -dCt value in number (Y-axis). Gene expression data was normalised with the plant reference gene ‘actin’ as an internal control. The values represented are the mean of two biological and three technical replicates, standard error is shown above the bar. For statistical significance, student t -test was performed and asterisk above the graph means significant differences from WT at P ≤ 0.05. Four days old seedlings were transferred to MS media supplemented with ( d ) 0 or ( e ) 25 µM of ABA. After 7 days of growth, pictures were taken and ( f ) root length was measured. Error bars represent the standard deviation (n = 15). For statistical significance, student’s t -test was performed and asterisk above the graph means significant differences from WT at P ≤ 0.05.

    Techniques Used: Transgenic Assay, Mass Spectrometry, Expressing, Quantitative RT-PCR, Isolation, Standard Deviation

    Growth of seedlings of contrasting Brassica genotypes in response to high salinity and ABA, and transcript abundance for various SOS pathway members. Nine days old hydroponically grown Brassica seedlings were subjected to salinity (200 mM NaCl) or ABA (100 µM). qRT-PCR was performed using the cDNA prepared from total RNA isolated from shoots of the seedlings subjected to salinity stress (200 mM NaCl); or ABA (100 µM) for 8 h and 24 h time period. ( a ). Representative photograph of seedlings of Brassica genotypes after 24 h of salinity (200 mM NaCl) or ABA (100 µM). Relative expression of ( b ) BjSOS1 , ( c ) BjSOS2 and ( d ) BjSOS3 . Bar graphs were plotted between stress duration (X-axis) and log 2 -ddCt value in number (Y-axis). Gene expression data was normalised with the plant reference gene ‘actin’ as an internal control. Relative expression of genes was plotted against the expression of B. nigra control. The values represented are the mean of two biological and three technical replicates, standard error is shown above the bar. For statistical significance, student t -test was performed and asterisk above the graph means significant differences from their respective control (Con) at P ≤ 0.05. ( For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper. )
    Figure Legend Snippet: Growth of seedlings of contrasting Brassica genotypes in response to high salinity and ABA, and transcript abundance for various SOS pathway members. Nine days old hydroponically grown Brassica seedlings were subjected to salinity (200 mM NaCl) or ABA (100 µM). qRT-PCR was performed using the cDNA prepared from total RNA isolated from shoots of the seedlings subjected to salinity stress (200 mM NaCl); or ABA (100 µM) for 8 h and 24 h time period. ( a ). Representative photograph of seedlings of Brassica genotypes after 24 h of salinity (200 mM NaCl) or ABA (100 µM). Relative expression of ( b ) BjSOS1 , ( c ) BjSOS2 and ( d ) BjSOS3 . Bar graphs were plotted between stress duration (X-axis) and log 2 -ddCt value in number (Y-axis). Gene expression data was normalised with the plant reference gene ‘actin’ as an internal control. Relative expression of genes was plotted against the expression of B. nigra control. The values represented are the mean of two biological and three technical replicates, standard error is shown above the bar. For statistical significance, student t -test was performed and asterisk above the graph means significant differences from their respective control (Con) at P ≤ 0.05. ( For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper. )

    Techniques Used: Quantitative RT-PCR, Isolation, Expressing

    46) Product Images from "Molecular Types and Genetic Profiles of Staphylococcus aureus Strains Isolated from Bovine Intramammary Infections and Extramammary Sites ▿"

    Article Title: Molecular Types and Genetic Profiles of Staphylococcus aureus Strains Isolated from Bovine Intramammary Infections and Extramammary Sites ▿

    Journal:

    doi: 10.1128/JCM.00769-08

    Agarose gel electrophoresis analysis showing PCR amplification products for the fnbB gene of S. aureus . Lanes: MP, DNA molecular size markers; 1 and 2, fnbB -positive isolates 774 and 196; 3, fnbB variant (teat orifice isolate); 4, fnbB -positive control
    Figure Legend Snippet: Agarose gel electrophoresis analysis showing PCR amplification products for the fnbB gene of S. aureus . Lanes: MP, DNA molecular size markers; 1 and 2, fnbB -positive isolates 774 and 196; 3, fnbB variant (teat orifice isolate); 4, fnbB -positive control

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Variant Assay, Positive Control

    47) Product Images from "Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells"

    Article Title: Multiplex polymerase chain reaction (PCR) with color-tagged module-shuffling primers for comparing gene expression levels in various cells

    Journal: Nucleic Acids Research

    doi:

    Reproducibility of PCR. The same cDNA (100 species) in the two mixtures was amplified independently. The two samples had the same cDNA groups and different cDNA mixtures from different sources, therefore they had different amount of co-existing DNAs. Different axes plot the fluorescence intensities obtained from different PCRs.
    Figure Legend Snippet: Reproducibility of PCR. The same cDNA (100 species) in the two mixtures was amplified independently. The two samples had the same cDNA groups and different cDNA mixtures from different sources, therefore they had different amount of co-existing DNAs. Different axes plot the fluorescence intensities obtained from different PCRs.

    Techniques Used: Polymerase Chain Reaction, Amplification, Fluorescence

    Comparative analysis of expressed genes by one-tube PCR (using MSPs) of the cDNA fragments mixed from different sources.
    Figure Legend Snippet: Comparative analysis of expressed genes by one-tube PCR (using MSPs) of the cDNA fragments mixed from different sources.

    Techniques Used: Polymerase Chain Reaction

    PCR amplification efficiency of each MSP. The sample was a mixture of cDNA fragments ligated with different tags for the three MSPs. The ORF YAL062W in the mixture was simultaneously amplified by MSP-A, B and C (symbolized by blue circles, green triangles and red squares, respectively).
    Figure Legend Snippet: PCR amplification efficiency of each MSP. The sample was a mixture of cDNA fragments ligated with different tags for the three MSPs. The ORF YAL062W in the mixture was simultaneously amplified by MSP-A, B and C (symbolized by blue circles, green triangles and red squares, respectively).

    Techniques Used: Polymerase Chain Reaction, Amplification

    Reproducibility of the MSP-PCR method. 200 cDNA fragments were measured. The cDNA of a S.cerevisiae ligated with two tags and simultaneously amplified by MSP-A (horizontal axis, labeled with FAM) and B (vertical axis, labeled with HEX). Thermal cycling was carried out 30 times. The mixture contains different cDNA fragments derived from different sources as co-existent DNA factors. The cDNA ligated with two tags in a mixture was amplified simultaneously in one reaction tube.
    Figure Legend Snippet: Reproducibility of the MSP-PCR method. 200 cDNA fragments were measured. The cDNA of a S.cerevisiae ligated with two tags and simultaneously amplified by MSP-A (horizontal axis, labeled with FAM) and B (vertical axis, labeled with HEX). Thermal cycling was carried out 30 times. The mixture contains different cDNA fragments derived from different sources as co-existent DNA factors. The cDNA ligated with two tags in a mixture was amplified simultaneously in one reaction tube.

    Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Derivative Assay

    48) Product Images from "LsaA, an Antigen Involved in Cell Attachment and Invasion, Is Expressed by Lawsonia intracellularis during Infection In Vitro and In Vivo "

    Article Title: LsaA, an Antigen Involved in Cell Attachment and Invasion, Is Expressed by Lawsonia intracellularis during Infection In Vitro and In Vivo

    Journal: Infection and Immunity

    doi: 10.1128/IAI.70.6.2899-2907.2002

    Transcriptional analysis of 16S rRNA and lsaA during infection in vivo. (a and b) Conventional PCR amplification of 16S rRNA (a) and lsaA (b) was performed with DNA extracted from ileum of L. intracellularis -infected (lanes 1 to 5) and uninfected (lanes 6 to 9) animals. Amplification products of 600 and 300 bp for 16S rRNA and/or lsaA , respectively, were evident only in DNA from infected animals. (c to f) Transcriptional analysis of L. intracellularis 16S rRNA (c and d) and lsaA (e and f) in RNA extracted from L. intracellularis -infected animals L1 (lanes 1 and 2), L2 (lanes 3 and 4), L3 (lanes 5 and 6), L4 (lanes 7 and 8), and L5 (lanes 9 and 10). Odd-numbered lanes indicate RT-PCRs in which reverse transcriptase was incorporated, while even-numbered lanes represent the reaction without reverse transcriptase (control). Products were separated by polyacrylamide gel electrophoresis. Only the relevant regions of the gels are shown. RT-PCR with primers specific for 16S rRNA (c) was confirmed by Southern blotting with a 16S rRNA probe (d) and that with primers specific for lsaA (e) was confirmed by Southern blotting with an lsaA probe (f).
    Figure Legend Snippet: Transcriptional analysis of 16S rRNA and lsaA during infection in vivo. (a and b) Conventional PCR amplification of 16S rRNA (a) and lsaA (b) was performed with DNA extracted from ileum of L. intracellularis -infected (lanes 1 to 5) and uninfected (lanes 6 to 9) animals. Amplification products of 600 and 300 bp for 16S rRNA and/or lsaA , respectively, were evident only in DNA from infected animals. (c to f) Transcriptional analysis of L. intracellularis 16S rRNA (c and d) and lsaA (e and f) in RNA extracted from L. intracellularis -infected animals L1 (lanes 1 and 2), L2 (lanes 3 and 4), L3 (lanes 5 and 6), L4 (lanes 7 and 8), and L5 (lanes 9 and 10). Odd-numbered lanes indicate RT-PCRs in which reverse transcriptase was incorporated, while even-numbered lanes represent the reaction without reverse transcriptase (control). Products were separated by polyacrylamide gel electrophoresis. Only the relevant regions of the gels are shown. RT-PCR with primers specific for 16S rRNA (c) was confirmed by Southern blotting with a 16S rRNA probe (d) and that with primers specific for lsaA (e) was confirmed by Southern blotting with an lsaA probe (f).

    Techniques Used: Infection, In Vivo, Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Southern Blot

    Schematic representation of the L. intracellularis lsaA gene. Relative positions and directions of primers used for PCR amplifications are shown with arrows. Primers DOP-for and DOP-rev were used for DOP-PCR. Primers LI02 and LI05 were used in RT-PCR and the first stage of semirandom PCR chromosome walking in combination with random primer AP1 or AP2 (not shown). Primers LI15 and LI14 were used as nested primers with SPI in second-stage semirandom PCR chromosome walking. Primers LI19-for and LI-end1 were used to amplify complete the lsaA gene. Primer sequences and amplification conditions are indicated in the text.
    Figure Legend Snippet: Schematic representation of the L. intracellularis lsaA gene. Relative positions and directions of primers used for PCR amplifications are shown with arrows. Primers DOP-for and DOP-rev were used for DOP-PCR. Primers LI02 and LI05 were used in RT-PCR and the first stage of semirandom PCR chromosome walking in combination with random primer AP1 or AP2 (not shown). Primers LI15 and LI14 were used as nested primers with SPI in second-stage semirandom PCR chromosome walking. Primers LI19-for and LI-end1 were used to amplify complete the lsaA gene. Primer sequences and amplification conditions are indicated in the text.

    Techniques Used: Polymerase Chain Reaction, Degenerate Oligonucleotide–primed Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Chromosome Walking, Amplification

    Transcriptional analysis of 16S rRNA (odd-numbered lanes) and lsaA (even-numbered lanes) in L. intracellularis grown in vitro. (a) The presence of L. intracellularis in infected IEC18 cells was confirmed by conventional PCR amplification of 16S rRNA and lsaA from extracted DNA. (b) RT-PCR with 16S rRNA- or lsaA -specific primers detected transcripts for both 16S rRNA and lsaA in RNA extracted from IEC18 cells infected with L. intracellularis . (c and d) Products obtained from RT-PCR amplification of 16S rRNA and lsaA were separated by electrophoresis and transferred to a nylon membrane for Southern blotting. Blotting was performed with digoxigenin-labeled 16S rRNA-specific (c) or lsaA -specific (d) probes, and the presence of single bands in each panel confirmed the specificity of the RT-PCR.
    Figure Legend Snippet: Transcriptional analysis of 16S rRNA (odd-numbered lanes) and lsaA (even-numbered lanes) in L. intracellularis grown in vitro. (a) The presence of L. intracellularis in infected IEC18 cells was confirmed by conventional PCR amplification of 16S rRNA and lsaA from extracted DNA. (b) RT-PCR with 16S rRNA- or lsaA -specific primers detected transcripts for both 16S rRNA and lsaA in RNA extracted from IEC18 cells infected with L. intracellularis . (c and d) Products obtained from RT-PCR amplification of 16S rRNA and lsaA were separated by electrophoresis and transferred to a nylon membrane for Southern blotting. Blotting was performed with digoxigenin-labeled 16S rRNA-specific (c) or lsaA -specific (d) probes, and the presence of single bands in each panel confirmed the specificity of the RT-PCR.

    Techniques Used: In Vitro, Infection, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Southern Blot, Labeling

    49) Product Images from "PBOV1 Is a Human De Novo Gene with Tumor-Specific Expression That Is Associated with a Positive Clinical Outcome of Cancer"

    Article Title: PBOV1 Is a Human De Novo Gene with Tumor-Specific Expression That Is Associated with a Positive Clinical Outcome of Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056162

    Expression profiling of PBOV1 and GAPDH (positive control) measured by PCR in cDNA panels from human normal tissues shows the lack of PBOV1 expression in adult and fetal normal tissures. A . Human MTC Panel I (1–8), Human MTC Panel II (9–16): 1 – brain, 2 – heart, 3 – kidney, 4 – liver, 5 – lung, 6 – pancreas, 7 – placenta, 8 – skeletal muscle, 9 – colon, 10 – ovary, 11 – peripheral blood leukocyte, 12 – prostate, 13 – small intestine, 14 – spleen, 15 – testis, 16 – thymus; Full size images of gels are shown on Figure S1 and Figure S2 in File S1. B . Human Digestive System MTC Panel: 1 – cecum, 2 – colon, ascending 3 – colon, descending 4 – colon, transverse 5 – duodenum, 6 – esophagus, 7 – ileocecum, 8 – ileum, 9 – jejunum, 10 – liver, 11 – rectum, 12 – stomach. Full-sized images of gels are presented on Figure S5 and Figure S6 in File S1. C . Human Immune System MTC Panel (1–7), Human Fetal MTC Panel(8–15): 1 – bone marrow, 2 – fetal liver, 3 – lymph node, 4 – peripheral blood leukocyte, 5 – spleen, 6 – thymus, 7 – tonsil, 8 – fetal brain, 9 – fetal heart, 10 – fetal kidney, 11 – fetal liver, 12 – fetal lung, 13 – fetal skeletal muscle, 14 – fetal spleen, 15 – fetal thymus; A–C: NC – PCR with no template, PC – PCR with human DNA. Full size images of gels are shown on Figure S3 and Figure S4 in File S1.
    Figure Legend Snippet: Expression profiling of PBOV1 and GAPDH (positive control) measured by PCR in cDNA panels from human normal tissues shows the lack of PBOV1 expression in adult and fetal normal tissures. A . Human MTC Panel I (1–8), Human MTC Panel II (9–16): 1 – brain, 2 – heart, 3 – kidney, 4 – liver, 5 – lung, 6 – pancreas, 7 – placenta, 8 – skeletal muscle, 9 – colon, 10 – ovary, 11 – peripheral blood leukocyte, 12 – prostate, 13 – small intestine, 14 – spleen, 15 – testis, 16 – thymus; Full size images of gels are shown on Figure S1 and Figure S2 in File S1. B . Human Digestive System MTC Panel: 1 – cecum, 2 – colon, ascending 3 – colon, descending 4 – colon, transverse 5 – duodenum, 6 – esophagus, 7 – ileocecum, 8 – ileum, 9 – jejunum, 10 – liver, 11 – rectum, 12 – stomach. Full-sized images of gels are presented on Figure S5 and Figure S6 in File S1. C . Human Immune System MTC Panel (1–7), Human Fetal MTC Panel(8–15): 1 – bone marrow, 2 – fetal liver, 3 – lymph node, 4 – peripheral blood leukocyte, 5 – spleen, 6 – thymus, 7 – tonsil, 8 – fetal brain, 9 – fetal heart, 10 – fetal kidney, 11 – fetal liver, 12 – fetal lung, 13 – fetal skeletal muscle, 14 – fetal spleen, 15 – fetal thymus; A–C: NC – PCR with no template, PC – PCR with human DNA. Full size images of gels are shown on Figure S3 and Figure S4 in File S1.

