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 tae buffer
    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/tae buffer/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tae buffer - by Bioz Stars, 2021-04
    99/100 stars

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    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: 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: 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).

    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, 2021-04
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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

    The strand-exchange assay with human RAD51. ( A ) A schematic diagram of the strand-exchange assay. ( B ) The strand exchange activity of RAD51 under the three different conditions. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA. The DNA products were then deproteinized, and 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) staining. Joint molecules and nicked circular DNA are indicated by jm and nc, respectively. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate experiments with RAD51 in the absence and presence of 0.2 M KCl, respectively. Lane 4 indicates an experiment with 5% methanol in the absence of 0.2 M KCl.

    Journal: Nucleic Acids Research

    Article Title: DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange

    doi: 10.1093/nar/gkp200

    Figure Lengend Snippet: The strand-exchange assay with human RAD51. ( A ) A schematic diagram of the strand-exchange assay. ( B ) The strand exchange activity of RAD51 under the three different conditions. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA. The DNA products were then deproteinized, and 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) staining. Joint molecules and nicked circular DNA are indicated by jm and nc, respectively. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate experiments with RAD51 in the absence and presence of 0.2 M KCl, respectively. Lane 4 indicates an experiment with 5% methanol in the absence of 0.2 M KCl.

    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.

    Techniques: Activity Assay, Incubation, Recombinase Polymerase Amplification, Agarose Gel Electrophoresis, Staining, Negative Control

    Effects of the DIDS reaction order on RAD51-mediated strand exchange. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA containing 2 μM 32 P-labeled ϕX174 linear dsDNA, in the absence of 0.2 M KCl. The reactions were stopped and deproteinized at the indicated times, and the products were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The jm products and dsDNA labeled by 32 P were visualized and quantified using an FLA-7000 imaging analyzer (Fujifilm). ( A ) RAD51 was incubated with DIDS (10 μM) at 37°C for 10 min. After the addition of the ϕX174 circular ssDNA, RPA was added to the reaction mixture. The ϕX174 linear dsDNA was then added to initiate the reaction. The reactions were continued for the indicated times. Lanes 1–4 indicate positive control experiments with RAD51 and 5% methanol. Lanes 5–8 indicate experiments with RAD51 and DIDS (and 5% methanol). Reaction times were 0 min (lanes 1 and 5), 30 min (lanes 2 and 6), 60 min (lanes 3 and 7) and 90 min (lanes 4 and 8). ( B ) Graphic representation of the experiments shown in (A). Closed and open circles indicate experiments with and without DIDS, respectively. ( C ) RAD51 was incubated with the ϕX174 circular ssDNA at 37°C for 10 min. After this incubation, DIDS (10 μM) was added, followed by the addition of RPA. The ϕX174 linear dsDNA was then added to initiate the reaction. ( D ) Graphic representation of the experiments shown in (C). Closed and open circles indicate experiments with and without DIDS, respectively. ( E ) RAD51 was incubated with the ϕX174 circular ssDNA at 37°C for 10 min, and RPA was added. DIDS was added, and then ϕX174 linear dsDNA was added to initiate the reaction. ( F ) Graphic representation of the experiments shown in (E). Closed and open circles indicate experiments with and without DIDS, respectively.

    Journal: Nucleic Acids Research

    Article Title: DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange

    doi: 10.1093/nar/gkp200

    Figure Lengend Snippet: Effects of the DIDS reaction order on RAD51-mediated strand exchange. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA containing 2 μM 32 P-labeled ϕX174 linear dsDNA, in the absence of 0.2 M KCl. The reactions were stopped and deproteinized at the indicated times, and the products were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The jm products and dsDNA labeled by 32 P were visualized and quantified using an FLA-7000 imaging analyzer (Fujifilm). ( A ) RAD51 was incubated with DIDS (10 μM) at 37°C for 10 min. After the addition of the ϕX174 circular ssDNA, RPA was added to the reaction mixture. The ϕX174 linear dsDNA was then added to initiate the reaction. The reactions were continued for the indicated times. Lanes 1–4 indicate positive control experiments with RAD51 and 5% methanol. Lanes 5–8 indicate experiments with RAD51 and DIDS (and 5% methanol). Reaction times were 0 min (lanes 1 and 5), 30 min (lanes 2 and 6), 60 min (lanes 3 and 7) and 90 min (lanes 4 and 8). ( B ) Graphic representation of the experiments shown in (A). Closed and open circles indicate experiments with and without DIDS, respectively. ( C ) RAD51 was incubated with the ϕX174 circular ssDNA at 37°C for 10 min. After this incubation, DIDS (10 μM) was added, followed by the addition of RPA. The ϕX174 linear dsDNA was then added to initiate the reaction. ( D ) Graphic representation of the experiments shown in (C). Closed and open circles indicate experiments with and without DIDS, respectively. ( E ) RAD51 was incubated with the ϕX174 circular ssDNA at 37°C for 10 min, and RPA was added. DIDS was added, and then ϕX174 linear dsDNA was added to initiate the reaction. ( F ) Graphic representation of the experiments shown in (E). Closed and open circles indicate experiments with and without DIDS, respectively.

