standard tris acetate edta tae buffer  (Thermo Fisher)


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    Name:
    TAE Buffer Tris acetate EDTA 50X
    Description:
    Thermo Scientific 50X TAE Buffer Tris acetate EDTA is used for electrophoresis of nucleic acids in agarose and polyacrylamide gels You can use this buffer for both genomic and large supercoiled DNA and you can also use this as both a running and a gel preparation buffer Applications• Electrophoresis of nucleic acids in agarose and polyacrylamide gels• Used both as a running buffer and as a gel preparation buffer• Filtered through a 0 22 µm membrane• Recommended for resolution of RNA and DNA fragments larger than 1500 bp for genomic DNA and for large supercoiled DNANoteTAE buffer has a relatively low buffering capacity Therefore periodic replacement of the buffer during prolonged electrophoresis is recommended
    Catalog Number:
    B49
    Price:
    None
    Category:
    Lab Reagents and Chemicals
    Applications:
    Agarose Gel Electrophoresis|DNA & RNA Purification & Analysis|Nucleic Acid Gel Electrophoresis & Blotting
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    Structured Review

    Thermo Fisher standard tris acetate edta tae buffer
    Real-time PCR reaction products checked on agarose gel stained with ethidium bromide in standard <t>tris-acetate-EDTA</t> (TAE) buffer: serial 10-fold dilutions of recombinant plasmid. Specific bands of approximately 168 bp were visualised for all replicates of recombinant plasmid dilutions, except the concentration of 1 × 10 −1 copies/ μ L. For recombinant plasmid dilution with a concentration of 1 × 10 −1 copies/ μ L only one specific amplicon to three replicates was visualised. MK: MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada). a, b, c, d, and e: recombinant plasmid dilutions with concentrations of 1 × 10 3 , 1 × 10 2 , 1 × 10 1 , 1 × 10 0 , and 1 × 10 −1 copies/ μ L, respectively. W: no template control (water).
    Thermo Scientific 50X TAE Buffer Tris acetate EDTA is used for electrophoresis of nucleic acids in agarose and polyacrylamide gels You can use this buffer for both genomic and large supercoiled DNA and you can also use this as both a running and a gel preparation buffer Applications• Electrophoresis of nucleic acids in agarose and polyacrylamide gels• Used both as a running buffer and as a gel preparation buffer• Filtered through a 0 22 µm membrane• Recommended for resolution of RNA and DNA fragments larger than 1500 bp for genomic DNA and for large supercoiled DNANoteTAE buffer has a relatively low buffering capacity Therefore periodic replacement of the buffer during prolonged electrophoresis is recommended
    https://www.bioz.com/result/standard tris acetate edta tae buffer/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    standard tris acetate edta tae buffer - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys"

    Article Title: A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

    Journal: The Scientific World Journal

    doi: 10.1100/2012/989514

    Real-time PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: serial 10-fold dilutions of recombinant plasmid. Specific bands of approximately 168 bp were visualised for all replicates of recombinant plasmid dilutions, except the concentration of 1 × 10 −1 copies/ μ L. For recombinant plasmid dilution with a concentration of 1 × 10 −1 copies/ μ L only one specific amplicon to three replicates was visualised. MK: MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada). a, b, c, d, and e: recombinant plasmid dilutions with concentrations of 1 × 10 3 , 1 × 10 2 , 1 × 10 1 , 1 × 10 0 , and 1 × 10 −1 copies/ μ L, respectively. W: no template control (water).
    Figure Legend Snippet: Real-time PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: serial 10-fold dilutions of recombinant plasmid. Specific bands of approximately 168 bp were visualised for all replicates of recombinant plasmid dilutions, except the concentration of 1 × 10 −1 copies/ μ L. For recombinant plasmid dilution with a concentration of 1 × 10 −1 copies/ μ L only one specific amplicon to three replicates was visualised. MK: MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada). a, b, c, d, and e: recombinant plasmid dilutions with concentrations of 1 × 10 3 , 1 × 10 2 , 1 × 10 1 , 1 × 10 0 , and 1 × 10 −1 copies/ μ L, respectively. W: no template control (water).