    Techniques Used: Expressing, Positive Control, Polymerase Chain Reaction

    PBOV1 expression profiling by PCR in cDNA panels from human tumors shows that PBOV1 is expressed in multiple tumor types. A . Tumor cDNA Panel (BioChain Institute, USA): 1 – Brain medulloblastoma, with glioma, 2 – Lung squamous cell carcinoma, 3 – Kidney granular cell carcinoma, 4 – Kidney clear cell carcinoma, 5 – Liver cholangiocellular carcinoma, 6 – Hepatocellular carcinoma, 7 – Gallbladder adenocarcinoma, 8 – Esophagus squamous cell carcinoma, 9 – Stomach signet ring cell carcinoma, 10 – Small Intestine adenocarcinoma, 11 – Colon papillary adenocarcinoma, 12 – Rectum adenocarcinoma, 13 – Breast fibroadenoma, 14 – Ovary serous cystoadenocarcinoma, 15 – Fallopian tube medullary carcinoma, 16 – Uterus adenocarcinoma, 17 – Ureter papillary transitional cell carcinoma, 18 – Bladder transitional cell carcinoma, 19 – Testis seminoma, 20 – Prostate adenocarcinoma, 21 – Malignant melanoma, 22 – Skeletal Muscle malignancy fibrous histocytoma, 23 – Adrenal pheochromocytoma, 24 – Non-Hodgkin's lymphoma, 25 – Thyroid papillary adenocarcinoma, 26 – Parotid mixed tumor, 27 – Pancreas adenocarcinoma, 28 – Thymus seminoma, 29 – Spleen serous adenocarcinoma, 30 – Hodgkin's lymphoma, 31 – T cell Hodgkin's lymphoma, 32 – Malignant lymphoma. NC – PCR with no template, PC – PCR with human DNA. DNA contamination was controlled using gDNA-CTR primers. Full-sized images of gels are presented on Figure S7 and Figure S8 in File S1. B . PBOV1 expression in clinical tumor samples (see Materials and Methods for full description of samples). PBOV1 is expressed in breast cancer (9–250), ovary cancer (1, 6), cervical cancer (2, 13), endometrial cancer (156, 270), lung cancer (12, 14, 17), seminoma (7), meningioma (63), non-Hodgkin lymphomas (67, 82, 92, 102, 113) Full-sized images of gels are presented on Figure S9 and Figure S10 in File S1.
    Figure Legend Snippet: PBOV1 expression profiling by PCR in cDNA panels from human tumors shows that PBOV1 is expressed in multiple tumor types. A . Tumor cDNA Panel (BioChain Institute, USA): 1 – Brain medulloblastoma, with glioma, 2 – Lung squamous cell carcinoma, 3 – Kidney granular cell carcinoma, 4 – Kidney clear cell carcinoma, 5 – Liver cholangiocellular carcinoma, 6 – Hepatocellular carcinoma, 7 – Gallbladder adenocarcinoma, 8 – Esophagus squamous cell carcinoma, 9 – Stomach signet ring cell carcinoma, 10 – Small Intestine adenocarcinoma, 11 – Colon papillary adenocarcinoma, 12 – Rectum adenocarcinoma, 13 – Breast fibroadenoma, 14 – Ovary serous cystoadenocarcinoma, 15 – Fallopian tube medullary carcinoma, 16 – Uterus adenocarcinoma, 17 – Ureter papillary transitional cell carcinoma, 18 – Bladder transitional cell carcinoma, 19 – Testis seminoma, 20 – Prostate adenocarcinoma, 21 – Malignant melanoma, 22 – Skeletal Muscle malignancy fibrous histocytoma, 23 – Adrenal pheochromocytoma, 24 – Non-Hodgkin's lymphoma, 25 – Thyroid papillary adenocarcinoma, 26 – Parotid mixed tumor, 27 – Pancreas adenocarcinoma, 28 – Thymus seminoma, 29 – Spleen serous adenocarcinoma, 30 – Hodgkin's lymphoma, 31 – T cell Hodgkin's lymphoma, 32 – Malignant lymphoma. NC – PCR with no template, PC – PCR with human DNA. DNA contamination was controlled using gDNA-CTR primers. Full-sized images of gels are presented on Figure S7 and Figure S8 in File S1. B . PBOV1 expression in clinical tumor samples (see Materials and Methods for full description of samples). PBOV1 is expressed in breast cancer (9–250), ovary cancer (1, 6), cervical cancer (2, 13), endometrial cancer (156, 270), lung cancer (12, 14, 17), seminoma (7), meningioma (63), non-Hodgkin lymphomas (67, 82, 92, 102, 113) Full-sized images of gels are presented on Figure S9 and Figure S10 in File S1.

    Techniques Used: Expressing, Polymerase Chain Reaction

    50) Product Images from "Interleukin-17A is involved in mechanical hyperalgesia but not in the severity of murine antigen-induced arthritis"

    Article Title: Interleukin-17A is involved in mechanical hyperalgesia but not in the severity of murine antigen-induced arthritis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-10509-5

    PCR and RT-PCR of IL-17 cytokine family members in knee joint tissues at day 2 of AIA. ( a ) Representative PCR-ScreenTape (Agilent 2200 TapeStation) of IL-17 cytokine family members in tissues of WT and IL-17AKO mice. PCR analysis does not give information about expression quantity. Upper (magenta) and lower (green) markers are used as internal references to determine the molecular weight size of the sample. DNA ladder (25–1500 bp) on the left. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) serves as a housekeeping control gene. ( b ) Quantitative reverse transcriptase (RT)-PCR in tissues of sympathectomised IL-17AKO mice and IL-17AKO AIA control mice (n = 5 per group). mRNA level after sympathectomy are given in relation to non-sympathectomised control mice. Sympathectomy significantly (p = 0.012) decreases IL-17D-mRNA. Values are mean ± SEM. *p
    Figure Legend Snippet: PCR and RT-PCR of IL-17 cytokine family members in knee joint tissues at day 2 of AIA. ( a ) Representative PCR-ScreenTape (Agilent 2200 TapeStation) of IL-17 cytokine family members in tissues of WT and IL-17AKO mice. PCR analysis does not give information about expression quantity. Upper (magenta) and lower (green) markers are used as internal references to determine the molecular weight size of the sample. DNA ladder (25–1500 bp) on the left. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) serves as a housekeeping control gene. ( b ) Quantitative reverse transcriptase (RT)-PCR in tissues of sympathectomised IL-17AKO mice and IL-17AKO AIA control mice (n = 5 per group). mRNA level after sympathectomy are given in relation to non-sympathectomised control mice. Sympathectomy significantly (p = 0.012) decreases IL-17D-mRNA. Values are mean ± SEM. *p

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Expressing, Molecular Weight

    51) Product Images from "Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR"

    Article Title: Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR

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

    doi:

    FCS analyses over successive rounds of PCR amplification of a 106-bp segment of IS6110 from M. tuberculosis . A standard PCR mixture with primers S1 and S2 and 10 4 target genomes in the presence of 50 ng/μl carrier DNA was supplemented with 10 nM probe PR1 to monitor specific amplification by probe extension. ( Left ) The obtained autocorrelation functions G ( t ) after the indicated numbers of thermal cycles. ( Right ) The linearized function [ G ( t = 0)]/[ G ( t )].
    Figure Legend Snippet: FCS analyses over successive rounds of PCR amplification of a 106-bp segment of IS6110 from M. tuberculosis . A standard PCR mixture with primers S1 and S2 and 10 4 target genomes in the presence of 50 ng/μl carrier DNA was supplemented with 10 nM probe PR1 to monitor specific amplification by probe extension. ( Left ) The obtained autocorrelation functions G ( t ) after the indicated numbers of thermal cycles. ( Right ) The linearized function [ G ( t = 0)]/[ G ( t )].

    Techniques Used: Polymerase Chain Reaction, Amplification

    Gel analysis of amplification and probe extension products. The top panels show ethidium bromide stained, 11% nondenaturing polyacrylamide gels of 106-bp PCR products from amplification of a IS6110 sequence from M. tuberculosis , using primer pair S1/S2. PCR was performed for the indicated number of cycles in the presence of 20 nM probes PR1 ( A ) and HS1 ( B ), respectively. Lanes M contain 2 μg DNA marker V (Boehringer Mannheim). ( C and D ) Corresponding analyses of the probe extension products on 6% sequencing gels using an automated fluorescence sequencer.
    Figure Legend Snippet: Gel analysis of amplification and probe extension products. The top panels show ethidium bromide stained, 11% nondenaturing polyacrylamide gels of 106-bp PCR products from amplification of a IS6110 sequence from M. tuberculosis , using primer pair S1/S2. PCR was performed for the indicated number of cycles in the presence of 20 nM probes PR1 ( A ) and HS1 ( B ), respectively. Lanes M contain 2 μg DNA marker V (Boehringer Mannheim). ( C and D ) Corresponding analyses of the probe extension products on 6% sequencing gels using an automated fluorescence sequencer.

    Techniques Used: Amplification, Staining, Polymerase Chain Reaction, Sequencing, Marker, Fluorescence

    52) Product Images from "Virus-Induced Silencing of a Plant Cellulose Synthase Gene"

    Article Title: Virus-Induced Silencing of a Plant Cellulose Synthase Gene

    Journal: The Plant Cell

    doi:

    Alignments of Isolated cDNAs against Plant CesA Genes. (A) Positions and lengths of the three cDNAs ( NtCesA-1a , NtCesA-1b, and NtCesA-2 ) from N. tabacum are shown in relation to the regions of plant CesA . CRP denotes conserved plant-specific insertions, HVR denotes hypervariable plant-specific insertions, HR denotes homologous regions of all CesA ). Note that the cDNAs start at different points at their 5′ ends but finish at the same point at their 3′ ends. The position of the fragment amplified during reverse transcription–PCR for estimation of mRNA abundance is also shown. (B) Nucleotide sequence alignments of the N. tabacum cDNAs NtCesA-1a , NtCesA-1b , and NtCesA-2 with the corresponding sequences of CesA genes from Arabidopsis ( AtCesA-1 ) and G. hirsutum ( GhCesA-1 ). The regions HR2, HVR2, CRP4, and HR3 are as described in (A) . The primers used for nested, anchor-ligated PCR of the region immediately 5′ to the NtCesA-1a cDNA fragment are highlighted in green. The sequence of the 3′ PCR primer included at the 3′ ends of the three Nicotiana ). White letters on black background denote nucleotides conserved in at least three of the five sequences. Dots denote gaps introduced by the alignment program.
    Figure Legend Snippet: Alignments of Isolated cDNAs against Plant CesA Genes. (A) Positions and lengths of the three cDNAs ( NtCesA-1a , NtCesA-1b, and NtCesA-2 ) from N. tabacum are shown in relation to the regions of plant CesA . CRP denotes conserved plant-specific insertions, HVR denotes hypervariable plant-specific insertions, HR denotes homologous regions of all CesA ). Note that the cDNAs start at different points at their 5′ ends but finish at the same point at their 3′ ends. The position of the fragment amplified during reverse transcription–PCR for estimation of mRNA abundance is also shown. (B) Nucleotide sequence alignments of the N. tabacum cDNAs NtCesA-1a , NtCesA-1b , and NtCesA-2 with the corresponding sequences of CesA genes from Arabidopsis ( AtCesA-1 ) and G. hirsutum ( GhCesA-1 ). The regions HR2, HVR2, CRP4, and HR3 are as described in (A) . The primers used for nested, anchor-ligated PCR of the region immediately 5′ to the NtCesA-1a cDNA fragment are highlighted in green. The sequence of the 3′ PCR primer included at the 3′ ends of the three Nicotiana ). White letters on black background denote nucleotides conserved in at least three of the five sequences. Dots denote gaps introduced by the alignment program.

    Techniques Used: Isolation, Amplification, Polymerase Chain Reaction, Sequencing

    53) Product Images from "Trichoderma asperellumChi42 Genes Encode Chitinase"

    Article Title: Trichoderma asperellumChi42 Genes Encode Chitinase

    Journal: Mycobiology

    doi: 10.5941/MYCO.2011.39.3.182

    Agarose gel electrophoresis (0.8% w/v) of PCR products from ITS region of Trichoderma strains CH2, SH16, PQ34, and TN42. SM, DNA size marker (λDNA/ Hin d III).
    Figure Legend Snippet: Agarose gel electrophoresis (0.8% w/v) of PCR products from ITS region of Trichoderma strains CH2, SH16, PQ34, and TN42. SM, DNA size marker (λDNA/ Hin d III).

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker

    Agarose gel electrophoresis (0.8% w/v) of PCR products of CHI-F and CHI-R primers from Trichoderma strains CH2, SH16, PQ34 and TN42. SM, DNA size marker (λDNA/ Hin d III).
    Figure Legend Snippet: Agarose gel electrophoresis (0.8% w/v) of PCR products of CHI-F and CHI-R primers from Trichoderma strains CH2, SH16, PQ34 and TN42. SM, DNA size marker (λDNA/ Hin d III).

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker

    54) Product Images from "The First Case of Antibiotic-associated Colitis by Clostridium difficile PCR Ribotype 027 in Korea"

    Article Title: The First Case of Antibiotic-associated Colitis by Clostridium difficile PCR Ribotype 027 in Korea

    Journal: Journal of Korean Medical Science

    doi: 10.3346/jkms.2009.24.3.520

    PCR products and restriction patterns of C. difficile ribotype 027 isolates from the patient. ( A ) Identical PCR ribotype profiles obtained from both the first and the last isolates. Lanes: M, 100 bp DNA ladder; 1, the last isolate; 2, the first isolate. ( B ) Types of restriction patterns by B1 and A3 PCR showing toxinotype III. Lanes: M, DNA size marker; 1, 4, Acc I restriction patterns of B1 fragment; 2, 5, Hinc II restriction patterns of B1 fragment; 3, 6, EcoR I restriction patterns of A3 fragment.
    Figure Legend Snippet: PCR products and restriction patterns of C. difficile ribotype 027 isolates from the patient. ( A ) Identical PCR ribotype profiles obtained from both the first and the last isolates. Lanes: M, 100 bp DNA ladder; 1, the last isolate; 2, the first isolate. ( B ) Types of restriction patterns by B1 and A3 PCR showing toxinotype III. Lanes: M, DNA size marker; 1, 4, Acc I restriction patterns of B1 fragment; 2, 5, Hinc II restriction patterns of B1 fragment; 3, 6, EcoR I restriction patterns of A3 fragment.

    Techniques Used: Polymerase Chain Reaction, Marker

    55) Product Images from "GS-5806 Inhibits a Broad Range of Respiratory Syncytial Virus Clinical Isolates by Blocking the Virus-Cell Fusion Process"

    Article Title: GS-5806 Inhibits a Broad Range of Respiratory Syncytial Virus Clinical Isolates by Blocking the Virus-Cell Fusion Process

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01497-15

    GS-5806 inhibits RSV fusion, but not attachment, to HEp-2 cells. (A) HEp-2 cells were infected with RSV A2 at 4°C for 3 h, washed to remove unbound virus, and then shifted to 37°C for 30 h to facilitate fusion; 50 μg/ml heparin, 1.25 nM GS-5806, 20 nM VP-14637, or 100 nM BMS-433771 was added to the cells either throughout the experiment, only during viral attachment, or only during viral fusion. Intercellular RSV RNA was quantified by quantitative PCR (qPCR) and compared to an uninhibited DMSO-treated infection. The error bars indicate standard deviations. (B) GS-5806 (10 nM) inhibits syncytium formation in 293T cells transiently expressing the RSV F protein.
    Figure Legend Snippet: GS-5806 inhibits RSV fusion, but not attachment, to HEp-2 cells. (A) HEp-2 cells were infected with RSV A2 at 4°C for 3 h, washed to remove unbound virus, and then shifted to 37°C for 30 h to facilitate fusion; 50 μg/ml heparin, 1.25 nM GS-5806, 20 nM VP-14637, or 100 nM BMS-433771 was added to the cells either throughout the experiment, only during viral attachment, or only during viral fusion. Intercellular RSV RNA was quantified by quantitative PCR (qPCR) and compared to an uninhibited DMSO-treated infection. The error bars indicate standard deviations. (B) GS-5806 (10 nM) inhibits syncytium formation in 293T cells transiently expressing the RSV F protein.

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Expressing

    56) Product Images from "Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages"

    Article Title: Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages

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

    doi: 10.1073/pnas.0710950105

    B. anthracis NO protects bacteria from the macrophage-inflicted oxidative damage. bNOS-dependent bacterial DNA protection during macrophage infection. Chromosomal damage was monitored by qPCR. A representative agarose gel shows a 3.6-kb PCR fragment amplified from the Sterne or Δ nos chromosome. Genomic DNA was isolated from J774A.1 macrophages at 2 h after infection. Control DNA was isolated from bacteria grown exponentially in BHI media without (lanes 3 and 4) or with (lanes 5 and 6) 10 mM H 2 O 2 treatment. M, 1 kb DNA marker. % indicates the fraction of the full size PCR products. Values are the mean ± SD from three experiments.
    Figure Legend Snippet: B. anthracis NO protects bacteria from the macrophage-inflicted oxidative damage. bNOS-dependent bacterial DNA protection during macrophage infection. Chromosomal damage was monitored by qPCR. A representative agarose gel shows a 3.6-kb PCR fragment amplified from the Sterne or Δ nos chromosome. Genomic DNA was isolated from J774A.1 macrophages at 2 h after infection. Control DNA was isolated from bacteria grown exponentially in BHI media without (lanes 3 and 4) or with (lanes 5 and 6) 10 mM H 2 O 2 treatment. M, 1 kb DNA marker. % indicates the fraction of the full size PCR products. Values are the mean ± SD from three experiments.