    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.

    Techniques: Incubation, Recombinase Polymerase Amplification, Labeling, Agarose Gel Electrophoresis, Imaging, Positive Control

    DIDS efficiently inhibits RAD51-mediated strand exchange. ( A ) DIDS titration experiments in the absence of 0.2 M KCl. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) in the presence of DIDS at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA. The DNA products were then deproteinized, and 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) staining. Joint molecule is indicated by jm. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 and 5% methanol in the absence of DIDS. DIDS concentrations were 0.01 μM (lane 3), 0.1 μM (lane 4), 1 μM (lane 5) and 10 μM (lane 6). Lane 7 indicates an experiment with 10 μM DIDS in the absence of RAD51. ( B ) Graphic representation of the experiments shown in (A). The band intensities of the jm product were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (A, lane 2) were plotted against the DIDS concentration. ( C ) The strand-exchange assay in the presence of 0.2 M KCl. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate control experiments without DIDS with RAD51 in the absence and presence of 5% methanol, respectively. DIDS concentrations were 0.01 μM (lane 4), 0.1 μM (lane 5), 1 μM (lane 6) and 10 μM (lane 7). Lane 8 indicates an experiment with 10 μM DIDS in the absence of RAD51. ( D ) Graphic representation of the experiments shown in (C). The band intensities of the jm products were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (C, lane 3) were plotted against the DIDS concentration.

    Journal: Nucleic Acids Research

    Article Title: DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange

    doi: 10.1093/nar/gkp200

    Figure Lengend Snippet: DIDS efficiently inhibits RAD51-mediated strand exchange. ( A ) DIDS titration experiments in the absence of 0.2 M KCl. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) in the presence of DIDS at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA. The DNA products were then deproteinized, and 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) staining. Joint molecule is indicated by jm. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 and 5% methanol in the absence of DIDS. DIDS concentrations were 0.01 μM (lane 3), 0.1 μM (lane 4), 1 μM (lane 5) and 10 μM (lane 6). Lane 7 indicates an experiment with 10 μM DIDS in the absence of RAD51. ( B ) Graphic representation of the experiments shown in (A). The band intensities of the jm product were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (A, lane 2) were plotted against the DIDS concentration. ( C ) The strand-exchange assay in the presence of 0.2 M KCl. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate control experiments without DIDS with RAD51 in the absence and presence of 5% methanol, respectively. DIDS concentrations were 0.01 μM (lane 4), 0.1 μM (lane 5), 1 μM (lane 6) and 10 μM (lane 7). Lane 8 indicates an experiment with 10 μM DIDS in the absence of RAD51. ( D ) Graphic representation of the experiments shown in (C). The band intensities of the jm products were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (C, lane 3) were plotted against the DIDS concentration.

    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.

    Techniques: Titration, Incubation, Recombinase Polymerase Amplification, Agarose Gel Electrophoresis, Staining, Negative Control, Concentration Assay