    Techniques Used: Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Recombinant, Plasmid Preparation, Concentration Assay, Amplification

    Real time-PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: positive samples detected by developed real-time PCR. Specific bands of approximately 168 bp were visualised for all replicates of the detected positive samples. In this figure is represented the 2% (w/v) agarose gel electrophoresis of five of the 11 positive samples detected in bats belonging to sampling area A (San Cesario sul Panaro, MO). Pl: amplicon of the recombinant plasmid with 1 × 10 4 copies/ μ L; 1, 2, 3, 4, and 5: amplicons of the five positive samples belonging to sampling area A, each with three repetitions.
    Figure Legend Snippet: Real time-PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: positive samples detected by developed real-time PCR. Specific bands of approximately 168 bp were visualised for all replicates of the detected positive samples. In this figure is represented the 2% (w/v) agarose gel electrophoresis of five of the 11 positive samples detected in bats belonging to sampling area A (San Cesario sul Panaro, MO). Pl: amplicon of the recombinant plasmid with 1 × 10 4 copies/ μ L; 1, 2, 3, 4, and 5: amplicons of the five positive samples belonging to sampling area A, each with three repetitions.

    Techniques Used: Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Sampling, Amplification, Recombinant, Plasmid Preparation

    2) Product Images from "A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys"

    Article Title: A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

    Journal: The Scientific World Journal

    doi: 10.1100/2012/989514

    Real-time PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: serial 10-fold dilutions of recombinant plasmid. Specific bands of approximately 168 bp were visualised for all replicates of recombinant plasmid dilutions, except the concentration of 1 × 10 −1 copies/ μ L. For recombinant plasmid dilution with a concentration of 1 × 10 −1 copies/ μ L only one specific amplicon to three replicates was visualised. MK: MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada). a, b, c, d, and e: recombinant plasmid dilutions with concentrations of 1 × 10 3 , 1 × 10 2 , 1 × 10 1 , 1 × 10 0 , and 1 × 10 −1 copies/ μ L, respectively. W: no template control (water).
    Figure Legend Snippet: Real-time PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: serial 10-fold dilutions of recombinant plasmid. Specific bands of approximately 168 bp were visualised for all replicates of recombinant plasmid dilutions, except the concentration of 1 × 10 −1 copies/ μ L. For recombinant plasmid dilution with a concentration of 1 × 10 −1 copies/ μ L only one specific amplicon to three replicates was visualised. MK: MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada). a, b, c, d, and e: recombinant plasmid dilutions with concentrations of 1 × 10 3 , 1 × 10 2 , 1 × 10 1 , 1 × 10 0 , and 1 × 10 −1 copies/ μ L, respectively. W: no template control (water).

    Techniques Used: Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Recombinant, Plasmid Preparation, Concentration Assay, Amplification

    Real time-PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: positive samples detected by developed real-time PCR. Specific bands of approximately 168 bp were visualised for all replicates of the detected positive samples. In this figure is represented the 2% (w/v) agarose gel electrophoresis of five of the 11 positive samples detected in bats belonging to sampling area A (San Cesario sul Panaro, MO). Pl: amplicon of the recombinant plasmid with 1 × 10 4 copies/ μ L; 1, 2, 3, 4, and 5: amplicons of the five positive samples belonging to sampling area A, each with three repetitions.
    Figure Legend Snippet: Real time-PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: positive samples detected by developed real-time PCR. Specific bands of approximately 168 bp were visualised for all replicates of the detected positive samples. In this figure is represented the 2% (w/v) agarose gel electrophoresis of five of the 11 positive samples detected in bats belonging to sampling area A (San Cesario sul Panaro, MO). Pl: amplicon of the recombinant plasmid with 1 × 10 4 copies/ μ L; 1, 2, 3, 4, and 5: amplicons of the five positive samples belonging to sampling area A, each with three repetitions.

    Techniques Used: Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Sampling, Amplification, Recombinant, Plasmid Preparation

    Related Articles

    SDS Page:

    Article Title: SLLISWD Sequence in the 10FNIII Domain Initiates Fibronectin Fibrillogenesis
    Article Snippet: Following incubation, samples were analyzed by non-reducing SDS-PAGE analysis. .. Samples were prepared in NuPAGE lithium dodecyl sulfate non-reducing sample buffer and resolved on 3–8% Tris acetate SDS-PAGE gels (Invitrogen). .. Fluorescent gel images were scanned on a Typhoon FLA 9000 with a RITC filter (GE Healthcare), and densitometric analysis was performed using ImageJ (National Institutes of Health).