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Isolation, Marker

    57) Product Images from "Metabolomic profiling of Burkholderia pseudomallei using UHPLC-ESI-Q-TOF-MS reveals specific biomarkers including 4-methyl-5-thiazoleethanol and unique thiamine degradation pathway"

    Article Title: Metabolomic profiling of Burkholderia pseudomallei using UHPLC-ESI-Q-TOF-MS reveals specific biomarkers including 4-methyl-5-thiazoleethanol and unique thiamine degradation pathway

    Journal: Cell & Bioscience

    doi: 10.1186/s13578-015-0018-x

    PCR for hydroxyethyl thiazole kinase genes and RT-PCR for mRNA detection in B. pseudomallei and B. thailandensis. a PCR of thiM from genomic DNA: thiM is present in the genomes of the three B. thailandensis strains, Bt1, Bt6 and Bt7, and 12 of 15 B. pseudomallei strains, but absent in the genomes of three B. pseudomallei strains, B24, B27 and VG550A (D10) with the highest 4-methyl-5-thiazoleethanol levels. b RT-PCR of thiM from mRNA of B. thailandensis : thiM is expressed in B. thailandensis strains Bt1, Bt6 and Bt7, but not expressed in the 12 B. pseudomallei strains that possessed thiM gene. Housekeeping gene apoB is used for normalization
    Figure Legend Snippet: PCR for hydroxyethyl thiazole kinase genes and RT-PCR for mRNA detection in B. pseudomallei and B. thailandensis. a PCR of thiM from genomic DNA: thiM is present in the genomes of the three B. thailandensis strains, Bt1, Bt6 and Bt7, and 12 of 15 B. pseudomallei strains, but absent in the genomes of three B. pseudomallei strains, B24, B27 and VG550A (D10) with the highest 4-methyl-5-thiazoleethanol levels. b RT-PCR of thiM from mRNA of B. thailandensis : thiM is expressed in B. thailandensis strains Bt1, Bt6 and Bt7, but not expressed in the 12 B. pseudomallei strains that possessed thiM gene. Housekeeping gene apoB is used for normalization

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    58) Product Images from "Droplet barcoding for massively parallel single-molecule deep sequencing"

    Article Title: Droplet barcoding for massively parallel single-molecule deep sequencing

    Journal: Nature Communications

    doi: 10.1038/ncomms11784

    Microfluidic workflow for generating barcoded DNA fragments. Left: schematics and false-colored microscope images of microfluidic devices. Right: schematic of molecular biology reactions occurring inside droplets. ( a ) A flow focus drop maker is used to encapsulate single templates into droplets. Inside droplets, PCR or MDA is used to clonally amplify the single template. ( b ) The splitmerger device is used to add transposases into template drops while achieving a 10 × dilution of the templates. The template droplets are injected on the left side, split at junction (1) so that 1/10th of the droplet continues on to pair with a reagent droplet generated on chip at (2) and the pair merges at the channel widening (3). The transposase reaction inside droplets fragments templates while adding adaptors to each fragment. ( c ) The microfluidic device used for attaching barcodes to DNA fragments. Templates droplets (1) and barcode droplets (2) are injected into the device where they pair with each other and a large PCR reagent droplet generated on chip (3). The three droplets merge at the electrode (4) and are split into smaller droplets for thermal cycling (5). Barcodes are spliced onto fragments by overlap-extension PCR. Scale bars, 100 μm.
    Figure Legend Snippet: Microfluidic workflow for generating barcoded DNA fragments. Left: schematics and false-colored microscope images of microfluidic devices. Right: schematic of molecular biology reactions occurring inside droplets. ( a ) A flow focus drop maker is used to encapsulate single templates into droplets. Inside droplets, PCR or MDA is used to clonally amplify the single template. ( b ) The splitmerger device is used to add transposases into template drops while achieving a 10 × dilution of the templates. The template droplets are injected on the left side, split at junction (1) so that 1/10th of the droplet continues on to pair with a reagent droplet generated on chip at (2) and the pair merges at the channel widening (3). The transposase reaction inside droplets fragments templates while adding adaptors to each fragment. ( c ) The microfluidic device used for attaching barcodes to DNA fragments. Templates droplets (1) and barcode droplets (2) are injected into the device where they pair with each other and a large PCR reagent droplet generated on chip (3). The three droplets merge at the electrode (4) and are split into smaller droplets for thermal cycling (5). Barcodes are spliced onto fragments by overlap-extension PCR. Scale bars, 100 μm.

    Techniques Used: Microscopy, Flow Cytometry, Polymerase Chain Reaction, Multiple Displacement Amplification, Injection, Generated, Chromatin Immunoprecipitation

    59) Product Images from "Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir"

    Article Title: Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir

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

    doi: 10.1073/pnas.0908074106

    Trex1 degrades retroviral cDNA. ( A ) Schematic of circularization of retroelement. As a nonfunctional by-product, retroelement cDNA is circularized by the cell's DNA repair system. The circles are covalently closed. Red and green arrows represent PCR primers
    Figure Legend Snippet: Trex1 degrades retroviral cDNA. ( A ) Schematic of circularization of retroelement. As a nonfunctional by-product, retroelement cDNA is circularized by the cell's DNA repair system. The circles are covalently closed. Red and green arrows represent PCR primers

    Techniques Used: Polymerase Chain Reaction

    60) Product Images from "SSBP2 is an in vivo tumor suppressor and regulator of LDB1 stability"

    Article Title: SSBP2 is an in vivo tumor suppressor and regulator of LDB1 stability

    Journal: Oncogene

    doi: 10.1038/onc.2010.78

    Decreased LDB1 half life in Ssbp2 −/− thymocytes underlies impaired T cell differentiation (A) LDB1 levels are decreased in the thymi of Ssbp2 −/− mice. Nuclear proteins from 4 weeks old wild type, Ssbp2 −/− , Trp53 −/− and Ssbp2 −/− Trp53 −/− mice thymi were separated on a 4–12% NuPage gel, transferred to nitrocellulose and probed with Ldb1 antibody. Lower panel shows Ponceau staining to denote equal loading. The signals were analyzed by IMAGE analysis software and the ratio for each sample is denoted. Representative samples from triplicate experiments are shown. (B) Ldb1 transcript levels are not altered in Ssbp2 −/− thymocytes Ssbp2 transcripts in four weeks old thymi were estimated by Real-time PCR. Results show that Ldb1 transcript levels were similar between all the four genotypes tested. Data represent average of two separate experiments each with triplicate reactions. (C) LDB1 half life is shortened in the absence of Ssbp2 Short term cultured thymocytes from wild type and Ssbp2 −/− mice were treated with cycloheximide for the indicated lengths of time and whole cell lysates isolated were examined for LDB1 levels by immunoblotting. Protein loading was quantified by Ponceau staining. The signals were analyzed by IMAGE analysis software and the ratios at various time points plotted to determine LDB1 half life. (D) preTα expression is decreased in Ssbp2 −/− thymus. Thymocytes from four weeks old mice were divided into seven subsets based on maturity. Real-time PCRs were performed on purified cell populations using specific primers for pTα. For each cDNA pool, transcript levels were normalized against 18sRNA within that sample. Two separate experiments, each with triplicate reactions were done.
    Figure Legend Snippet: Decreased LDB1 half life in Ssbp2 −/− thymocytes underlies impaired T cell differentiation (A) LDB1 levels are decreased in the thymi of Ssbp2 −/− mice. Nuclear proteins from 4 weeks old wild type, Ssbp2 −/− , Trp53 −/− and Ssbp2 −/− Trp53 −/− mice thymi were separated on a 4–12% NuPage gel, transferred to nitrocellulose and probed with Ldb1 antibody. Lower panel shows Ponceau staining to denote equal loading. The signals were analyzed by IMAGE analysis software and the ratio for each sample is denoted. Representative samples from triplicate experiments are shown. (B) Ldb1 transcript levels are not altered in Ssbp2 −/− thymocytes Ssbp2 transcripts in four weeks old thymi were estimated by Real-time PCR. Results show that Ldb1 transcript levels were similar between all the four genotypes tested. Data represent average of two separate experiments each with triplicate reactions. (C) LDB1 half life is shortened in the absence of Ssbp2 Short term cultured thymocytes from wild type and Ssbp2 −/− mice were treated with cycloheximide for the indicated lengths of time and whole cell lysates isolated were examined for LDB1 levels by immunoblotting. Protein loading was quantified by Ponceau staining. The signals were analyzed by IMAGE analysis software and the ratios at various time points plotted to determine LDB1 half life. (D) preTα expression is decreased in Ssbp2 −/− thymus. Thymocytes from four weeks old mice were divided into seven subsets based on maturity. Real-time PCRs were performed on purified cell populations using specific primers for pTα. For each cDNA pool, transcript levels were normalized against 18sRNA within that sample. Two separate experiments, each with triplicate reactions were done.

    Techniques Used: Cell Differentiation, Mouse Assay, Staining, Software, Real-time Polymerase Chain Reaction, Cell Culture, Isolation, Expressing, Purification

    61) Product Images from "Basolateral proteinase-activated receptor (PAR-2) induces chloride secretion in M-1 mouse renal cortical collecting duct cells"

    Article Title: Basolateral proteinase-activated receptor (PAR-2) induces chloride secretion in M-1 mouse renal cortical collecting duct cells

    Journal: The Journal of Physiology

    doi: 10.1111/j.1469-7793.1999.00003.x

    RT-PCR and Northern blot evidence for PAR-2 expression in M-1 CCD cells A , agarose gel electrophoresis of products from RT-PCR reactions performed with primers based on mouse PAR-2 and using RNA from M-1 cells, mouse kidney and colon. Products of the predicted size of 1200 bp were detected except in the negative control in which water was used instead of RNA. B , Northern blot analysis using a cDNA probe to mouse PAR-2 and RNA isolated from M-1 cells, mouse kidney and colon. A primary transcript of the predicted size of approximately 3 kb was detected in M-1 cells, as well as in intact kidney and colon.
    Figure Legend Snippet: RT-PCR and Northern blot evidence for PAR-2 expression in M-1 CCD cells A , agarose gel electrophoresis of products from RT-PCR reactions performed with primers based on mouse PAR-2 and using RNA from M-1 cells, mouse kidney and colon. Products of the predicted size of 1200 bp were detected except in the negative control in which water was used instead of RNA. B , Northern blot analysis using a cDNA probe to mouse PAR-2 and RNA isolated from M-1 cells, mouse kidney and colon. A primary transcript of the predicted size of approximately 3 kb was detected in M-1 cells, as well as in intact kidney and colon.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot, Expressing, Agarose Gel Electrophoresis, Negative Control, Isolation

    62) Product Images from "A Novel Dirofilaria Species Causing Human and Canine Infections in Hong Kong"

    Article Title: A Novel Dirofilaria Species Causing Human and Canine Infections in Hong Kong

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.01590-12

    PCR and DNA sequencing.
    Figure Legend Snippet: PCR and DNA sequencing.

    Techniques Used: Polymerase Chain Reaction, DNA Sequencing

    63) Product Images from "Revisiting the human polypeptide GalNAc-T1 and T13 paralogs"

    Article Title: Revisiting the human polypeptide GalNAc-T1 and T13 paralogs

    Journal: Glycobiology

    doi: 10.1093/glycob/cww111

    GalNAc-T13 splice variants. ( A ) Reverse transcription-polymerase chain reaction (RT-PCR) was performed with GalNT13_Fw and T13_XhoI_Rev primers on four different cancer cell lines and on human brain tissue. Several bands of unexpected size were observed after ethidium bromide gel staining. MW: molecular weight (100 bp DNA ladder); -: negative control (water); IGR-N-91 PTX: neuroblastoma cell line derived from a primary tumor; IGR-N-91 BM: metastatic neuroblastoma cell line; CNS: central nervous system tissue (brain); HeLa: cervical cancer cell line; IMR-32: neuroblastoma cell line. The β2M (β2-microglobulin) gene was amplified to verify cDNA quality (except for CNS, where we used GalNAc-T9 as a control). ( B ) Screening of GalNAc-T13 splice variants by colony PCR. Splice variants were screened by RT-PCR with T13_XhoI_Fw and T13_XhoI_Rev primers. The product was cloned into the pGEM-T Easy plasmid and analyzed by colony PCR and digestion with restriction enzymes Hind III and Eco RV. The expected product sizes were 732, 562 and 377 bp. In the illustrative gel (representative gel of 9 of more than 300 analyzed clones), the black arrow indicates digestion products of the previously characterized positive control (pGEM-T with the GalNAc-T13 coding region, confirmed by sequencing), whereas the white arrows mark clones with alternative digestion patterns. These clones were subsequently analyzed by sequencing. ( C ) Exon organization of the newly identified GalNAc-T13 splice variants. The transmembrane (TM), catalytic and lectin domains of GalNAc-Ts are indicated, as well as the three lectin subdomains α, β and γ. The amino acid (AA) extension of each variant is indicated on the right. Skipped exons are shown in white, Ex10b is shown in black and stop codons are marked by arrows. Dashed lines indicate regions where exon skipping introduces a change in the reading frame, resulting in a different AA sequence.
    Figure Legend Snippet: GalNAc-T13 splice variants. ( A ) Reverse transcription-polymerase chain reaction (RT-PCR) was performed with GalNT13_Fw and T13_XhoI_Rev primers on four different cancer cell lines and on human brain tissue. Several bands of unexpected size were observed after ethidium bromide gel staining. MW: molecular weight (100 bp DNA ladder); -: negative control (water); IGR-N-91 PTX: neuroblastoma cell line derived from a primary tumor; IGR-N-91 BM: metastatic neuroblastoma cell line; CNS: central nervous system tissue (brain); HeLa: cervical cancer cell line; IMR-32: neuroblastoma cell line. The β2M (β2-microglobulin) gene was amplified to verify cDNA quality (except for CNS, where we used GalNAc-T9 as a control). ( B ) Screening of GalNAc-T13 splice variants by colony PCR. Splice variants were screened by RT-PCR with T13_XhoI_Fw and T13_XhoI_Rev primers. The product was cloned into the pGEM-T Easy plasmid and analyzed by colony PCR and digestion with restriction enzymes Hind III and Eco RV. The expected product sizes were 732, 562 and 377 bp. In the illustrative gel (representative gel of 9 of more than 300 analyzed clones), the black arrow indicates digestion products of the previously characterized positive control (pGEM-T with the GalNAc-T13 coding region, confirmed by sequencing), whereas the white arrows mark clones with alternative digestion patterns. These clones were subsequently analyzed by sequencing. ( C ) Exon organization of the newly identified GalNAc-T13 splice variants. The transmembrane (TM), catalytic and lectin domains of GalNAc-Ts are indicated, as well as the three lectin subdomains α, β and γ. The amino acid (AA) extension of each variant is indicated on the right. Skipped exons are shown in white, Ex10b is shown in black and stop codons are marked by arrows. Dashed lines indicate regions where exon skipping introduces a change in the reading frame, resulting in a different AA sequence.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Staining, Molecular Weight, Negative Control, Derivative Assay, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Positive Control, Sequencing, Variant Assay

    64) Product Images from "Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE) Genotyping Method for Genomic DNA, Guthrie Blood Spot, and Whole Blood"

    Article Title: Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE) Genotyping Method for Genomic DNA, Guthrie Blood Spot, and Whole Blood

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0153593

    Representative gel electrophoresis of PCR amplicon using sequencing primers from different DNA preparations. (A) PCR amplicon generated from genomic DNA purified from peripheral blood cells. (B) PCR amplicon generated from Guthrie blood spot. (C) PCR amplicon generated from a crude lysate of peripheral blood cells (C). M: DNA markers; the arrow indicates the PCR amplicon with the size of 307 bp. W: indicates water blank as a negative control.
    Figure Legend Snippet: Representative gel electrophoresis of PCR amplicon using sequencing primers from different DNA preparations. (A) PCR amplicon generated from genomic DNA purified from peripheral blood cells. (B) PCR amplicon generated from Guthrie blood spot. (C) PCR amplicon generated from a crude lysate of peripheral blood cells (C). M: DNA markers; the arrow indicates the PCR amplicon with the size of 307 bp. W: indicates water blank as a negative control.