    DIDS inhibits DNA binding by RAD51. ( A ) The ssDNA-binding experiments in the presence of ATP. The ϕX174 circular ssDNA (40 μM) was incubated with RAD51 (2 μM) in the presence of DIDS at 37°C for 15 min. The samples were analyzed by 0.8% agarose gel electrophoresis in 1× TAE buffer. The bands were visualized by ethidium bromide staining. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 alone. Lane 3 indicates an experiment with RAD51 and 5% methanol. DIDS concentrations were 0.1 μM (lane 4), 1 μM (lane 5), 10 μM (lane 6) and 20 μM (lane 7). Lane 8 indicates an experiment with 20 μM DIDS in the absence of RAD51. ( B ) The dsDNA-binding experiments in the presence of ATP. The linear ϕX174 dsDNA (10 μM) was incubated with RAD51 (1 μM) in the presence of DIDS at 37°C for 15 min. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 alone. Lane 3 indicates an experiment with RAD51 and 5% methanol. DIDS concentrations were 0.01 μM (lane 4), 0.1 μM (lane 5), 1 μM (lane 6) and 10 μM (lane 7). Lane 8 indicates an experiment with 10 μM DIDS in the absence of RAD51. ( C ) The ssDNA-binding experiments in the presence of ATP, AMPPNP and ATPγS. The ϕX174 circular ssDNA (40 μM) was incubated with RAD51 (2 μM) at 37°C for 15 min. Lanes 1–4, lanes 5–8 and lanes 9–12 represent experiments with ATP, AMPPNP and ATPγS, respectively. Lanes 1, 5 and 9 indicate negative control experiments without RAD51. Lanes 2, 6 and 10 indicate experiments with RAD51 alone. Lanes 3, 7 and 11 indicate experiments with RAD51 and 5% methanol. Lanes 4, 8 and 12 indicate experiments with RAD51 and DIDS (20 μM). ( D ) The dsDNA-binding experiments in the presence of ATP, AMPPNP and ATPγS. The linear ϕX174 dsDNA (10 μM) was incubated with RAD51 (1 μM) in the presence of DIDS at 37°C for 15 min. Lanes correspond to those in (C). The DIDS concentration was 10 μM. ( E ) Effect of DIDS on the ssDNA binding of RPA. The ϕX174 circular ssDNA (40 μM) was incubated with RPA (0.5 μM) in the presence of DIDS at 37°C for 15 min. Lanes 1 and 8 indicate negative control experiments without RPA. Lanes 2 and 3 indicate experiments with RPA in the absence and presence of 5% methanol, respectively. The DIDS concentrations were 0.1 μM (lane 4), 1 μM (lane 5), 10 μM (lane 6) and 20 μM (lanes 7 and 8). ( F ) The ssDNA-binding and dsDNA-binding experiments with RAD51 were performed in the presence of methanol. Lanes 1–6 and lanes 7–12 indicate experiments with ssDNA and dsDNA, respectively. Lanes 1, 6, 7 and 12 are negative controls without RAD51, and lanes 2 and 8 are positive controls with RAD51 in the absence of methanol. Methanol concentrations were 2.5% (lanes 3 and 9), 5% (lanes 4 and 10) and 10% (lanes 5, 6, 11 and 12).