    Agarose Gel Electrophoresis:

    Article Title: A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys
    Article Snippet: Detection of Coronavirus in Bat Faecal Samples Using SYBR Green Real-Time PCR After optimisation of the SYBR Green real-time PCR assay, for each run, duplicates of six 10-fold dilutions of the standard plasmid, triplicates of the viral reverse-transcribed RNA of the bat samples, and a no template control were simultaneously subjected to analysis. .. To confirm the expected size of the reaction products, five microliters of the amplicons were electrophoresed in 2% (w/v) agarose gel stained with ethidium bromide in 1x standard tris-acetate-EDTA (TAE) buffer and visualised by ultravioltet (UV) light; MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada) was used to check the size of DNA fragments. .. Development and Validation of SYBR Green Real-Time PCR The linearity and efficiency of the SYBR Green real-time PCR were determined by generating a standard curve in which serial 10-fold dilutions of recombinant plasmid were tested.

    Article Title: DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange
    Article Snippet: .. The reactions were stopped by the addition of 0.1% SDS and 1.97 mg/ml proteinase K (Roche Applied Science, Basel, Switzerland), and were further incubated at 37°C for 20 min. After adding 6-fold loading dye, the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen, Carlsbad, CA, USA) staining. .. When the reactions were performed with the 32 P-labeled dsDNA, the gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan).

    Article Title: GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination
    Article Snippet: .. After an incubation at 37°C for 30 min, the reactions were stopped by the addition of 0.1% SDS and 1.97 mg/ml proteinase K (Roche Applied Science), and were further incubated at 37°C for 15 min. After adding 6-fold loading dye, the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 1.3 V/cm at 4°C for 16 h. The products were visualized by SYBR Gold (Invitrogen) staining. .. Cloning of chicken GEMIN2 cDNA The full-length cDNA encoding for the GEMIN2 proteins was obtained from the NCBI data bank.

    Article Title: A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys
    Article Snippet: To verify the specificity of the reaction, a melting curve analysis and electrophoresis on agarose gel were carried out for the products of the SYBR Green real-time PCR reaction. .. Five microliters of the amplicons were electrophoresed in 2% (w/v) agarose gel stained with ethidium bromide in 1x standard tris-acetate-EDTA (TAE) buffer and visualised by ultravioltet (UV) light; MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada) was used to check that amplicons of the expected size were present. ..

    Staining:

    Article Title: A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys
    Article Snippet: Detection of Coronavirus in Bat Faecal Samples Using SYBR Green Real-Time PCR After optimisation of the SYBR Green real-time PCR assay, for each run, duplicates of six 10-fold dilutions of the standard plasmid, triplicates of the viral reverse-transcribed RNA of the bat samples, and a no template control were simultaneously subjected to analysis. .. To confirm the expected size of the reaction products, five microliters of the amplicons were electrophoresed in 2% (w/v) agarose gel stained with ethidium bromide in 1x standard tris-acetate-EDTA (TAE) buffer and visualised by ultravioltet (UV) light; MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada) was used to check the size of DNA fragments. .. Development and Validation of SYBR Green Real-Time PCR The linearity and efficiency of the SYBR Green real-time PCR were determined by generating a standard curve in which serial 10-fold dilutions of recombinant plasmid were tested.

    Article Title: DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange
    Article Snippet: .. The reactions were stopped by the addition of 0.1% SDS and 1.97 mg/ml proteinase K (Roche Applied Science, Basel, Switzerland), and were further incubated at 37°C for 20 min. After adding 6-fold loading dye, the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen, Carlsbad, CA, USA) staining. .. When the reactions were performed with the 32 P-labeled dsDNA, the gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan).

    Article Title: GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination
    Article Snippet: .. After an incubation at 37°C for 30 min, the reactions were stopped by the addition of 0.1% SDS and 1.97 mg/ml proteinase K (Roche Applied Science), and were further incubated at 37°C for 15 min. After adding 6-fold loading dye, the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 1.3 V/cm at 4°C for 16 h. The products were visualized by SYBR Gold (Invitrogen) staining. .. Cloning of chicken GEMIN2 cDNA The full-length cDNA encoding for the GEMIN2 proteins was obtained from the NCBI data bank.

    Article Title: A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys
    Article Snippet: To verify the specificity of the reaction, a melting curve analysis and electrophoresis on agarose gel were carried out for the products of the SYBR Green real-time PCR reaction. .. Five microliters of the amplicons were electrophoresed in 2% (w/v) agarose gel stained with ethidium bromide in 1x standard tris-acetate-EDTA (TAE) buffer and visualised by ultravioltet (UV) light; MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada) was used to check that amplicons of the expected size were present. ..