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification, Sequencing, Generated, Purification, Negative Control

    65) Product Images from "Transcript Profiling Reveals the Presence of Abiotic Stress and Developmental Stage Specific Ascorbate Oxidase Genes in Plants"

    Article Title: Transcript Profiling Reveals the Presence of Abiotic Stress and Developmental Stage Specific Ascorbate Oxidase Genes in Plants

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2017.00198

    Expression pattern of AAO genes in rice based on RT-PCR and qRT-PCR analysis. (A) EtBr stained agarose gel depicting the expression of AAO genes in root and shoot tissue of rice seedling. Individual lanes show amplicons corresponding to OsAAO1 – OsAAO5 amplified from rice root and shoot tissue using gene specific real-time PCR primers. M corresponds to 50 bp ladder. (B) Histogram representing fold change of OsAAO1, OsAAO2, OsAAO3 , and OsAAO4 in 1 and 24 h stress treated shoot and root tissue of rice seedling based on qRT-PCR analysis. OsAAO5 could not be amplified hence it was not included in real-time analysis. Real-time PCR was done with cDNA template synthesized from shoot and root tissue of 10 days old control or stressed (salinity 200 mM NaCl and drought) rice seedlings.
    Figure Legend Snippet: Expression pattern of AAO genes in rice based on RT-PCR and qRT-PCR analysis. (A) EtBr stained agarose gel depicting the expression of AAO genes in root and shoot tissue of rice seedling. Individual lanes show amplicons corresponding to OsAAO1 – OsAAO5 amplified from rice root and shoot tissue using gene specific real-time PCR primers. M corresponds to 50 bp ladder. (B) Histogram representing fold change of OsAAO1, OsAAO2, OsAAO3 , and OsAAO4 in 1 and 24 h stress treated shoot and root tissue of rice seedling based on qRT-PCR analysis. OsAAO5 could not be amplified hence it was not included in real-time analysis. Real-time PCR was done with cDNA template synthesized from shoot and root tissue of 10 days old control or stressed (salinity 200 mM NaCl and drought) rice seedlings.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Staining, Agarose Gel Electrophoresis, Amplification, Real-time Polymerase Chain Reaction, Synthesized

    66) Product Images from "Evidence for Distinct Functions of MRE11 in Arabidopsis Meiosis "

    Article Title: Evidence for Distinct Functions of MRE11 in Arabidopsis Meiosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078760

    Molecular analysis and the effect of the T-DNA insertion in mre11 mutant lines. a ) Schematic representation of the mre11-4 allele with the T-DNA disruption located in the 18 th intron (right border, NPT-1) and the left border (LBc-1) oriented toward 3´ end of the MRE11 gene. Vertical arrows indicate the T-DNA insertion sites for mre11-2 and mre11-3 alleles, previously characterized [ 21 , 35 ]. Green boxes represent exons. MRE11 gene specific primers are shown by short horizontal arrows. ( b ) Reverse transcriptase (RT)-PCR of MRE11 transcripts in wild-type and three mre11 mutants. The full-length transcripts were not produced in the three mre11 mutants. Primers spanning different regions of MRE11 transcripts used in the second round of RT-PCR are indicated at the top of each column. Glyceraldehyde-3-phosphate dehydrogenase A (GAPA) was used as control for cDNA amount and quality. c ) Schematic representation of the predicted full-length MRE11 protein (wt) and putative truncated MRE11 proteins: mre11-3 mutant lacks 461 amino acids, mre11-4 lacks 221 amino acids and mre11-2 lacks 191 amino acids. Arrows indicate the T-DNA disruption sites of the MRE11 gene with respect to the full-length protein. The various putative protein domains are marked according to [ 8 , 36 ]; the phosphoesterase motifs (I to IV) with red boxes and two DNA binding domains (blue boxes) as well as the regions important for NBS1 and RAD50 interaction. Ideograms are drawn roughly in scale. Scale bars indicate 100 amino acids. d ) Sequence analysis of the junction between the T-DNA and MRE11 gene obtained via sequencing in the mre11-4 mutants. The top line shows the genomic sequence, exon sequence is shown in uppercase letters, intron sequence is shown in lowercase italic letters, the filler DNA nucleotides are shown in small red uppercase letters and the nucleotides derived from the T-DNA insertion are shown in uppercase boldface letters. The bottom lines show the predicted amino acid sequence as a result of the T-DNA insertion. If the truncated intron 18 is not spliced out, hypothetically, 35 amino acids (ARRYRFS CLITFFNSGLLFQTGTTLNPFSGYSFDL) could be derived from the intron, filler DNA and T-DNA and form the C terminus of the predicted protein in the mre11-4 line. The predicted STOP codon is indicated by *.
    Figure Legend Snippet: Molecular analysis and the effect of the T-DNA insertion in mre11 mutant lines. a ) Schematic representation of the mre11-4 allele with the T-DNA disruption located in the 18 th intron (right border, NPT-1) and the left border (LBc-1) oriented toward 3´ end of the MRE11 gene. Vertical arrows indicate the T-DNA insertion sites for mre11-2 and mre11-3 alleles, previously characterized [ 21 , 35 ]. Green boxes represent exons. MRE11 gene specific primers are shown by short horizontal arrows. ( b ) Reverse transcriptase (RT)-PCR of MRE11 transcripts in wild-type and three mre11 mutants. The full-length transcripts were not produced in the three mre11 mutants. Primers spanning different regions of MRE11 transcripts used in the second round of RT-PCR are indicated at the top of each column. Glyceraldehyde-3-phosphate dehydrogenase A (GAPA) was used as control for cDNA amount and quality. c ) Schematic representation of the predicted full-length MRE11 protein (wt) and putative truncated MRE11 proteins: mre11-3 mutant lacks 461 amino acids, mre11-4 lacks 221 amino acids and mre11-2 lacks 191 amino acids. Arrows indicate the T-DNA disruption sites of the MRE11 gene with respect to the full-length protein. The various putative protein domains are marked according to [ 8 , 36 ]; the phosphoesterase motifs (I to IV) with red boxes and two DNA binding domains (blue boxes) as well as the regions important for NBS1 and RAD50 interaction. Ideograms are drawn roughly in scale. Scale bars indicate 100 amino acids. d ) Sequence analysis of the junction between the T-DNA and MRE11 gene obtained via sequencing in the mre11-4 mutants. The top line shows the genomic sequence, exon sequence is shown in uppercase letters, intron sequence is shown in lowercase italic letters, the filler DNA nucleotides are shown in small red uppercase letters and the nucleotides derived from the T-DNA insertion are shown in uppercase boldface letters. The bottom lines show the predicted amino acid sequence as a result of the T-DNA insertion. If the truncated intron 18 is not spliced out, hypothetically, 35 amino acids (ARRYRFS CLITFFNSGLLFQTGTTLNPFSGYSFDL) could be derived from the intron, filler DNA and T-DNA and form the C terminus of the predicted protein in the mre11-4 line. The predicted STOP codon is indicated by *.

    Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Produced, Binding Assay, Sequencing, Derivative Assay

    67) Product Images from "A toolkit for rapid gene mapping in the nematode Caenorhabditis briggsae"

    Article Title: A toolkit for rapid gene mapping in the nematode Caenorhabditis briggsae

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-236

    Sub-chromosomal localization of mutations by medium and small indel-based mapping . The gel images show non-mutant (wild type, W) and mutant (M) pool of PCR amplified DNA from F2 worms. The histograms show ULVs for various indels. (A) dpy(sy5001) is most strongly linked to the medium indel cb-m197 (located roughly in the middle of chromosome X, ChrX-M) compared to flanking indels cb-m204 (left arm, ChrX-L) and cb-m136 (right arm, ChrX-R). (B) dpy(s1272), unc(sa972) , and lin(bh20) are located on chromosome 3. While dpy(s1272) and unc(sa972) are strongly linked to bhP14 and bhP18 and appear to be on the left arm, lin(bh20) maps closer to bhP40 (center right region). (C) unc(sy5422) is tightly linked to indels bhP9 and bhP16 on the right arm of chromosome 4.
    Figure Legend Snippet: Sub-chromosomal localization of mutations by medium and small indel-based mapping . The gel images show non-mutant (wild type, W) and mutant (M) pool of PCR amplified DNA from F2 worms. The histograms show ULVs for various indels. (A) dpy(sy5001) is most strongly linked to the medium indel cb-m197 (located roughly in the middle of chromosome X, ChrX-M) compared to flanking indels cb-m204 (left arm, ChrX-L) and cb-m136 (right arm, ChrX-R). (B) dpy(s1272), unc(sa972) , and lin(bh20) are located on chromosome 3. While dpy(s1272) and unc(sa972) are strongly linked to bhP14 and bhP18 and appear to be on the left arm, lin(bh20) maps closer to bhP40 (center right region). (C) unc(sy5422) is tightly linked to indels bhP9 and bhP16 on the right arm of chromosome 4.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification

    Mutation mapping by polymorphisms . The indels were used to map dpy(sy5001) (A) and dpy(sy5148) (B, C) and snip-SNP (cb650) was used to map lin(bh25) (D). (A) Mapping of X-linked mutation dpy(sy5001) using six medium indels (one per chromosome). W, non-mutant (phenotypically wild type) pool; M, mutant pool. (B) dpy(sy5148) localization on chromosome 2 by small indels (Chr 1: bhP19, Chr 2: bhP21, Chr 3: bhP12, Chr 4: bhP11 and Chr X: bhP26). (C) ULVs for dpy(sy5148) show linkage to bhP21. X-axis shows chromosomes whereas Y-axis linkage values. The dotted line shows the baseline for unlinked chromosomes. (D) Sac I digested PCR amplified genomic DNA of wild-type controls (A: AF16, H: HK104) and lin(bh25) mutant (M) and non-mutant (W) categories. There is a clear bias towards AF16 DNA (uncut) in the mutant pool compared to the non-mutant pool, demonstrating that lin(bh25) is linked to cb650 (chr. 1).
    Figure Legend Snippet: Mutation mapping by polymorphisms . The indels were used to map dpy(sy5001) (A) and dpy(sy5148) (B, C) and snip-SNP (cb650) was used to map lin(bh25) (D). (A) Mapping of X-linked mutation dpy(sy5001) using six medium indels (one per chromosome). W, non-mutant (phenotypically wild type) pool; M, mutant pool. (B) dpy(sy5148) localization on chromosome 2 by small indels (Chr 1: bhP19, Chr 2: bhP21, Chr 3: bhP12, Chr 4: bhP11 and Chr X: bhP26). (C) ULVs for dpy(sy5148) show linkage to bhP21. X-axis shows chromosomes whereas Y-axis linkage values. The dotted line shows the baseline for unlinked chromosomes. (D) Sac I digested PCR amplified genomic DNA of wild-type controls (A: AF16, H: HK104) and lin(bh25) mutant (M) and non-mutant (W) categories. There is a clear bias towards AF16 DNA (uncut) in the mutant pool compared to the non-mutant pool, demonstrating that lin(bh25) is linked to cb650 (chr. 1).

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification

    68) Product Images from "Effect of sample size and P-value filtering techniques on the detection of transcriptional changes induced in rat neuroblastoma (NG108) cells by mefloquine"

    Article Title: Effect of sample size and P-value filtering techniques on the detection of transcriptional changes induced in rat neuroblastoma (NG108) cells by mefloquine

    Journal: Malaria Journal

    doi: 10.1186/1475-2875-2-4

    Semi-quantitative RT-PCR of tubulin, pJunB, EST cJun (AI175959), IkappaB and GADD153 mRNA from DMSO or mefloquine-treated NG108 rat neuronal cell cultures. cDNA was synthesized using oligo-(dT) primers and amplified using gene specific primers. Results are expressed relative to mean optical density for DMSO samples for each gene. (* P value
    Figure Legend Snippet: Semi-quantitative RT-PCR of tubulin, pJunB, EST cJun (AI175959), IkappaB and GADD153 mRNA from DMSO or mefloquine-treated NG108 rat neuronal cell cultures. cDNA was synthesized using oligo-(dT) primers and amplified using gene specific primers. Results are expressed relative to mean optical density for DMSO samples for each gene. (* P value

    Techniques Used: Quantitative RT-PCR, Synthesized, Amplification

    69) Product Images from "The KDM3A–KLF2–IRF4 axis maintains myeloma cell survival"

    Article Title: The KDM3A–KLF2–IRF4 axis maintains myeloma cell survival

    Journal: Nature Communications

    doi: 10.1038/ncomms10258

    KDM3A directly regulates KLF2 and IRF4 expression through H3K9 demethylation at their promoters in MM cells. ( a ) Heatmap depicting the relative gene expression in RPMI8226 cells transduced with two independent shRNAs targeting KDM3A (shKDM3A #1 and shKDM3A #2) or shLuc. A total of 305 probes were selected based on ≥1.5-fold downregulation in KDM3A -knockdown cells and clustered. ( b – d ) Quantitative real-time PCR ( b ) and immunoblot ( c , d ) analysis of KLF2 and IRF4 in RPMI8226 cells transduced with either shKDM3A or shLuc. After 3 days of infection, which is defined as day 0 in Fig. 2 , cells were harvested for isolation of total RNA or whole-cell lysates. ( b ) Values represent the amount of mRNA relative to shLuc, defined as 1. ( c ) Arrowhead represents KLF2. ( d ) Signal intensity of each immunoblot was quantified using the ImageJ software. Results were normalized by Actin and are shown as relative signal intensity (shLuc=1). Error bars represent s.e.m of three independent experiments. ( e , f ) Quantitative real-time PCR ( e ) and immunoblot ( f ) analysis of KLF2 and IRF4 in MM.1S and U266 cells transduced with either shKDM3A or shLuc. After 3 days of infection, cells were harvested for isolation of total RNA or whole-cell lysates. ( g ) ChIP analysis showing KDM3A occupancy on KLF2 and IRF4 core promoters in RPMI8226 cells. Results were normalized to control IgG. The MYOD1 promoter region was used as negative control. ( h ) KDM3A occupancy is abrogated by KDM3A knockdown on KLF2 and IRF4 promoter regions in RPMI8226 cells. RPMI8226 cells transduced with either shKDM3A or shLuc were used for ChIP, followed by quantitative real-time PCR. ( i ) Enrichment of H3K9 methylation by KDM3A knockdown on KLF2 and IRF4 promoter regions in RPMI8226 cells. ChIP assays were performed with RPMI8226 cells transduced with either shKDM3A or shLuc. The relative enrichment over the input was assessed. Results are shown as fold enrichment compared with control shLuc. For b , e , g – i , error bars represent s.d. of triplicate measurements. For b – i , data are representative of at least two independent experiments. ** P
    Figure Legend Snippet: KDM3A directly regulates KLF2 and IRF4 expression through H3K9 demethylation at their promoters in MM cells. ( a ) Heatmap depicting the relative gene expression in RPMI8226 cells transduced with two independent shRNAs targeting KDM3A (shKDM3A #1 and shKDM3A #2) or shLuc. A total of 305 probes were selected based on ≥1.5-fold downregulation in KDM3A -knockdown cells and clustered. ( b – d ) Quantitative real-time PCR ( b ) and immunoblot ( c , d ) analysis of KLF2 and IRF4 in RPMI8226 cells transduced with either shKDM3A or shLuc. After 3 days of infection, which is defined as day 0 in Fig. 2 , cells were harvested for isolation of total RNA or whole-cell lysates. ( b ) Values represent the amount of mRNA relative to shLuc, defined as 1. ( c ) Arrowhead represents KLF2. ( d ) Signal intensity of each immunoblot was quantified using the ImageJ software. Results were normalized by Actin and are shown as relative signal intensity (shLuc=1). Error bars represent s.e.m of three independent experiments. ( e , f ) Quantitative real-time PCR ( e ) and immunoblot ( f ) analysis of KLF2 and IRF4 in MM.1S and U266 cells transduced with either shKDM3A or shLuc. After 3 days of infection, cells were harvested for isolation of total RNA or whole-cell lysates. ( g ) ChIP analysis showing KDM3A occupancy on KLF2 and IRF4 core promoters in RPMI8226 cells. Results were normalized to control IgG. The MYOD1 promoter region was used as negative control. ( h ) KDM3A occupancy is abrogated by KDM3A knockdown on KLF2 and IRF4 promoter regions in RPMI8226 cells. RPMI8226 cells transduced with either shKDM3A or shLuc were used for ChIP, followed by quantitative real-time PCR. ( i ) Enrichment of H3K9 methylation by KDM3A knockdown on KLF2 and IRF4 promoter regions in RPMI8226 cells. ChIP assays were performed with RPMI8226 cells transduced with either shKDM3A or shLuc. The relative enrichment over the input was assessed. Results are shown as fold enrichment compared with control shLuc. For b , e , g – i , error bars represent s.d. of triplicate measurements. For b – i , data are representative of at least two independent experiments. ** P

    Techniques Used: Expressing, Transduction, Real-time Polymerase Chain Reaction, Infection, Isolation, Software, Chromatin Immunoprecipitation, Negative Control, Methylation

    70) Product Images from "A Salt Overly Sensitive Pathway Member from Brassica juncea BjSOS3 Can Functionally Complement ΔAtsos3 in Arabidopsis"

    Article Title: A Salt Overly Sensitive Pathway Member from Brassica juncea BjSOS3 Can Functionally Complement ΔAtsos3 in Arabidopsis

    Journal: Current Genomics

    doi: 10.2174/1389202918666170228133621

    Transcript abundance of SOS3 members in BjSOS3 complemented transgenic arabidopsis and their ABA stress response. Transgenic arabidopsis seedlings were grown on MS agar plate and samples were harvested for expression analysis. qRT-PCR was performed using the cDNA prepared from total RNA isolated from whole seedlings. Relative expression of ( a ) BjSOS3 , ( b ) AtSOS1 ( c ) AtSOS2 . Bar graphs were plotted between stress duration (X-axis) and log 2 -dCt value in number (Y-axis). Gene expression data was normalised with the plant reference gene ‘actin’ as an internal control. The values represented are the mean of two biological and three technical replicates, standard error is shown above the bar. For statistical significance, student t -test was performed and asterisk above the graph means significant differences from WT at P ≤ 0.05. Four days old seedlings were transferred to MS media supplemented with ( d ) 0 or ( e ) 25 µM of ABA. After 7 days of growth, pictures were taken and ( f ) root length was measured. Error bars represent the standard deviation (n = 15). For statistical significance, student’s t -test was performed and asterisk above the graph means significant differences from WT at P ≤ 0.05.
    Figure Legend Snippet: Transcript abundance of SOS3 members in BjSOS3 complemented transgenic arabidopsis and their ABA stress response. Transgenic arabidopsis seedlings were grown on MS agar plate and samples were harvested for expression analysis. qRT-PCR was performed using the cDNA prepared from total RNA isolated from whole seedlings. Relative expression of ( a ) BjSOS3 , ( b ) AtSOS1 ( c ) AtSOS2 . Bar graphs were plotted between stress duration (X-axis) and log 2 -dCt value in number (Y-axis). Gene expression data was normalised with the plant reference gene ‘actin’ as an internal control. The values represented are the mean of two biological and three technical replicates, standard error is shown above the bar. For statistical significance, student t -test was performed and asterisk above the graph means significant differences from WT at P ≤ 0.05. Four days old seedlings were transferred to MS media supplemented with ( d ) 0 or ( e ) 25 µM of ABA. After 7 days of growth, pictures were taken and ( f ) root length was measured. Error bars represent the standard deviation (n = 15). For statistical significance, student’s t -test was performed and asterisk above the graph means significant differences from WT at P ≤ 0.05.