    Journal: Nucleic Acids Research

    Article Title: DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange

    doi: 10.1093/nar/gkp200

    Figure Lengend Snippet: DIDS inhibits DNA binding by RAD51. ( A ) The ssDNA-binding experiments in the presence of ATP. The ϕX174 circular ssDNA (40 μM) was incubated with RAD51 (2 μM) in the presence of DIDS at 37°C for 15 min. The samples were analyzed by 0.8% agarose gel electrophoresis in 1× TAE buffer. The bands were visualized by ethidium bromide staining. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 alone. Lane 3 indicates an experiment with RAD51 and 5% methanol. DIDS concentrations were 0.1 μM (lane 4), 1 μM (lane 5), 10 μM (lane 6) and 20 μM (lane 7). Lane 8 indicates an experiment with 20 μM DIDS in the absence of RAD51. ( B ) The dsDNA-binding experiments in the presence of ATP. The linear ϕX174 dsDNA (10 μM) was incubated with RAD51 (1 μM) in the presence of DIDS at 37°C for 15 min. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 alone. Lane 3 indicates an experiment with RAD51 and 5% methanol. DIDS concentrations were 0.01 μM (lane 4), 0.1 μM (lane 5), 1 μM (lane 6) and 10 μM (lane 7). Lane 8 indicates an experiment with 10 μM DIDS in the absence of RAD51. ( C ) The ssDNA-binding experiments in the presence of ATP, AMPPNP and ATPγS. The ϕX174 circular ssDNA (40 μM) was incubated with RAD51 (2 μM) at 37°C for 15 min. Lanes 1–4, lanes 5–8 and lanes 9–12 represent experiments with ATP, AMPPNP and ATPγS, respectively. Lanes 1, 5 and 9 indicate negative control experiments without RAD51. Lanes 2, 6 and 10 indicate experiments with RAD51 alone. Lanes 3, 7 and 11 indicate experiments with RAD51 and 5% methanol. Lanes 4, 8 and 12 indicate experiments with RAD51 and DIDS (20 μM). ( D ) The dsDNA-binding experiments in the presence of ATP, AMPPNP and ATPγS. The linear ϕX174 dsDNA (10 μM) was incubated with RAD51 (1 μM) in the presence of DIDS at 37°C for 15 min. Lanes correspond to those in (C). The DIDS concentration was 10 μM. ( E ) Effect of DIDS on the ssDNA binding of RPA. The ϕX174 circular ssDNA (40 μM) was incubated with RPA (0.5 μM) in the presence of DIDS at 37°C for 15 min. Lanes 1 and 8 indicate negative control experiments without RPA. Lanes 2 and 3 indicate experiments with RPA in the absence and presence of 5% methanol, respectively. The DIDS concentrations were 0.1 μM (lane 4), 1 μM (lane 5), 10 μM (lane 6) and 20 μM (lanes 7 and 8). ( F ) The ssDNA-binding and dsDNA-binding experiments with RAD51 were performed in the presence of methanol. Lanes 1–6 and lanes 7–12 indicate experiments with ssDNA and dsDNA, respectively. Lanes 1, 6, 7 and 12 are negative controls without RAD51, and lanes 2 and 8 are positive controls with RAD51 in the absence of methanol. Methanol concentrations were 2.5% (lanes 3 and 9), 5% (lanes 4 and 10) and 10% (lanes 5, 6, 11 and 12).

    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.

    Techniques: Binding Assay, Incubation, Agarose Gel Electrophoresis, Staining, Negative Control, Concentration Assay, Recombinase Polymerase Amplification

    GEMIN2 enhances the homologous-pairing and strand-exchange activities of RAD51. ( A ) GEMIN2 stimulates the RAD51-mediated homologous pairing. RAD51 and GEMIN2 were incubated at 37°C for 5 min. After this incubation, a 32 P-labeled 50-mer oligonucleotide (1 µM) was added, and the samples were further incubated at 37°C for 5 min. The reactions were then initiated by the addition of the pB5Sarray superhelical dsDNA (20 µM), and were continued at 37°C for 30 min. The reactions were stopped by the addition of SDS and proteinase K, and the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1× TAE buffer. The gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan). The reactions were conducted with 100 nM RAD51 in the presence of increasing amounts of GEMIN2. A schematic representation of the homologous pairing is presented on the top of the panel. ( B ) Graphic representation of the experiments shown in panel A. Amounts of D-loops relative to that of the RAD51 alone are plotted. The average values of three independent experiments are shown with the SD values. ( C ) Schematic representations of strand-exchange reactions. (i) The RAD51-ssDNA complexes are formed before the RPA addition. (ii) The RPA-ssDNA complexes are formed before the RAD51 addition. ( D ) Strand-exchange reactions where RPA was added to ϕX174 circular ssDNA (20 µM), after [lanes 1–4, panel C(i)] or before [lanes 5–8, panel C(ii)] incubation of the ssDNA with RAD51. Strand-exchange reactions were initiated by the addition of ϕX174 linear dsDNA (20 µM) and (NH 4 ) 2 SO 4 (100 mM), and incubated for 30 min. The deproteinized products of the reaction mixtures were separated using 1% agarose gel electrophoresis and were visualized by SYBR Gold (Invitrogen) staining. ( E ) GEMIN2 enhances strand exchange. ssDNA was incubated with RPA and then with RAD51 [panel C(ii)]. The indicated amounts of GEMIN2 were pre-incubated with RAD51, and subsequently added to the reaction mixture containing the ssDNA and RPA. ( F ) Quantification of panel E. The band intensities of the joint molecule (jm) products were quantified as the percentage of the entire input of the ssDNA and dsDNA molecules. Average values of three independent experiments are shown with standard deviation values.