    Incubation:

    Article Title: DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange
    Article Snippet: .. The reactions were stopped by the addition of 0.1% SDS and 1.97 mg/ml proteinase K (Roche Applied Science, Basel, Switzerland), and were further incubated at 37°C for 20 min. After adding 6-fold loading dye, the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen, Carlsbad, CA, USA) staining. .. When the reactions were performed with the 32 P-labeled dsDNA, the gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan).

    Article Title: BmILF and i-motif structure are involved in transcriptional regulation of BmPOUM2 in Bombyx mori
    Article Snippet: The protein concentration was determined using the BCA Protein Assay Reagent Kit (Thermo Scientific, USA). .. DNA oligonucleotide sequences of G4 structure were heated at 95°C for 10 min in 50 mM Tris buffer at pH 7.5 and slowly cooled to room temperature over 4 h. DNA oligonucleotide sequences of i-motif structure were heated at 95°C for 10 min in 50 mM Tris-acetate buffer at pH 4.1 and slowly cooled to room temperature over 4 h. Single-stranded biotinylated DNA oligonucleotide (20 μg) was incubated with 100 μg streptavidin-coated Dynabeads (Life Technologies, USA) in 400 μl binding buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 1 M NaCl, 0.003% NP40) for 30 min at room temperature with constant and slow rotation. .. After twice washing with binding buffer, the immobilized DNA was incubated for 30 min in 400 μl blocking buffer (2.5 mg/ml BSA, 10 mM HEPES, pH 7.6, 100 mM potassium glutamate, 2.5 mM DTT, 10 mM magnesium acetate, 5 mM EGTA, 3.5% glycerol with 0.003% NP40 and 5 mg/ml polyvinylpyrrolidone).

    Article Title: GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination
    Article Snippet: .. After an incubation at 37°C for 30 min, the reactions were stopped by the addition of 0.1% SDS and 1.97 mg/ml proteinase K (Roche Applied Science), and were further incubated at 37°C for 15 min. After adding 6-fold loading dye, the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 1.3 V/cm at 4°C for 16 h. The products were visualized by SYBR Gold (Invitrogen) staining. .. Cloning of chicken GEMIN2 cDNA The full-length cDNA encoding for the GEMIN2 proteins was obtained from the NCBI data bank.

    Binding Assay:

    Article Title: BmILF and i-motif structure are involved in transcriptional regulation of BmPOUM2 in Bombyx mori
    Article Snippet: The protein concentration was determined using the BCA Protein Assay Reagent Kit (Thermo Scientific, USA). .. DNA oligonucleotide sequences of G4 structure were heated at 95°C for 10 min in 50 mM Tris buffer at pH 7.5 and slowly cooled to room temperature over 4 h. DNA oligonucleotide sequences of i-motif structure were heated at 95°C for 10 min in 50 mM Tris-acetate buffer at pH 4.1 and slowly cooled to room temperature over 4 h. Single-stranded biotinylated DNA oligonucleotide (20 μg) was incubated with 100 μg streptavidin-coated Dynabeads (Life Technologies, USA) in 400 μl binding buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 1 M NaCl, 0.003% NP40) for 30 min at room temperature with constant and slow rotation. .. After twice washing with binding buffer, the immobilized DNA was incubated for 30 min in 400 μl blocking buffer (2.5 mg/ml BSA, 10 mM HEPES, pH 7.6, 100 mM potassium glutamate, 2.5 mM DTT, 10 mM magnesium acetate, 5 mM EGTA, 3.5% glycerol with 0.003% NP40 and 5 mg/ml polyvinylpyrrolidone).

    Isolation:

    Article Title: Serine-scanning mutagenesis studies of the C-terminal heptad repeats in the SARS coronavirus S glycoprotein highlight the important role of the short helical region
    Article Snippet: A soluble fraction was prepared by centrifugation at 15,000×g for 30 min at 4 °C. .. The expressed S glycoproteins were isolated using the Spep affinity tag and S-protein agarose (Novagen) and resolved using NuPAGE 3–8% Tris–acetate gels (Invitrogen) and the recommended sample buffer containing LDS and reducing agent. .. Molecular weight markers included [14 C]-methylated Rainbow proteins (Amersham Pharmacia Biotech) and HiMark standards (Invitrogen).