    Techniques Used: Transgenic Assay, Mass Spectrometry, Expressing, Quantitative RT-PCR, Isolation, Standard Deviation

    Growth of seedlings of contrasting Brassica genotypes in response to high salinity and ABA, and transcript abundance for various SOS pathway members. Nine days old hydroponically grown Brassica seedlings were subjected to salinity (200 mM NaCl) or ABA (100 µM). qRT-PCR was performed using the cDNA prepared from total RNA isolated from shoots of the seedlings subjected to salinity stress (200 mM NaCl); or ABA (100 µM) for 8 h and 24 h time period. ( a ). Representative photograph of seedlings of Brassica genotypes after 24 h of salinity (200 mM NaCl) or ABA (100 µM). Relative expression of ( b ) BjSOS1 , ( c ) BjSOS2 and ( d ) BjSOS3 . Bar graphs were plotted between stress duration (X-axis) and log 2 -ddCt value in number (Y-axis). Gene expression data was normalised with the plant reference gene ‘actin’ as an internal control. Relative expression of genes was plotted against the expression of B. nigra control. The values represented are the mean of two biological and three technical replicates, standard error is shown above the bar. For statistical significance, student t -test was performed and asterisk above the graph means significant differences from their respective control (Con) at P ≤ 0.05. ( For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper. )
    Figure Legend Snippet: Growth of seedlings of contrasting Brassica genotypes in response to high salinity and ABA, and transcript abundance for various SOS pathway members. Nine days old hydroponically grown Brassica seedlings were subjected to salinity (200 mM NaCl) or ABA (100 µM). qRT-PCR was performed using the cDNA prepared from total RNA isolated from shoots of the seedlings subjected to salinity stress (200 mM NaCl); or ABA (100 µM) for 8 h and 24 h time period. ( a ). Representative photograph of seedlings of Brassica genotypes after 24 h of salinity (200 mM NaCl) or ABA (100 µM). Relative expression of ( b ) BjSOS1 , ( c ) BjSOS2 and ( d ) BjSOS3 . Bar graphs were plotted between stress duration (X-axis) and log 2 -ddCt value in number (Y-axis). Gene expression data was normalised with the plant reference gene ‘actin’ as an internal control. Relative expression of genes was plotted against the expression of B. nigra control. The values represented are the mean of two biological and three technical replicates, standard error is shown above the bar. For statistical significance, student t -test was performed and asterisk above the graph means significant differences from their respective control (Con) at P ≤ 0.05. ( For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper. )

    Techniques Used: Quantitative RT-PCR, Isolation, Expressing

    71) Product Images from "Sequence of the Escherichia coli O121 O-Antigen Gene Cluster and Detection of Enterohemorrhagic E. coli O121 by PCR Amplification of the wzx and wzy Genes"

    Article Title: Sequence of the Escherichia coli O121 O-Antigen Gene Cluster and Detection of Enterohemorrhagic E. coli O121 by PCR Amplification of the wzx and wzy Genes

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.41.7.3379-3383.2003

    Agarose gel showing PCR results of DNA from seven E. coli O121-positive swine fecal samples using two primer sets for the wzx gene and two primer sets for the wzy gene. (I) Lanes 1 to 7 and 8 to 14, PCR products using DNA from isolates K84-9 O121:H10, K84-11 O121:H10, K84-12 O121:H10, K84-36 O121:H10, K84-40 O121:H10, K102-27 O121:H − , and K150-1 O121:H − , respectively, and primer sets O121wzx1F-O121wzx1R (A) and O121wzx2F-O121wzx2R (B). Lanes 15 to 20 and 21 to 26, PCR products using DNA from E. coli O103:H3 93-0626, C. freundii ATCC 33128, C. braakii ATCC 43162, E. coli O111:NM 91.0130, S. flexneri ATCC 12022, and E. coli O121:H19 96-1585, respectively, and primer sets O121wzx2F-O121wzx2R and O121wzx1F-O121wzx1R, respectively. (II) Lanes 1 to 7 and 8 to 14, PCR products using DNA from the same swine fecal isolates described above for panel I and primer sets O121wzy1F-O121wzy1R (A) and O121wzy2F-O121wzy2R (B), respectively. Lanes 15 to 20 and 21 to 26, PCR products using DNA from E. coli O121:H19 96-1585, E. coli O103:H3 93-0626, C. freundii ATCC 33128, C. braakii ATCC 43162, E. coli O111:NM 91.0130, and S. flexneri ATCC 12022, respectively, and primer sets O121wzy1F-O121wzy1R and O121wzy2F-O121wzy2R, respectively. Lanes M, 100-bp ladder molecular size standards (Invitrogen).
    Figure Legend Snippet: Agarose gel showing PCR results of DNA from seven E. coli O121-positive swine fecal samples using two primer sets for the wzx gene and two primer sets for the wzy gene. (I) Lanes 1 to 7 and 8 to 14, PCR products using DNA from isolates K84-9 O121:H10, K84-11 O121:H10, K84-12 O121:H10, K84-36 O121:H10, K84-40 O121:H10, K102-27 O121:H − , and K150-1 O121:H − , respectively, and primer sets O121wzx1F-O121wzx1R (A) and O121wzx2F-O121wzx2R (B). Lanes 15 to 20 and 21 to 26, PCR products using DNA from E. coli O103:H3 93-0626, C. freundii ATCC 33128, C. braakii ATCC 43162, E. coli O111:NM 91.0130, S. flexneri ATCC 12022, and E. coli O121:H19 96-1585, respectively, and primer sets O121wzx2F-O121wzx2R and O121wzx1F-O121wzx1R, respectively. (II) Lanes 1 to 7 and 8 to 14, PCR products using DNA from the same swine fecal isolates described above for panel I and primer sets O121wzy1F-O121wzy1R (A) and O121wzy2F-O121wzy2R (B), respectively. Lanes 15 to 20 and 21 to 26, PCR products using DNA from E. coli O121:H19 96-1585, E. coli O103:H3 93-0626, C. freundii ATCC 33128, C. braakii ATCC 43162, E. coli O111:NM 91.0130, and S. flexneri ATCC 12022, respectively, and primer sets O121wzy1F-O121wzy1R and O121wzy2F-O121wzy2R, respectively. Lanes M, 100-bp ladder molecular size standards (Invitrogen).

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction

    72) Product Images from "Allele-Specific rpoB PCR Assays for Detection of Rifampin-Resistant Mycobacterium tuberculosis in Sputum Smears"

    Article Title: Allele-Specific rpoB PCR Assays for Detection of Rifampin-Resistant Mycobacterium tuberculosis in Sputum Smears

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.47.7.2231-2235.2003

    Profiles generated by single-step MAS-PCR assay with purified DNA preparations from clinical M. tuberculosis strains targeting three rpoB codons: codon 531 (A), codon 526 (B), and codon 516 (C). Lanes: 1, H37Rv strain; 2 and 3, strains with rpoB531 mutant alleles (TCG and TTG); 4 to 6, strains with rpoB526 mutant alleles (GAC, TAC, and CTC); 7 to 9, strains with rpoB516 mutant alleles (GTC, TAC, and GGC); M, 100-bp DNA ladder (Amersham Bioscience).
    Figure Legend Snippet: Profiles generated by single-step MAS-PCR assay with purified DNA preparations from clinical M. tuberculosis strains targeting three rpoB codons: codon 531 (A), codon 526 (B), and codon 516 (C). Lanes: 1, H37Rv strain; 2 and 3, strains with rpoB531 mutant alleles (TCG and TTG); 4 to 6, strains with rpoB526 mutant alleles (GAC, TAC, and CTC); 7 to 9, strains with rpoB516 mutant alleles (GTC, TAC, and GGC); M, 100-bp DNA ladder (Amersham Bioscience).

    Techniques Used: Generated, Polymerase Chain Reaction, Purification, Mutagenesis

    Profiles generated by two-step nested allele-specific PCR assays with sputum slides DNA preparations. (A) First-step PCR with outer rpoB derived primers. (B to D) Analysis of three rpoB codons, codons 531, 526, and 516, respectively, by allele-specific PCR assays. Lanes: 1, H37Rv strain; 2 and 3, strains with rpoB531 mutant alleles (TCG and TTG); 4 to 6, strains with rpoB526 mutant alleles (GAC, TAC, and CTC), 7 to 9, strains with rpoB516 mutant alleles (GTC, TAC, and GGC); M, 100-bp DNA ladder (Amersham Bioscience).
    Figure Legend Snippet: Profiles generated by two-step nested allele-specific PCR assays with sputum slides DNA preparations. (A) First-step PCR with outer rpoB derived primers. (B to D) Analysis of three rpoB codons, codons 531, 526, and 516, respectively, by allele-specific PCR assays. Lanes: 1, H37Rv strain; 2 and 3, strains with rpoB531 mutant alleles (TCG and TTG); 4 to 6, strains with rpoB526 mutant alleles (GAC, TAC, and CTC), 7 to 9, strains with rpoB516 mutant alleles (GTC, TAC, and GGC); M, 100-bp DNA ladder (Amersham Bioscience).

    Techniques Used: Generated, Polymerase Chain Reaction, Derivative Assay, Mutagenesis

    73) Product Images from "Recombinant Modified Vaccinia Virus Ankara Generating Excess Early Double-Stranded RNA Transiently Activates Protein Kinase R and Triggers Enhanced Innate Immune Responses"

    Article Title: Recombinant Modified Vaccinia Virus Ankara Generating Excess Early Double-Stranded RNA Transiently Activates Protein Kinase R and Triggers Enhanced Innate Immune Responses

    Journal: Journal of Virology

    doi: 10.1128/JVI.02082-14

    MVA recombinants expressing excess early dsRNA from neo or EGFP transgenes induce increased IFN-β expression. (A) Schematic representation of the two types of MVA recombinants generating excess early dsRNA either from two neo inserts (top) or from two EGFP inserts (bottom), each with the corresponding control and reference constructs. IGR, intergenic region. (B) Total RNA from murine BALB/3T3-A31 cells infected with the indicated viruses (MOI 10) or mock infected for 6 h was digested with RNase A/T1 (ssRNase digest) or RNase A/T1/V1 (ss+dsRNase digest) or not digested, and duplicate RT-qPCR quantification of total EGFP transcript (both sense and antisense) was performed as described in Materials and Methods. The mean of the fold induction values of EGFP or C7L transcripts over mock in undigested samples was set to 100%, and the mean percentage of the remaining qPCR signals after the indicated RNase digests was calculated for EGFP and C7L transcripts. Shown is one out two independent experiments. Where error bars are not visible, the standard error was negligible. (C) MEFs in 6-well plates were mock infected or infected with crude stocks of the indicated MVA recombinants at an MOI of 10 in duplicate. Fold induction of IFN-β mRNA over mock was determined by duplicate RT-qPCR per sample using total RNA isolated from cells at 6 h p.i. using a commercially available TaqMan assay (Life Technologies) for the murine IFN-β gene. Poly(I·C) was transfected using Fugene HD at 2 μg/well. 18S rRNA served as the endogenous control in all RT-qPCR analyses. Where error bars are not visible, the standard error was negligible. (D) IFN-β amounts in supernatants of MEF cultures infected in parallel to those shown in panel C were determined at 14 h p.i. by ELISA. (E) Murine A31 cells were either preincubated with 40 μg/ml of AraC for 1 h or left untreated and infected in duplicate at an MOI of 10 with the indicated MVAs with either 40 μg/ml AraC throughout infection or without AraC. Cells were harvested at 6 h p.i. for isolation of total RNA. Messenger RNAs for murine IFN-β and the late F17R VACV gene were quantified by qRT-PCR analysis as described above.
    Figure Legend Snippet: MVA recombinants expressing excess early dsRNA from neo or EGFP transgenes induce increased IFN-β expression. (A) Schematic representation of the two types of MVA recombinants generating excess early dsRNA either from two neo inserts (top) or from two EGFP inserts (bottom), each with the corresponding control and reference constructs. IGR, intergenic region. (B) Total RNA from murine BALB/3T3-A31 cells infected with the indicated viruses (MOI 10) or mock infected for 6 h was digested with RNase A/T1 (ssRNase digest) or RNase A/T1/V1 (ss+dsRNase digest) or not digested, and duplicate RT-qPCR quantification of total EGFP transcript (both sense and antisense) was performed as described in Materials and Methods. The mean of the fold induction values of EGFP or C7L transcripts over mock in undigested samples was set to 100%, and the mean percentage of the remaining qPCR signals after the indicated RNase digests was calculated for EGFP and C7L transcripts. Shown is one out two independent experiments. Where error bars are not visible, the standard error was negligible. (C) MEFs in 6-well plates were mock infected or infected with crude stocks of the indicated MVA recombinants at an MOI of 10 in duplicate. Fold induction of IFN-β mRNA over mock was determined by duplicate RT-qPCR per sample using total RNA isolated from cells at 6 h p.i. using a commercially available TaqMan assay (Life Technologies) for the murine IFN-β gene. Poly(I·C) was transfected using Fugene HD at 2 μg/well. 18S rRNA served as the endogenous control in all RT-qPCR analyses. Where error bars are not visible, the standard error was negligible. (D) IFN-β amounts in supernatants of MEF cultures infected in parallel to those shown in panel C were determined at 14 h p.i. by ELISA. (E) Murine A31 cells were either preincubated with 40 μg/ml of AraC for 1 h or left untreated and infected in duplicate at an MOI of 10 with the indicated MVAs with either 40 μg/ml AraC throughout infection or without AraC. Cells were harvested at 6 h p.i. for isolation of total RNA. Messenger RNAs for murine IFN-β and the late F17R VACV gene were quantified by qRT-PCR analysis as described above.