    Journal: Nucleic Acids Research

    Article Title: GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination

    doi: 10.1093/nar/gkq271

    Figure Lengend Snippet: GEMIN2 enhances the homologous-pairing and strand-exchange activities of RAD51. ( A ) GEMIN2 stimulates the RAD51-mediated homologous pairing. RAD51 and GEMIN2 were incubated at 37°C for 5 min. After this incubation, a 32 P-labeled 50-mer oligonucleotide (1 µM) was added, and the samples were further incubated at 37°C for 5 min. The reactions were then initiated by the addition of the pB5Sarray superhelical dsDNA (20 µM), and were continued at 37°C for 30 min. The reactions were stopped by the addition of SDS and proteinase K, and the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1× TAE buffer. The gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan). The reactions were conducted with 100 nM RAD51 in the presence of increasing amounts of GEMIN2. A schematic representation of the homologous pairing is presented on the top of the panel. ( B ) Graphic representation of the experiments shown in panel A. Amounts of D-loops relative to that of the RAD51 alone are plotted. The average values of three independent experiments are shown with the SD values. ( C ) Schematic representations of strand-exchange reactions. (i) The RAD51-ssDNA complexes are formed before the RPA addition. (ii) The RPA-ssDNA complexes are formed before the RAD51 addition. ( D ) Strand-exchange reactions where RPA was added to ϕX174 circular ssDNA (20 µM), after [lanes 1–4, panel C(i)] or before [lanes 5–8, panel C(ii)] incubation of the ssDNA with RAD51. Strand-exchange reactions were initiated by the addition of ϕX174 linear dsDNA (20 µM) and (NH 4 ) 2 SO 4 (100 mM), and incubated for 30 min. The deproteinized products of the reaction mixtures were separated using 1% agarose gel electrophoresis and were visualized by SYBR Gold (Invitrogen) staining. ( E ) GEMIN2 enhances strand exchange. ssDNA was incubated with RPA and then with RAD51 [panel C(ii)]. The indicated amounts of GEMIN2 were pre-incubated with RAD51, and subsequently added to the reaction mixture containing the ssDNA and RPA. ( F ) Quantification of panel E. The band intensities of the joint molecule (jm) products were quantified as the percentage of the entire input of the ssDNA and dsDNA molecules. Average values of three independent experiments are shown with standard deviation values.

    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.

    Techniques: Incubation, Labeling, Agarose Gel Electrophoresis, Imaging, Recombinase Polymerase Amplification, Staining, Standard Deviation

    Effects of TMPyP4 and TMPyP2 on the i-motif structure and promoter activity of BmPOUM2 . ( A ) The structure of the porphyrin compounds TMPyP2 and TMPyP4. ( B ) CD analysis of the i-motif structure in the presence of TMPyP4 or TMPyP2. ( C ) CD analysis of melting temperature of the i-motif structure in the presence of TMPyP4 or TMPyP2. The synthesized ssDNA oligonucleotides that contained the i-motif region (5 μM) 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 period to allow i-motif structure to form. TMPyP2 or TMPyP4 was then added into the solution at a final concentration of 25 μM and incubated overnight at 4°C, followed by CD analysis. The dot lines show the melting temperatures. ( D ) Determination of the promoter activity by the luciferase assay in the presence of TMPyP4 or TMPyP2. Data are means ± SEM ( n = 3). *** P

    Journal: Nucleic Acids Research

    Article Title: BmILF and i-motif structure are involved in transcriptional regulation of BmPOUM2 in Bombyx mori

    doi: 10.1093/nar/gkx1207

    Figure Lengend Snippet: Effects of TMPyP4 and TMPyP2 on the i-motif structure and promoter activity of BmPOUM2 . ( A ) The structure of the porphyrin compounds TMPyP2 and TMPyP4. ( B ) CD analysis of the i-motif structure in the presence of TMPyP4 or TMPyP2. ( C ) CD analysis of melting temperature of the i-motif structure in the presence of TMPyP4 or TMPyP2. The synthesized ssDNA oligonucleotides that contained the i-motif region (5 μM) 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 period to allow i-motif structure to form. TMPyP2 or TMPyP4 was then added into the solution at a final concentration of 25 μM and incubated overnight at 4°C, followed by CD analysis. The dot lines show the melting temperatures. ( D ) Determination of the promoter activity by the luciferase assay in the presence of TMPyP4 or TMPyP2. Data are means ± SEM ( n = 3). *** P

    Article Snippet: 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.

    Techniques: Activity Assay, Synthesized, Concentration Assay, Incubation, Luciferase