    Article Title: Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell–cell fusion but does not affect virion entry
    Article Snippet: In some studies, the glycoproteins were deglycosylated using peptide N-glycosidase (PNGase F; New England Biolabs). .. The isolated glycoproteins and deglycosylated polypeptides were resolved using NuPAGE 3–8% Tris–acetate gels (Invitrogen) and the recommended sample buffer containing lithium dodecyl sulfate and reducing agent. .. Molecular size markers are [14 C]-methylated Rainbow proteins (Amersham Pharmacia Biotech).

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    Thermo Fisher standard tris acetate edta tae buffer
    Real-time PCR reaction products checked on agarose gel stained with ethidium bromide in standard <t>tris-acetate-EDTA</t> (TAE) buffer: serial 10-fold dilutions of recombinant plasmid. Specific bands of approximately 168 bp were visualised for all replicates of recombinant plasmid dilutions, except the concentration of 1 × 10 −1 copies/ μ L. For recombinant plasmid dilution with a concentration of 1 × 10 −1 copies/ μ L only one specific amplicon to three replicates was visualised. MK: MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada). a, b, c, d, and e: recombinant plasmid dilutions with concentrations of 1 × 10 3 , 1 × 10 2 , 1 × 10 1 , 1 × 10 0 , and 1 × 10 −1 copies/ μ L, respectively. W: no template control (water).
    Standard Tris Acetate Edta Tae Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/standard tris acetate edta tae buffer/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    standard tris acetate edta tae buffer - by Bioz Stars, 2021-05
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    Real-time PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: serial 10-fold dilutions of recombinant plasmid. Specific bands of approximately 168 bp were visualised for all replicates of recombinant plasmid dilutions, except the concentration of 1 × 10 −1 copies/ μ L. For recombinant plasmid dilution with a concentration of 1 × 10 −1 copies/ μ L only one specific amplicon to three replicates was visualised. MK: MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada). a, b, c, d, and e: recombinant plasmid dilutions with concentrations of 1 × 10 3 , 1 × 10 2 , 1 × 10 1 , 1 × 10 0 , and 1 × 10 −1 copies/ μ L, respectively. W: no template control (water).

    Journal: The Scientific World Journal

    Article Title: A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

    doi: 10.1100/2012/989514

    Figure Lengend Snippet: Real-time PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: serial 10-fold dilutions of recombinant plasmid. Specific bands of approximately 168 bp were visualised for all replicates of recombinant plasmid dilutions, except the concentration of 1 × 10 −1 copies/ μ L. For recombinant plasmid dilution with a concentration of 1 × 10 −1 copies/ μ L only one specific amplicon to three replicates was visualised. MK: MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada). a, b, c, d, and e: recombinant plasmid dilutions with concentrations of 1 × 10 3 , 1 × 10 2 , 1 × 10 1 , 1 × 10 0 , and 1 × 10 −1 copies/ μ L, respectively. W: no template control (water).

    Article Snippet: Five microliters of the amplicons were electrophoresed in 2% (w/v) agarose gel stained with ethidium bromide in 1x standard tris-acetate-EDTA (TAE) buffer and visualised by ultravioltet (UV) light; MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada) was used to check that amplicons of the expected size were present.

    Techniques: Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Recombinant, Plasmid Preparation, Concentration Assay, Amplification

    Real time-PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: positive samples detected by developed real-time PCR. Specific bands of approximately 168 bp were visualised for all replicates of the detected positive samples. In this figure is represented the 2% (w/v) agarose gel electrophoresis of five of the 11 positive samples detected in bats belonging to sampling area A (San Cesario sul Panaro, MO). Pl: amplicon of the recombinant plasmid with 1 × 10 4 copies/ μ L; 1, 2, 3, 4, and 5: amplicons of the five positive samples belonging to sampling area A, each with three repetitions.

    Journal: The Scientific World Journal

    Article Title: A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

    doi: 10.1100/2012/989514

    Figure Lengend Snippet: Real time-PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: positive samples detected by developed real-time PCR. Specific bands of approximately 168 bp were visualised for all replicates of the detected positive samples. In this figure is represented the 2% (w/v) agarose gel electrophoresis of five of the 11 positive samples detected in bats belonging to sampling area A (San Cesario sul Panaro, MO). Pl: amplicon of the recombinant plasmid with 1 × 10 4 copies/ μ L; 1, 2, 3, 4, and 5: amplicons of the five positive samples belonging to sampling area A, each with three repetitions.