    Techniques Used: Expressing, Construct, Infection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Isolation, TaqMan Assay, Transfection, Enzyme-linked Immunosorbent Assay

    74) Product Images from "An Outbreak of Keratitis Caused by Mycobacterium immunogenum"

    Article Title: An Outbreak of Keratitis Caused by Mycobacterium immunogenum

    Journal:

    doi: 10.1128/JCM.00656-06

    (A) PFGE pattern of DraI digests of chromosomal DNA. Lanes: 1 and 6, lambda DNA standards (New England Biolabs); 2, 3, 4, and 5, outbreak isolates F1111, F1112, F1113, and F1114, respectively. (B) Electrophoretic patterns obtained by ERIC-PCR. Lanes:
    Figure Legend Snippet: (A) PFGE pattern of DraI digests of chromosomal DNA. Lanes: 1 and 6, lambda DNA standards (New England Biolabs); 2, 3, 4, and 5, outbreak isolates F1111, F1112, F1113, and F1114, respectively. (B) Electrophoretic patterns obtained by ERIC-PCR. Lanes:

    Techniques Used: Lambda DNA Preparation, Polymerase Chain Reaction

    75) Product Images from "Evolutionarily novel genes are expressed in transgenic fish tumors and their orthologs are involved in development of progressive traits in humans"

    Article Title: Evolutionarily novel genes are expressed in transgenic fish tumors and their orthologs are involved in development of progressive traits in humans

    Journal: Infectious Agents and Cancer

    doi: 10.1186/s13027-019-0262-5

    Expression of human orthologs of fish TT Rgr EEN genes in cDNA panels from human tumor tissues. LEP – leptin; NR2E1 – nuclear receptor subfamily 2 group E member 1. Tumor cDNA Panel: 1 – brain malignant meningioma moderately differentiated, 2 – breast invasive ductal carcinoma, 3 – colon adenocarcinoma, well differentiated, 4 – kidney renal cell carcinoma papillary, 5 – lung squamous cell carcinoma, well differentiated, 6 – ovary teratoma, 7 – pancreas adenocarcinoma, 8 – prostate adenocarcinoma, 9 – stomach adenocarcinoma, 10 – uterus leiomyoma. NC – no template control, PC – PCR with human DNA. Lower pane: GAPDH control
    Figure Legend Snippet: Expression of human orthologs of fish TT Rgr EEN genes in cDNA panels from human tumor tissues. LEP – leptin; NR2E1 – nuclear receptor subfamily 2 group E member 1. Tumor cDNA Panel: 1 – brain malignant meningioma moderately differentiated, 2 – breast invasive ductal carcinoma, 3 – colon adenocarcinoma, well differentiated, 4 – kidney renal cell carcinoma papillary, 5 – lung squamous cell carcinoma, well differentiated, 6 – ovary teratoma, 7 – pancreas adenocarcinoma, 8 – prostate adenocarcinoma, 9 – stomach adenocarcinoma, 10 – uterus leiomyoma. NC – no template control, PC – PCR with human DNA. Lower pane: GAPDH control

    Techniques Used: Expressing, Fluorescence In Situ Hybridization, Polymerase Chain Reaction

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    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water. .. The PCR amplification consisted of 1 cycle of enzyme activation for 2 min at 50°C followed by denaturation at 95°C for 20 s, and 40 cycles of denaturation at 95°C for 3 s, 30 s annealing at 60°C.

    Article Title: Non-Sulfate-Reducing, Syntrophic Bacteria Affiliated with Desulfotomaculum Cluster I Are Widely Distributed in Methanogenic Environments
    Article Snippet: Paragraph title: PCR amplification of dsrAB and apsA . ... Approximately 40 ng of template DNA was used in a 100-μl PCR mixture; the concentration of template DNA was measured by using the PicoGreen double-stranded DNA quantification kit (Molecular Probes) and a spectrofluorophotometer (RF-5300PC; Shimadzu).

    Article Title: Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT
    Article Snippet: Samples were analysed in triplicate and in the absence of RT enzyme or without sample RNA (replaced with DNase-/RNase-free H2 O; Invitrogen), to control for non-specific amplification of DNA and contamination, respectively. .. Complementary DNA was added to a PCR mix containing sequence-specific reverse and forward primers ( ) and 1 × TaqMan Universal PCR Master Mix (Invitrogen).

    Article Title: Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism
    Article Snippet: The PCR reactions were performed with approximately 50 ng of genomic DNA in a 25-μl PCR mixture, which included 1 μL (200 ng) of DNA template, 2.5 μL of buffer solution (containing the dNTPs and MgCl2 ), 1 μL of each 10 μM primer (F18S and F28S), 1 μL of Taq DNA polymerase (Invitrogen), and 18.5 μL of distilled water. .. The detection of amplified products (550 base pairs [bp]) was performed by electrophoresis of an aliquot of 5 μL of each amplicon in a 1% agarose gel with ethidium bromide 0.02% in 1 × Tris-acetate-EDTA (TAE) buffer.

    Article Title: Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus
    Article Snippet: Briefly, 25 μl of the PCR mixture consisted of 250 ng of DNA; a 0.2 μM concentration of each of the forward and reverse primers; a 200 μM concentration each of dATP, dCTP, dGTP, and dTTP; 2.5 mM MgCl2 ; and 2.5 U of DyNAzyme EXT (Finnzymes) in a 1× reaction buffer. .. One-tenth of the amplified reaction mixture was mixed with a gel-loading buffer and electrophoresed on a 0.8% (wt/vol) agarose gel; the reaction products were visualized by ethidium bromide staining.

    Article Title: Modeling of 5? Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica
    Article Snippet: The Salmonella 5′ nuclease PCR assay amplified a 0.12-kb amplicon that originated from the invA gene ( ). .. The PCR mixture for Ampli Taq Gold was composed of the following reagents and concentrations: 100 nM TaqMan probe, forward and reverse primers at concentrations of 900 nM each, 1× TaqMan Universal PCR Master Mix (including deoxynucleoside triphosphates [dNTPs; 200 μM dATP, 200 μM dCTP, 200 μM dGTP, and 400 μM dUTP]; Applied Biosystems), 0.01 U of uracil- N -glycosylase per μl, 2.5 mM MgCl2 , and 0.025 U of Ampli Taq Gold per μl.

    Article Title: Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay
    Article Snippet: To test the validity of each of the ASPE primers, we amplified the plasmid DNAs individually by PCR. .. One microliter of 10 ng/μl plasmid DNAs (equivalent to 1,618 viral DNA copies) was added to 100 μl of the PCR mixture, containing 0.2 μM primers M13F (5′-CCCAG TCACG ACGTT GTAAA ACG-3′) and M13R (5′-AGCGG ATAAC AATTT CACAC AGG-3′), 1× High Fidelity PCR buffer, 2.0 mM MgSO4 , and 2.0 U of Platinum Taq High Fidelity (Life Technologies, Bethesda, MD).

    Positive Control:

    Article Title: Non-Sulfate-Reducing, Syntrophic Bacteria Affiliated with Desulfotomaculum Cluster I Are Widely Distributed in Methanogenic Environments
    Article Snippet: Approximately 40 ng of template DNA was used in a 100-μl PCR mixture; the concentration of template DNA was measured by using the PicoGreen double-stranded DNA quantification kit (Molecular Probes) and a spectrofluorophotometer (RF-5300PC; Shimadzu). .. Genomic DNA from S. fumaroxidans was used as the positive control in all dsrAB - and apsA -targeted PCR experiments ( ).

    Polymerase Chain Reaction:

    Article Title: The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis
    Article Snippet: .. The complete coding DNA sequence (CDS) of VvibZIPC22 was amplified in a 12.5 µl PCR mix containing primers (200 pM each), 1U of Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), dNTPs (200 µM), PCR buffer (1×), and cDNA (diluted 10-fold). ..

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: .. The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water. .. The PCR amplification consisted of 1 cycle of enzyme activation for 2 min at 50°C followed by denaturation at 95°C for 20 s, and 40 cycles of denaturation at 95°C for 3 s, 30 s annealing at 60°C.

    Article Title: Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle
    Article Snippet: .. This DNA was used as a template in a PCR mixture containing 20 mM Tris (pH 8.4), 50 mM potassium chloride, 1.5 mM magnesium chloride, 1 mM deoxynucleoside triphosphates, and 2.5 U of Platinum Taq DNA polymerase (Gibco BRL) in the following reaction conditions: 94°C for 2 min for 1 cycle and 94°C for 30 s, 55°C for 30 s, and 72°C for 5 min for 30 cycles. ..

    Article Title: Considerations When Analyzing the Methylation Status of PTEN Tumor Suppressor Gene
    Article Snippet: .. For set III, the PCR mixture contained 1X PCR buffer, dNTPs (each at 1.28 mmol/L; Invitrogen), primers (4 ng/μl; Sigma Genosys), HotStarTaq DNA polymerase (1.5U; Qiagen), and bisulfite-modified DNA (∼100 ng) or unmodified DNA (50 to 100 ng) in a final volume of 25 μl. ..

    Article Title: Phase Variation in Myxococcus xanthus Yields Cells Specialized for Iron Sequestration
    Article Snippet: .. 5 µl of diluted cDNA sample was added to 20 µl of a PCR mixture prepared from Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), which contained each primer at a concentration of 160 nM. .. Primers, listed in , were chosen using Primer Express 3.0 software (Applied Biosystems) and were designed to amplify a region of about 150–200 bp within each transcript.

    Article Title: Non-Sulfate-Reducing, Syntrophic Bacteria Affiliated with Desulfotomaculum Cluster I Are Widely Distributed in Methanogenic Environments
    Article Snippet: .. Approximately 40 ng of template DNA was used in a 100-μl PCR mixture; the concentration of template DNA was measured by using the PicoGreen double-stranded DNA quantification kit (Molecular Probes) and a spectrofluorophotometer (RF-5300PC; Shimadzu). .. An approximately 1,900-bp fragment of dsrAB was amplified by using the primers DSR1Fdeg and DSR4Rdeg ( ).

    Article Title: Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT
    Article Snippet: .. Complementary DNA was added to a PCR mix containing sequence-specific reverse and forward primers ( ) and 1 × TaqMan Universal PCR Master Mix (Invitrogen). .. Samples were denatured for 2 min at 50 °C, Taq polymerase was activated by heating for 10 min at 95 °C and cDNA amplified using 40 cycles of 95 °C for 15 s and 60 °C for 1 min. mRNA expression was calculated using the comparative Ct method , relative to the housekeeping genes β-2-microglobulin ( ) and peptidylprolyl isomerase A (PPIA) ( ; ), in cell lines ( ) and primary ESFTs, respectively.

    Article Title: Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism
    Article Snippet: .. The PCR reactions were performed with approximately 50 ng of genomic DNA in a 25-μl PCR mixture, which included 1 μL (200 ng) of DNA template, 2.5 μL of buffer solution (containing the dNTPs and MgCl2 ), 1 μL of each 10 μM primer (F18S and F28S), 1 μL of Taq DNA polymerase (Invitrogen), and 18.5 μL of distilled water. .. The PCR reactions were performed using the iCycler thermocycler (Bio-Rad, Hercules, CA, USA) with the following cycling parameters: 1 cycle at 95°C for 3 minutes, followed by 45 cycles with a denaturation step at 95°C for 30 seconds, an annealing step at 55°C for 30 seconds, and an extension step at 68°C for 2 minutes.

    Article Title: cytb as a New Genetic Marker for Differentiation of Prototheca Species
    Article Snippet: .. Experimentally, 644-bp amplimers of the partial cytb gene, produced with the PCR mixture and cycling conditions identical to those described above, were doubly digested with FastDigest RsaI and TaiI (Thermo Fisher Scientific, Waltham, MA, USA). ..

    Article Title: Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus
    Article Snippet: .. Briefly, 25 μl of the PCR mixture consisted of 250 ng of DNA; a 0.2 μM concentration of each of the forward and reverse primers; a 200 μM concentration each of dATP, dCTP, dGTP, and dTTP; 2.5 mM MgCl2 ; and 2.5 U of DyNAzyme EXT (Finnzymes) in a 1× reaction buffer. ..

    Article Title: Modeling of 5? Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica
    Article Snippet: .. The PCR mixture for Ampli Taq Gold was composed of the following reagents and concentrations: 100 nM TaqMan probe, forward and reverse primers at concentrations of 900 nM each, 1× TaqMan Universal PCR Master Mix (including deoxynucleoside triphosphates [dNTPs; 200 μM dATP, 200 μM dCTP, 200 μM dGTP, and 400 μM dUTP]; Applied Biosystems), 0.01 U of uracil- N -glycosylase per μl, 2.5 mM MgCl2 , and 0.025 U of Ampli Taq Gold per μl. .. The PCR mixture for r Tth DNA polymerase had the following reagents and concentrations: 100 nM TaqMan probe, forward and reverse primers at concentrations of 900 nM each, 1× chelating buffer (N808-0098; Applied Biosytems), 0.05 U of r Tth DNA polymerase per μl, dNTPs (dATP, dCTP, dGTP, and dTTP, each at a concentration of 200 μM; Applied Biosystems), 2.5 mM MgCl2 , and 8% (vol/vol) glycerol (Merck).

    Article Title: Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay
    Article Snippet: .. One microliter of 10 ng/μl plasmid DNAs (equivalent to 1,618 viral DNA copies) was added to 100 μl of the PCR mixture, containing 0.2 μM primers M13F (5′-CCCAG TCACG ACGTT GTAAA ACG-3′) and M13R (5′-AGCGG ATAAC AATTT CACAC AGG-3′), 1× High Fidelity PCR buffer, 2.0 mM MgSO4 , and 2.0 U of Platinum Taq High Fidelity (Life Technologies, Bethesda, MD). ..

    Quantitative RT-PCR:

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: Paragraph title: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry ... The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water.

    Article Title: Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT
    Article Snippet: Paragraph title: Quantitative RT-PCR for MRP-1 and Pgp ... Complementary DNA was added to a PCR mix containing sequence-specific reverse and forward primers ( ) and 1 × TaqMan Universal PCR Master Mix (Invitrogen).

    Real-time Polymerase Chain Reaction:

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix. .. The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water.

    Article Title: Phase Variation in Myxococcus xanthus Yields Cells Specialized for Iron Sequestration
    Article Snippet: Paragraph title: Real-time PCR ... 5 µl of diluted cDNA sample was added to 20 µl of a PCR mixture prepared from Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), which contained each primer at a concentration of 160 nM.

    In Silico:

    Article Title: cytb as a New Genetic Marker for Differentiation of Prototheca Species
    Article Snippet: In silico restriction digestions were carried out, and predicted restriction patterns were determined for each species (genotype). .. Experimentally, 644-bp amplimers of the partial cytb gene, produced with the PCR mixture and cycling conditions identical to those described above, were doubly digested with FastDigest RsaI and TaiI (Thermo Fisher Scientific, Waltham, MA, USA).

    Expressing:

    Article Title: Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle
    Article Snippet: Paragraph title: Cloning and expression analysis of rAAV in vivo vector forms. ... This DNA was used as a template in a PCR mixture containing 20 mM Tris (pH 8.4), 50 mM potassium chloride, 1.5 mM magnesium chloride, 1 mM deoxynucleoside triphosphates, and 2.5 U of Platinum Taq DNA polymerase (Gibco BRL) in the following reaction conditions: 94°C for 2 min for 1 cycle and 94°C for 30 s, 55°C for 30 s, and 72°C for 5 min for 30 cycles.

    Article Title: Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT
    Article Snippet: Quantitative RT-PCR for MRP-1 and Pgp For the analysis of ABC transporter mRNA expression by quantitative RT-PCR (qRT-PCR), 10 ng or 1 ng per 1 μ g of cell line RNA was amplified for MRP-1 and Pgp, respectively; 5 ng of RNA from LCM primary tumour was amplified for MRP-1 and Pgp. .. Complementary DNA was added to a PCR mix containing sequence-specific reverse and forward primers ( ) and 1 × TaqMan Universal PCR Master Mix (Invitrogen).

    Modification:

    Article Title: Considerations When Analyzing the Methylation Status of PTEN Tumor Suppressor Gene
    Article Snippet: Genomic DNA (18 μl) obtained from tissues was treated with 5 mol/L sodium metabisulfite (Fisher Scientific Ltd., Ottawa, Canada) for 16 hours (protocol kindly provided by Peter Laird, University of Southern California), while cell line DNA (1 μg) was modified using the CpGenome DNA Modification Kit (Intergen Company, Purchase, NY). .. For set III, the PCR mixture contained 1X PCR buffer, dNTPs (each at 1.28 mmol/L; Invitrogen), primers (4 ng/μl; Sigma Genosys), HotStarTaq DNA polymerase (1.5U; Qiagen), and bisulfite-modified DNA (∼100 ng) or unmodified DNA (50 to 100 ng) in a final volume of 25 μl.

    Transformation Assay:

    Article Title: The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis
    Article Snippet: Paragraph title: Cloning of VvibZIPC22 and tobacco transformation ... The complete coding DNA sequence (CDS) of VvibZIPC22 was amplified in a 12.5 µl PCR mix containing primers (200 pM each), 1U of Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), dNTPs (200 µM), PCR buffer (1×), and cDNA (diluted 10-fold).

    Derivative Assay:

    Article Title: The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis
    Article Snippet: The VvibZIPC22 sequence considered in this study was derived from the 12Xv1 assembly of the Pinot Noir genome (VIT_07s0005g01450, from http://genomes.cribi.unipd.it/ DATA/V1, last accessed 11 December 2015) and confirmed by EST sequences (EE065899.1, EC946004.1, and EC945791.1). .. The complete coding DNA sequence (CDS) of VvibZIPC22 was amplified in a 12.5 µl PCR mix containing primers (200 pM each), 1U of Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), dNTPs (200 µM), PCR buffer (1×), and cDNA (diluted 10-fold).

