widemini subcell gt cell  (Bio-Rad)

Journal: bioRxiv

Article Title: The peroxiredoxin Tsa1 extends the lifespan of budding yeast by maintaining the stability of the ribosomal RNA gene cluster

doi: 10.1101/2024.03.14.585068

Figure Lengend Snippet: (A–D) PFGE analysis of the size heterogeneity of chr XII. The indicated genes that encode enzymes that remove ROS were deleted from WT cells. DNA was extracted from two independent clones of the WT and all mutant strains and separated by PFGE. DNA was stained with ethidium bromide. M indicates H. wingei chromosomal DNA markers. (E) ERC detection. DNA was isolated from two independent clones of the indicated strains and separated by agarose gel electrophoresis, followed by Southern blotting with rDNA probe 1, as indicated in . The chromosomal rDNA array and the supercoiled and relaxed forms of monomeric and dimeric ERCs are indicated. The sizes of lambda DNA-HindIII markers are indicated. (F) Quantitation of ERCs. The ERCs in (E) were quantified. The bars show the means ± s.e.m. Multiple comparisons were performed by one-way ANOVA, followed by Tukey’s multiple comparisons test; * indicates a statistically significant difference (p ≤ 0.05); ns indicates no significant difference.

Article Snippet: Second-dimensional electrophoresis was performed on a subcell GT electrophoresis system (Bio-Rad) in 1.5 L of 1× TBE containing 0.3 μg/mL EtBr at 6.0 V/cm for 6 h at 4°C with buffer circulation.

Techniques: Clone Assay, Mutagenesis, Staining, Isolation, Agarose Gel Electrophoresis, Southern Blot, Lambda DNA Preparation, Quantitation Assay

Journal: bioRxiv

Article Title: The peroxiredoxin Tsa1 extends the lifespan of budding yeast by maintaining the stability of the ribosomal RNA gene cluster

doi: 10.1101/2024.03.14.585068

Figure Lengend Snippet: (A) PFGE analysis of the size heterogeneity of chr XII. DNA was extracted from four independent clones of the indicated strains and separated by PFGE. DNA was stained with ethidium bromide. M indicates H. wingei chromosomal DNA markers. (B) ERC detection. DNA was isolated from three independent clones of the indicated strains and separated by agarose gel electrophoresis, followed by Southern blotting with rDNA probe 1, as shown in . The chromosomal rDNA array and the supercoiled and relaxed forms of monomeric and dimeric ERCs are indicated. The sizes of lambda DNA-HindIII markers are indicated. (C) Quantitation of ERCs. The ERCs in (B) were quantified. The bars show the means ± s.e.m. Multiple comparisons were performed by one-way ANOVA, followed by Tukey’s multiple comparisons test; * indicates a statistically significant difference (p ≤ 0.05); ns indicates no significant difference.

Article Snippet: Second-dimensional electrophoresis was performed on a subcell GT electrophoresis system (Bio-Rad) in 1.5 L of 1× TBE containing 0.3 μg/mL EtBr at 6.0 V/cm for 6 h at 4°C with buffer circulation.

Techniques: Clone Assay, Staining, Isolation, Agarose Gel Electrophoresis, Southern Blot, Lambda DNA Preparation, Quantitation Assay

Journal: bioRxiv

Article Title: The peroxiredoxin Tsa1 extends the lifespan of budding yeast by maintaining the stability of the ribosomal RNA gene cluster

doi: 10.1101/2024.03.14.585068

Figure Lengend Snippet: (A) Restriction map of the probe used for 2D analysis. The restriction sites for NheI (N) and the position of probe 1 are indicated. (B) 2D agarose gel electrophoresis. Genomic DNA from the indicated strains was digested with NheI and separated according to size in the first dimension and according to size and shape in the second dimension, followed by Southern blotting with the probe, as indicated in (A). The diagrams on the left and right show the expected migration patterns of different replications of the FOB1 and fob1 strains, respectively. 1N represents linear DNA, and 2N represents linear DNA that was nearly fully replicated. X structures that include recombination intermediates are indicated in pink. (C–F) Quantitation of DNA replication and recombination intermediates. The frequencies of bubbles, arcs (C), arrested forks (D), X structures in the FOB1 background (E) and X structures in the fob1 background (F) were determined by quantifying the signal of each intermediate relative to the total number of replication intermediates. The levels of different molecules in each mutant were normalized to the average of the WT clones (C-E) and fob1 clones (F). Bars indicate the range of two independent experiments.

Article Snippet: Second-dimensional electrophoresis was performed on a subcell GT electrophoresis system (Bio-Rad) in 1.5 L of 1× TBE containing 0.3 μg/mL EtBr at 6.0 V/cm for 6 h at 4°C with buffer circulation.

Techniques: Agarose Gel Electrophoresis, Southern Blot, Migration, Quantitation Assay, Mutagenesis, Clone Assay

Journal: bioRxiv

Article Title: The peroxiredoxin Tsa1 extends the lifespan of budding yeast by maintaining the stability of the ribosomal RNA gene cluster

doi: 10.1101/2024.03.14.585068

Figure Lengend Snippet: (A) Restriction map and positions of a probe used for SSB analyses. (B) SSB assay. Genomic DNA was isolated from three clones of the indicated strains, digested with BglII, and separated by agarose gel electrophoresis under denaturing conditions, followed by Southern blotting with rDNA probe 2, as shown in (A). Bands corresponding to DSB fragments at the RFB and SSBs are indicated.

Article Snippet: Second-dimensional electrophoresis was performed on a subcell GT electrophoresis system (Bio-Rad) in 1.5 L of 1× TBE containing 0.3 μg/mL EtBr at 6.0 V/cm for 6 h at 4°C with buffer circulation.

Techniques: Isolation, Clone Assay, Agarose Gel Electrophoresis, Southern Blot