    Article Snippet: Five microliters of the amplicons were electrophoresed in 2% (w/v) agarose gel stained with ethidium bromide in 1x standard tris-acetate-EDTA (TAE) buffer and visualised by ultravioltet (UV) light; MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada) was used to check that amplicons of the expected size were present.

    Techniques: Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Sampling, Amplification, Recombinant, Plasmid Preparation

    Serine mutation at R667, but not K672, reduces the ability of the S glycoprotein to mediate cell–cell fusion. (A) Wild-type SARS-CoV S glycoprotein and the R667S and K672S mutants were metabolically labeled in COS-7 cells and isolated using the C-terminal S-peptide (Spep) affinity tag ( Kim and Raines, 1993 ). The mock lane represents cells transfected without vTF7-3 infection. Proteins were resolved by SDS polyacrylamide gel electrophoresis in NuPAGE 3–8% Tris–acetate gels (Invitrogen) either as the isolated glycoproteins (above) or following deglycosylation using PNGase F (below). The Endo-H-resistant ( S r ) and Endo-H-sensitive ( S s ) forms of the S glycoprotein are indicated, as is the fully deglycosylated S polypeptide (S). [ 14 C]-methylated Rainbow molecular weight markers are indicated. The images are printed dark to highlight the absence of proteolytic cleavage. (B) The ability of the S glycoproteins to promote ACE2-dependent cell–cell fusion was detected using the recombinant vaccinia virus-based β-galactosidase reporter assay ( Nussbaum et al., 1994 , York et al., 2004 ). COS-7 cells expressing the wild-type and mutant glycoproteins were co-cultured with COS-7 cells transiently expressing the SARS-CoV cellular receptor ACE2 and infected with the fusion reporter recombinant vaccinia virus vCB21R-lacZ ( Nussbaum et al., 1994 , York et al., 2004 ). X-gal was used to detect β-galactosidase activity arising from cell–cell fusion. The number of blue syncytia is shown from two experiments, and error bars represent one standard deviation. Transfection efficiencies were comparable in all cases.

    Journal: Virology

    Article Title: Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell–cell fusion but does not affect virion entry

    doi: 10.1016/j.virol.2006.02.003

    Figure Lengend Snippet: Serine mutation at R667, but not K672, reduces the ability of the S glycoprotein to mediate cell–cell fusion. (A) Wild-type SARS-CoV S glycoprotein and the R667S and K672S mutants were metabolically labeled in COS-7 cells and isolated using the C-terminal S-peptide (Spep) affinity tag ( Kim and Raines, 1993 ). The mock lane represents cells transfected without vTF7-3 infection. Proteins were resolved by SDS polyacrylamide gel electrophoresis in NuPAGE 3–8% Tris–acetate gels (Invitrogen) either as the isolated glycoproteins (above) or following deglycosylation using PNGase F (below). The Endo-H-resistant ( S r ) and Endo-H-sensitive ( S s ) forms of the S glycoprotein are indicated, as is the fully deglycosylated S polypeptide (S). [ 14 C]-methylated Rainbow molecular weight markers are indicated. The images are printed dark to highlight the absence of proteolytic cleavage. (B) The ability of the S glycoproteins to promote ACE2-dependent cell–cell fusion was detected using the recombinant vaccinia virus-based β-galactosidase reporter assay ( Nussbaum et al., 1994 , York et al., 2004 ). COS-7 cells expressing the wild-type and mutant glycoproteins were co-cultured with COS-7 cells transiently expressing the SARS-CoV cellular receptor ACE2 and infected with the fusion reporter recombinant vaccinia virus vCB21R-lacZ ( Nussbaum et al., 1994 , York et al., 2004 ). X-gal was used to detect β-galactosidase activity arising from cell–cell fusion. The number of blue syncytia is shown from two experiments, and error bars represent one standard deviation. Transfection efficiencies were comparable in all cases.

    Article Snippet: The isolated glycoproteins and deglycosylated polypeptides were resolved using NuPAGE 3–8% Tris–acetate gels (Invitrogen) and the recommended sample buffer containing lithium dodecyl sulfate and reducing agent.

    Techniques: Mutagenesis, Metabolic Labelling, Labeling, Isolation, Transfection, Infection, Polyacrylamide Gel Electrophoresis, Methylation, Molecular Weight, Recombinant, Reporter Assay, Expressing, Cell Culture, Activity Assay, Standard Deviation