    Hybridization:

    Article Title: Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle
    Article Snippet: This DNA was used as a template in a PCR mixture containing 20 mM Tris (pH 8.4), 50 mM potassium chloride, 1.5 mM magnesium chloride, 1 mM deoxynucleoside triphosphates, and 2.5 U of Platinum Taq DNA polymerase (Gibco BRL) in the following reaction conditions: 94°C for 2 min for 1 cycle and 94°C for 30 s, 55°C for 30 s, and 72°C for 5 min for 30 cycles. .. Positive colonies were identified using a radioactive HCMV probe by a standard colony hybridization method ( ). rAAV vector fragments were released by Pst I digestion and self-ligated using T4 DNA ligase (New England Biolabs).

    Concentration Assay:

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water. .. Serial 10-fold dilutions of the cDNA transcripts from IVT RNA were prepared in nuclease free water starting with highest concentration of 1.7 x 108 copies/μl.

    Article Title: Phase Variation in Myxococcus xanthus Yields Cells Specialized for Iron Sequestration
    Article Snippet: .. 5 µl of diluted cDNA sample was added to 20 µl of a PCR mixture prepared from Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), which contained each primer at a concentration of 160 nM. .. Primers, listed in , were chosen using Primer Express 3.0 software (Applied Biosystems) and were designed to amplify a region of about 150–200 bp within each transcript.

    Article Title: Non-Sulfate-Reducing, Syntrophic Bacteria Affiliated with Desulfotomaculum Cluster I Are Widely Distributed in Methanogenic Environments
    Article Snippet: .. Approximately 40 ng of template DNA was used in a 100-μl PCR mixture; the concentration of template DNA was measured by using the PicoGreen double-stranded DNA quantification kit (Molecular Probes) and a spectrofluorophotometer (RF-5300PC; Shimadzu). .. An approximately 1,900-bp fragment of dsrAB was amplified by using the primers DSR1Fdeg and DSR4Rdeg ( ).

    Article Title: Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus
    Article Snippet: .. Briefly, 25 μl of the PCR mixture consisted of 250 ng of DNA; a 0.2 μM concentration of each of the forward and reverse primers; a 200 μM concentration each of dATP, dCTP, dGTP, and dTTP; 2.5 mM MgCl2 ; and 2.5 U of DyNAzyme EXT (Finnzymes) in a 1× reaction buffer. ..

    Article Title: Modeling of 5? Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica
    Article Snippet: The PCR mixture for Ampli Taq Gold was composed of the following reagents and concentrations: 100 nM TaqMan probe, forward and reverse primers at concentrations of 900 nM each, 1× TaqMan Universal PCR Master Mix (including deoxynucleoside triphosphates [dNTPs; 200 μM dATP, 200 μM dCTP, 200 μM dGTP, and 400 μM dUTP]; Applied Biosystems), 0.01 U of uracil- N -glycosylase per μl, 2.5 mM MgCl2 , and 0.025 U of Ampli Taq Gold per μl. .. The PCR mixture for r Tth DNA polymerase had the following reagents and concentrations: 100 nM TaqMan probe, forward and reverse primers at concentrations of 900 nM each, 1× chelating buffer (N808-0098; Applied Biosytems), 0.05 U of r Tth DNA polymerase per μl, dNTPs (dATP, dCTP, dGTP, and dTTP, each at a concentration of 200 μM; Applied Biosystems), 2.5 mM MgCl2 , and 8% (vol/vol) glycerol (Merck).

    Generated:

    Article Title: cytb as a New Genetic Marker for Differentiation of Prototheca Species
    Article Snippet: To develop a PCR-RFLP assay for species- or genotype-specific identification of Prototheca algae, single nucleotide polymorphisms (SNPs) were detected upon alignment of the cytb gene partial sequences generated from each Prototheca species (genotype), as described above. .. Experimentally, 644-bp amplimers of the partial cytb gene, produced with the PCR mixture and cycling conditions identical to those described above, were doubly digested with FastDigest RsaI and TaiI (Thermo Fisher Scientific, Waltham, MA, USA).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: The melting curve analysis was performed from 60°C to 95°C in 0.1°C/s increments to assess the specificity of the RT-PCR products. .. The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water.

    TaqMan Assay:

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: .. The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water. .. The PCR amplification consisted of 1 cycle of enzyme activation for 2 min at 50°C followed by denaturation at 95°C for 20 s, and 40 cycles of denaturation at 95°C for 3 s, 30 s annealing at 60°C.

    In Vivo:

    Article Title: Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle
    Article Snippet: Paragraph title: Cloning and expression analysis of rAAV in vivo vector forms. ... This DNA was used as a template in a PCR mixture containing 20 mM Tris (pH 8.4), 50 mM potassium chloride, 1.5 mM magnesium chloride, 1 mM deoxynucleoside triphosphates, and 2.5 U of Platinum Taq DNA polymerase (Gibco BRL) in the following reaction conditions: 94°C for 2 min for 1 cycle and 94°C for 30 s, 55°C for 30 s, and 72°C for 5 min for 30 cycles.

    Methylation:

    Article Title: Considerations When Analyzing the Methylation Status of PTEN Tumor Suppressor Gene
    Article Snippet: Paragraph title: Methylation-Specific Polymerase Chain Reaction (MSP) ... For set III, the PCR mixture contained 1X PCR buffer, dNTPs (each at 1.28 mmol/L; Invitrogen), primers (4 ng/μl; Sigma Genosys), HotStarTaq DNA polymerase (1.5U; Qiagen), and bisulfite-modified DNA (∼100 ng) or unmodified DNA (50 to 100 ng) in a final volume of 25 μl.

    Isolation:

    Article Title: Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle
    Article Snippet: One microgram of total muscle DNA, isolated from an rAAV/ova-injected mouse, was digested with Eco RI (an enzyme which does not cut within the rAAV vector) and treated with Plasmid-Safe DNase as described above. .. This DNA was used as a template in a PCR mixture containing 20 mM Tris (pH 8.4), 50 mM potassium chloride, 1.5 mM magnesium chloride, 1 mM deoxynucleoside triphosphates, and 2.5 U of Platinum Taq DNA polymerase (Gibco BRL) in the following reaction conditions: 94°C for 2 min for 1 cycle and 94°C for 30 s, 55°C for 30 s, and 72°C for 5 min for 30 cycles.

    Multiplex Assay:

    Article Title: Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay
    Article Snippet: Paragraph title: Development of the multiplex allele-specific HIVDR assay for HIV-1 subtype C. ... One microliter of 10 ng/μl plasmid DNAs (equivalent to 1,618 viral DNA copies) was added to 100 μl of the PCR mixture, containing 0.2 μM primers M13F (5′-CCCAG TCACG ACGTT GTAAA ACG-3′) and M13R (5′-AGCGG ATAAC AATTT CACAC AGG-3′), 1× High Fidelity PCR buffer, 2.0 mM MgSO4 , and 2.0 U of Platinum Taq High Fidelity (Life Technologies, Bethesda, MD).

    Labeling:

    Article Title: Modeling of 5? Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica
    Article Snippet: The TaqMan probe was labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye). .. The PCR mixture for Ampli Taq Gold was composed of the following reagents and concentrations: 100 nM TaqMan probe, forward and reverse primers at concentrations of 900 nM each, 1× TaqMan Universal PCR Master Mix (including deoxynucleoside triphosphates [dNTPs; 200 μM dATP, 200 μM dCTP, 200 μM dGTP, and 400 μM dUTP]; Applied Biosystems), 0.01 U of uracil- N -glycosylase per μl, 2.5 mM MgCl2 , and 0.025 U of Ampli Taq Gold per μl.

    Purification:

    Article Title: Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay
    Article Snippet: One microliter of 10 ng/μl plasmid DNAs (equivalent to 1,618 viral DNA copies) was added to 100 μl of the PCR mixture, containing 0.2 μM primers M13F (5′-CCCAG TCACG ACGTT GTAAA ACG-3′) and M13R (5′-AGCGG ATAAC AATTT CACAC AGG-3′), 1× High Fidelity PCR buffer, 2.0 mM MgSO4 , and 2.0 U of Platinum Taq High Fidelity (Life Technologies, Bethesda, MD). .. The PCR products were purified using the ExoSAP-IT PCR cleanup kit (USB Co., Cleveland, OH).

    Sequencing:

    Article Title: The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis
    Article Snippet: .. The complete coding DNA sequence (CDS) of VvibZIPC22 was amplified in a 12.5 µl PCR mix containing primers (200 pM each), 1U of Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), dNTPs (200 µM), PCR buffer (1×), and cDNA (diluted 10-fold). ..

    Article Title: Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT
    Article Snippet: .. Complementary DNA was added to a PCR mix containing sequence-specific reverse and forward primers ( ) and 1 × TaqMan Universal PCR Master Mix (Invitrogen). .. Samples were denatured for 2 min at 50 °C, Taq polymerase was activated by heating for 10 min at 95 °C and cDNA amplified using 40 cycles of 95 °C for 15 s and 60 °C for 1 min. mRNA expression was calculated using the comparative Ct method , relative to the housekeeping genes β-2-microglobulin ( ) and peptidylprolyl isomerase A (PPIA) ( ; ), in cell lines ( ) and primary ESFTs, respectively.

    Plasmid Preparation:

    Article Title: The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis
    Article Snippet: The complete coding DNA sequence (CDS) of VvibZIPC22 was amplified in a 12.5 µl PCR mix containing primers (200 pM each), 1U of Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), dNTPs (200 µM), PCR buffer (1×), and cDNA (diluted 10-fold). .. A PCR product of 438bp was obtained and subcloned into a pGEM-T Easy Vector (Promega, Mannheim, Germany) for sequencing.

    Article Title: Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle
    Article Snippet: Paragraph title: Cloning and expression analysis of rAAV in vivo vector forms. ... This DNA was used as a template in a PCR mixture containing 20 mM Tris (pH 8.4), 50 mM potassium chloride, 1.5 mM magnesium chloride, 1 mM deoxynucleoside triphosphates, and 2.5 U of Platinum Taq DNA polymerase (Gibco BRL) in the following reaction conditions: 94°C for 2 min for 1 cycle and 94°C for 30 s, 55°C for 30 s, and 72°C for 5 min for 30 cycles.

    Article Title: Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay
    Article Snippet: .. One microliter of 10 ng/μl plasmid DNAs (equivalent to 1,618 viral DNA copies) was added to 100 μl of the PCR mixture, containing 0.2 μM primers M13F (5′-CCCAG TCACG ACGTT GTAAA ACG-3′) and M13R (5′-AGCGG ATAAC AATTT CACAC AGG-3′), 1× High Fidelity PCR buffer, 2.0 mM MgSO4 , and 2.0 U of Platinum Taq High Fidelity (Life Technologies, Bethesda, MD). ..

    Software:

    Article Title: Phase Variation in Myxococcus xanthus Yields Cells Specialized for Iron Sequestration
    Article Snippet: 5 µl of diluted cDNA sample was added to 20 µl of a PCR mixture prepared from Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), which contained each primer at a concentration of 160 nM. .. Primers, listed in , were chosen using Primer Express 3.0 software (Applied Biosystems) and were designed to amplify a region of about 150–200 bp within each transcript.

    Article Title: cytb as a New Genetic Marker for Differentiation of Prototheca Species
    Article Snippet: To check if the SNPs located within the restriction enzyme recognition sites that would produce an RFLP, the sequences were screened with Clone Manager software, version 9.0 (Sci-Ed Software, Denver, CO, USA). .. Experimentally, 644-bp amplimers of the partial cytb gene, produced with the PCR mixture and cycling conditions identical to those described above, were doubly digested with FastDigest RsaI and TaiI (Thermo Fisher Scientific, Waltham, MA, USA).

    SYBR Green Assay:

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: Paragraph title: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry ... The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water.

    Article Title: Phase Variation in Myxococcus xanthus Yields Cells Specialized for Iron Sequestration
    Article Snippet: .. 5 µl of diluted cDNA sample was added to 20 µl of a PCR mixture prepared from Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), which contained each primer at a concentration of 160 nM. .. Primers, listed in , were chosen using Primer Express 3.0 software (Applied Biosystems) and were designed to amplify a region of about 150–200 bp within each transcript.

    Negative Control:

    Article Title: Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay
    Article Snippet: One microliter of 10 ng/μl plasmid DNAs (equivalent to 1,618 viral DNA copies) was added to 100 μl of the PCR mixture, containing 0.2 μM primers M13F (5′-CCCAG TCACG ACGTT GTAAA ACG-3′) and M13R (5′-AGCGG ATAAC AATTT CACAC AGG-3′), 1× High Fidelity PCR buffer, 2.0 mM MgSO4 , and 2.0 U of Platinum Taq High Fidelity (Life Technologies, Bethesda, MD). .. The purified PCR products of plasmids C-wt and C-mut1, a mixture of the two at a 1:1 ratio (wt/wt), and a no-target PCR negative control (NC) were used to validate each of the WT and Mut ASPE primers.

    Agarose Gel Electrophoresis:

    Article Title: Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism
    Article Snippet: The PCR reactions were performed with approximately 50 ng of genomic DNA in a 25-μl PCR mixture, which included 1 μL (200 ng) of DNA template, 2.5 μL of buffer solution (containing the dNTPs and MgCl2 ), 1 μL of each 10 μM primer (F18S and F28S), 1 μL of Taq DNA polymerase (Invitrogen), and 18.5 μL of distilled water. .. The detection of amplified products (550 base pairs [bp]) was performed by electrophoresis of an aliquot of 5 μL of each amplicon in a 1% agarose gel with ethidium bromide 0.02% in 1 × Tris-acetate-EDTA (TAE) buffer.

    Article Title: Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus
    Article Snippet: Briefly, 25 μl of the PCR mixture consisted of 250 ng of DNA; a 0.2 μM concentration of each of the forward and reverse primers; a 200 μM concentration each of dATP, dCTP, dGTP, and dTTP; 2.5 mM MgCl2 ; and 2.5 U of DyNAzyme EXT (Finnzymes) in a 1× reaction buffer. .. One-tenth of the amplified reaction mixture was mixed with a gel-loading buffer and electrophoresed on a 0.8% (wt/vol) agarose gel; the reaction products were visualized by ethidium bromide staining.

    In Vitro:

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix. .. The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water.

    Electrophoresis:

    Article Title: Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism
    Article Snippet: The PCR reactions were performed with approximately 50 ng of genomic DNA in a 25-μl PCR mixture, which included 1 μL (200 ng) of DNA template, 2.5 μL of buffer solution (containing the dNTPs and MgCl2 ), 1 μL of each 10 μM primer (F18S and F28S), 1 μL of Taq DNA polymerase (Invitrogen), and 18.5 μL of distilled water. .. The detection of amplified products (550 base pairs [bp]) was performed by electrophoresis of an aliquot of 5 μL of each amplicon in a 1% agarose gel with ethidium bromide 0.02% in 1 × Tris-acetate-EDTA (TAE) buffer.

    Laser Capture Microdissection:

    Article Title: Membrane expression of MRP-1, but not MRP-1 splicing or Pgp expression, predicts survival in patients with ESFT
    Article Snippet: Quantitative RT-PCR for MRP-1 and Pgp For the analysis of ABC transporter mRNA expression by quantitative RT-PCR (qRT-PCR), 10 ng or 1 ng per 1 μ g of cell line RNA was amplified for MRP-1 and Pgp, respectively; 5 ng of RNA from LCM primary tumour was amplified for MRP-1 and Pgp. .. Complementary DNA was added to a PCR mix containing sequence-specific reverse and forward primers ( ) and 1 × TaqMan Universal PCR Master Mix (Invitrogen).

    Produced:

    Article Title: cytb as a New Genetic Marker for Differentiation of Prototheca Species
    Article Snippet: .. Experimentally, 644-bp amplimers of the partial cytb gene, produced with the PCR mixture and cycling conditions identical to those described above, were doubly digested with FastDigest RsaI and TaiI (Thermo Fisher Scientific, Waltham, MA, USA). ..

    Activation Assay:

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
    Article Snippet: The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water. .. The PCR amplification consisted of 1 cycle of enzyme activation for 2 min at 50°C followed by denaturation at 95°C for 20 s, and 40 cycles of denaturation at 95°C for 3 s, 30 s annealing at 60°C.

    Staining:

    Article Title: Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism
    Article Snippet: Paragraph title: Gomori Methenamine Silver (GMS) Staining and PCR Detection for Fusarium Species ... The PCR reactions were performed with approximately 50 ng of genomic DNA in a 25-μl PCR mixture, which included 1 μL (200 ng) of DNA template, 2.5 μL of buffer solution (containing the dNTPs and MgCl2 ), 1 μL of each 10 μM primer (F18S and F28S), 1 μL of Taq DNA polymerase (Invitrogen), and 18.5 μL of distilled water.

    Article Title: Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus
    Article Snippet: Briefly, 25 μl of the PCR mixture consisted of 250 ng of DNA; a 0.2 μM concentration of each of the forward and reverse primers; a 200 μM concentration each of dATP, dCTP, dGTP, and dTTP; 2.5 mM MgCl2 ; and 2.5 U of DyNAzyme EXT (Finnzymes) in a 1× reaction buffer. .. One-tenth of the amplified reaction mixture was mixed with a gel-loading buffer and electrophoresed on a 0.8% (wt/vol) agarose gel; the reaction products were visualized by ethidium bromide staining.

    Hood:

    Article Title: Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism
    Article Snippet: The PCR reactions were performed with approximately 50 ng of genomic DNA in a 25-μl PCR mixture, which included 1 μL (200 ng) of DNA template, 2.5 μL of buffer solution (containing the dNTPs and MgCl2 ), 1 μL of each 10 μM primer (F18S and F28S), 1 μL of Taq DNA polymerase (Invitrogen), and 18.5 μL of distilled water. .. The DNA bands were visualized under UV illumination (Universal Hood II; Bio-Rad).

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  • 90
    Thermo Fisher sybr green i
    Correlation of detection of virus by Bio-Plex and RT-qPCR. Correlation between qPCR (Ct value) and Bio-Plex (MFI) detection of in-vitro grown ISAV standard curve by (A) <t>SYBR</t> <t>GREEN</t> I and (B)TaqMan; Correlation of detection of copy number of ISAV in plasma samples by (C) SYBR Green I (n = 13) and (D) TaqMan chemistries (n = 8).
    Sybr Green I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 803 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sybr green i/product/Thermo Fisher
    Average 90 stars, based on 803 article reviews
    Price from $9.99 to $1999.99
    sybr green i - by Bioz Stars, 2020-04
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    95
    Thermo Fisher qrt pcr reaction mixture
    Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by <t>qRT-PCR</t> with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P
    Qrt Pcr Reaction Mixture, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qrt pcr reaction mixture/product/Thermo Fisher
    Average 95 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    qrt pcr reaction mixture - by Bioz Stars, 2020-04
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    94
    Thermo Fisher dreamtaq green pcr master mix 2x
    BFR1 deletion does not affect PMT1 and PMT2 transcript localization. ( A ) and ( B ): JCY017 (wild type BFR1-3xHA) cells were grown in YPD medium, lysed and total cell extracts were subjected to one step ultracentrifugation. ( A ) Western blot analysis of total cell lysates (TCL), soluble and membrane fractions (SF and MF respectively) upon one step ultracentrifugation. Equivalents to 0.25 OD 600 were resolved on a 12% PAA gel and detection was performed with the indicated antibodies. Sec61 served as a membrane marker and G6PDH as a cytosolic marker. Bfr1-3xHA was detected using the HA-tag. ( B ) <t>RT-PCR</t> analysis of PMT2 mRNA from soluble and membrane fractions upon one step ultracentrifugation. Total RNA was extracted from respective fractions and cDNA was prepared. PMT2 mRNA from each fraction was normalized to ACT1 mRNA. Results show the average membrane to soluble PMT2 mRNA ratio from three independent experiments and error bars represent the confidence interval. For statistical significance one-sample t -test was performed on log2 −ΔΔCt . ( C ), ( D ), and ( E ): JCY017 (wild type BFR1-3xHA) and bfr1 Δ cells were grown in YPD medium, lysed and total cell extracts were subjected to sucrose step gradient centrifugation. ( C ) Absorbance 260 profile of fractions collected upon sucrose step gradient centrifugation (upper panel) and agarose gel electrophoresis of equivalent amounts of each fraction (lower panel). F5, F10, and F16 indicate fractions selected for further analysis. Black and grey arrows next to the agarose gel depict ribosomal subunits 60S and 40S. ( D ) Western blot analysis of total cell lysates (TCL) and selected sucrose gradient fractions from JCY017 (wild type BFR1-3xHA) cells. 0.25 OD 600 units of total cell extract and equivalents of selected fractions were resolved on a 12% PAA gel and detection was performed with the indicated antibodies. Sec61 and G6PDH were detected exclusively in fractions F16 and F5 respectively confirming successful fractionation. The ribosomal protein Rpl5 was mainly detected in fractions F10 and F16 that represent cytoplasmic and membrane bound polysomes respectively. The weaker Rpl5 signal detected in fraction F5 probably emanates from free cytosolic ribosomes. ( E ) Semi-quantitative PCR analysis of PMT1 and PMT2 mRNA from sucrose gradient fractions F10 and F16 from JCY017 (wild type BFR1-3xHA) and bfr1 Δ cells. Total RNA was extracted from respective fractions, cDNA was prepared and a 1:20 dilution was used as template in a standard <t>DreamTaq</t> PCR reaction. ACT1 that served as a loading control also shows strong engagement in the ER membrane containing fraction F16 in line with reports that the ER is a general translation hub even for cytosolic proteins [ 53 ]. Results are representative of two independent fractionations. N.s. = not significant.
    Dreamtaq Green Pcr Master Mix 2x, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher f13a1 gene pcr mix
    Gradient <t>PCR</t> for <t>F13A1</t> (F13) gene. Gradient PCR for F13 gene was carried out using specific primers as outlined in the M M section. Lane 1: 50 °C, lane 2: 51 °C, lane 3:52.9 °C, lane 4: 55.7 °C, lane 5: 59.1 °C, lane 6: 62 °C, lane 7 63.8 °C, lane 8: 65 °C. The F13 PCR showed maximum signal between 50–52.9 °C.M: DNA molecular weight maker (75 bp–10 Kb).
    F13a1 Gene Pcr Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Correlation of detection of virus by Bio-Plex and RT-qPCR. Correlation between qPCR (Ct value) and Bio-Plex (MFI) detection of in-vitro grown ISAV standard curve by (A) SYBR GREEN I and (B)TaqMan; Correlation of detection of copy number of ISAV in plasma samples by (C) SYBR Green I (n = 13) and (D) TaqMan chemistries (n = 8).

    Journal: PLoS ONE

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology

    doi: 10.1371/journal.pone.0159155

    Figure Lengend Snippet: Correlation of detection of virus by Bio-Plex and RT-qPCR. Correlation between qPCR (Ct value) and Bio-Plex (MFI) detection of in-vitro grown ISAV standard curve by (A) SYBR GREEN I and (B)TaqMan; Correlation of detection of copy number of ISAV in plasma samples by (C) SYBR Green I (n = 13) and (D) TaqMan chemistries (n = 8).

    Article Snippet: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix.

    Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, In Vitro, SYBR Green Assay

    RT-qPCR standard curves. Standard curve relating viral copy number to Ct value from RT-Polymerase chain reaction using 10-fold dilutions of in-vitro transcribed RNA using (a) SYBR ® Green I and (b) TaqMan chemistries.

    Journal: PLoS ONE

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology

    doi: 10.1371/journal.pone.0159155

    Figure Lengend Snippet: RT-qPCR standard curves. Standard curve relating viral copy number to Ct value from RT-Polymerase chain reaction using 10-fold dilutions of in-vitro transcribed RNA using (a) SYBR ® Green I and (b) TaqMan chemistries.

    Article Snippet: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix.

    Techniques: Quantitative RT-PCR, Polymerase Chain Reaction, In Vitro, SYBR Green Assay

    Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by qRT-PCR with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Journal: Frontiers in Plant Science

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    doi: 10.3389/fpls.2018.00942

    Figure Lengend Snippet: Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by qRT-PCR with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

    Techniques: Over Expression, Expressing, Plasmid Preparation, Construct, Quantitative RT-PCR, High Performance Liquid Chromatography

    Analysis of gene expression in transgenic G(G)PPS and GES C. roseus plants. qRT-PCR analysis of G(G)PPS (gray bar) (A) and G(G)PPS (gray bar)/ GES (black bar) (B) in transgenic C. roseus plants. Expression levels of genes were normalized to the endogenous reference gene CrN227 and are represented relative to the wild type (WT) controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Journal: Frontiers in Plant Science

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    doi: 10.3389/fpls.2018.00942

    Figure Lengend Snippet: Analysis of gene expression in transgenic G(G)PPS and GES C. roseus plants. qRT-PCR analysis of G(G)PPS (gray bar) (A) and G(G)PPS (gray bar)/ GES (black bar) (B) in transgenic C. roseus plants. Expression levels of genes were normalized to the endogenous reference gene CrN227 and are represented relative to the wild type (WT) controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

    Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR

    PRX1 expression in transgenic C. roseus plants. Analysis of PRX1 expression by qRT-PCR in G(G)PPS (A) and G(G)PPS + GES (B) overexpressing transgenic C. roseus plants. Expression levels of PRX1 were normalized to the endogenous reference gene CrN227 and are represented relative to the WT controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Journal: Frontiers in Plant Science

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    doi: 10.3389/fpls.2018.00942

    Figure Lengend Snippet: PRX1 expression in transgenic C. roseus plants. Analysis of PRX1 expression by qRT-PCR in G(G)PPS (A) and G(G)PPS + GES (B) overexpressing transgenic C. roseus plants. Expression levels of PRX1 were normalized to the endogenous reference gene CrN227 and are represented relative to the WT controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

    Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR

    BFR1 deletion does not affect PMT1 and PMT2 transcript localization. ( A ) and ( B ): JCY017 (wild type BFR1-3xHA) cells were grown in YPD medium, lysed and total cell extracts were subjected to one step ultracentrifugation. ( A ) Western blot analysis of total cell lysates (TCL), soluble and membrane fractions (SF and MF respectively) upon one step ultracentrifugation. Equivalents to 0.25 OD 600 were resolved on a 12% PAA gel and detection was performed with the indicated antibodies. Sec61 served as a membrane marker and G6PDH as a cytosolic marker. Bfr1-3xHA was detected using the HA-tag. ( B ) RT-PCR analysis of PMT2 mRNA from soluble and membrane fractions upon one step ultracentrifugation. Total RNA was extracted from respective fractions and cDNA was prepared. PMT2 mRNA from each fraction was normalized to ACT1 mRNA. Results show the average membrane to soluble PMT2 mRNA ratio from three independent experiments and error bars represent the confidence interval. For statistical significance one-sample t -test was performed on log2 −ΔΔCt . ( C ), ( D ), and ( E ): JCY017 (wild type BFR1-3xHA) and bfr1 Δ cells were grown in YPD medium, lysed and total cell extracts were subjected to sucrose step gradient centrifugation. ( C ) Absorbance 260 profile of fractions collected upon sucrose step gradient centrifugation (upper panel) and agarose gel electrophoresis of equivalent amounts of each fraction (lower panel). F5, F10, and F16 indicate fractions selected for further analysis. Black and grey arrows next to the agarose gel depict ribosomal subunits 60S and 40S. ( D ) Western blot analysis of total cell lysates (TCL) and selected sucrose gradient fractions from JCY017 (wild type BFR1-3xHA) cells. 0.25 OD 600 units of total cell extract and equivalents of selected fractions were resolved on a 12% PAA gel and detection was performed with the indicated antibodies. Sec61 and G6PDH were detected exclusively in fractions F16 and F5 respectively confirming successful fractionation. The ribosomal protein Rpl5 was mainly detected in fractions F10 and F16 that represent cytoplasmic and membrane bound polysomes respectively. The weaker Rpl5 signal detected in fraction F5 probably emanates from free cytosolic ribosomes. ( E ) Semi-quantitative PCR analysis of PMT1 and PMT2 mRNA from sucrose gradient fractions F10 and F16 from JCY017 (wild type BFR1-3xHA) and bfr1 Δ cells. Total RNA was extracted from respective fractions, cDNA was prepared and a 1:20 dilution was used as template in a standard DreamTaq PCR reaction. ACT1 that served as a loading control also shows strong engagement in the ER membrane containing fraction F16 in line with reports that the ER is a general translation hub even for cytosolic proteins [ 53 ]. Results are representative of two independent fractionations. N.s. = not significant.

    Journal: International Journal of Molecular Sciences

    Article Title: Translational Regulation of Pmt1 and Pmt2 by Bfr1 Affects Unfolded Protein O-Mannosylation

    doi: 10.3390/ijms20246220

    Figure Lengend Snippet: BFR1 deletion does not affect PMT1 and PMT2 transcript localization. ( A ) and ( B ): JCY017 (wild type BFR1-3xHA) cells were grown in YPD medium, lysed and total cell extracts were subjected to one step ultracentrifugation. ( A ) Western blot analysis of total cell lysates (TCL), soluble and membrane fractions (SF and MF respectively) upon one step ultracentrifugation. Equivalents to 0.25 OD 600 were resolved on a 12% PAA gel and detection was performed with the indicated antibodies. Sec61 served as a membrane marker and G6PDH as a cytosolic marker. Bfr1-3xHA was detected using the HA-tag. ( B ) RT-PCR analysis of PMT2 mRNA from soluble and membrane fractions upon one step ultracentrifugation. Total RNA was extracted from respective fractions and cDNA was prepared. PMT2 mRNA from each fraction was normalized to ACT1 mRNA. Results show the average membrane to soluble PMT2 mRNA ratio from three independent experiments and error bars represent the confidence interval. For statistical significance one-sample t -test was performed on log2 −ΔΔCt . ( C ), ( D ), and ( E ): JCY017 (wild type BFR1-3xHA) and bfr1 Δ cells were grown in YPD medium, lysed and total cell extracts were subjected to sucrose step gradient centrifugation. ( C ) Absorbance 260 profile of fractions collected upon sucrose step gradient centrifugation (upper panel) and agarose gel electrophoresis of equivalent amounts of each fraction (lower panel). F5, F10, and F16 indicate fractions selected for further analysis. Black and grey arrows next to the agarose gel depict ribosomal subunits 60S and 40S. ( D ) Western blot analysis of total cell lysates (TCL) and selected sucrose gradient fractions from JCY017 (wild type BFR1-3xHA) cells. 0.25 OD 600 units of total cell extract and equivalents of selected fractions were resolved on a 12% PAA gel and detection was performed with the indicated antibodies. Sec61 and G6PDH were detected exclusively in fractions F16 and F5 respectively confirming successful fractionation. The ribosomal protein Rpl5 was mainly detected in fractions F10 and F16 that represent cytoplasmic and membrane bound polysomes respectively. The weaker Rpl5 signal detected in fraction F5 probably emanates from free cytosolic ribosomes. ( E ) Semi-quantitative PCR analysis of PMT1 and PMT2 mRNA from sucrose gradient fractions F10 and F16 from JCY017 (wild type BFR1-3xHA) and bfr1 Δ cells. Total RNA was extracted from respective fractions, cDNA was prepared and a 1:20 dilution was used as template in a standard DreamTaq PCR reaction. ACT1 that served as a loading control also shows strong engagement in the ER membrane containing fraction F16 in line with reports that the ER is a general translation hub even for cytosolic proteins [ 53 ]. Results are representative of two independent fractionations. N.s. = not significant.

    Article Snippet: Semi-quantitative PCR was performed using the DreamTaq Green PCR master mix (#K1081, Thermo Fisher Scientific; Waltham, MA, USA) according to manufacturer´s instructions.

    Techniques: Western Blot, Marker, Reverse Transcription Polymerase Chain Reaction, Gradient Centrifugation, Agarose Gel Electrophoresis, Fractionation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Gradient PCR for F13A1 (F13) gene. Gradient PCR for F13 gene was carried out using specific primers as outlined in the M M section. Lane 1: 50 °C, lane 2: 51 °C, lane 3:52.9 °C, lane 4: 55.7 °C, lane 5: 59.1 °C, lane 6: 62 °C, lane 7 63.8 °C, lane 8: 65 °C. The F13 PCR showed maximum signal between 50–52.9 °C.M: DNA molecular weight maker (75 bp–10 Kb).

    Journal: MethodsX

    Article Title: Use of coagulation factor XIII (F13) gene as an internal control for normalization of genomic DNA’s for HLA typing

    doi: 10.1016/j.mex.2018.07.020

    Figure Lengend Snippet: Gradient PCR for F13A1 (F13) gene. Gradient PCR for F13 gene was carried out using specific primers as outlined in the M M section. Lane 1: 50 °C, lane 2: 51 °C, lane 3:52.9 °C, lane 4: 55.7 °C, lane 5: 59.1 °C, lane 6: 62 °C, lane 7 63.8 °C, lane 8: 65 °C. The F13 PCR showed maximum signal between 50–52.9 °C.M: DNA molecular weight maker (75 bp–10 Kb).

    Article Snippet: The F13A1 gene PCR mix comprised of 40 ng of human gDNA with 1 U of Taq DNA polymerase, 2.5 μl of 10 X ammonium sulfate buffer, pH 8.3 (Thermo Fisher Scientific, USA), 0.2 mM dNTP’s, 2% Tween-20, 0.32% BSA, 5% DMSO and 2.5 mM MgCl2 in a final volume of 25 μl master mix.

    Techniques: Polymerase Chain Reaction, Molecular Weight