Journal: FEMS microbiology ecology

Article Title: The use of PCR for the identification and characterisation of bacteriocin genes from bacterial strains isolated from rumen or caecal contents of cattle and sheep.

doi: 10.1016/j.femsec.2004.01.003

Figure Lengend Snippet: Fig. 4. PFGE profiles of Streptococcus spp. possessing the bovicin 255 gene. Lane 1, S. gallolyticus LRC0255; 2, S. bovis 7–2; 3, S. bovis 7–25; 4, S. bovis 7–26; 5, S. bovis GKF1; 6, S. bovis 22/01 F; 7, S. bovis 24/01 B; 8, S. bovis 24/01 H; 9, S. bovis 4b; 10, S. bovis 13a; and 11, S. bovis 13b.

Article Snippet: DNA embedded in agarose was digested with SmaI (New England Biolabs, Beverly, MA), loaded into the wells of 1% agarose gels (pulsed field certified agarose, Bio-Rad Laboratories, Hercules, CA), and run at 5.0 V cm 1 for 20 h at 14 C in 0.5 Tris–borate buffer using a CHEF DR III PFGE apparatus cooled using a model 1000 mini chiller (Bio-Rad).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells

doi: 10.1083/jcb.201406099

Figure Lengend Snippet: RAD51 is present at forks upon mild genotoxic stress and modulates fork progression and integrity. (A) Linear regression analysis shows strict direct correlation (P < 0.0001) between accumulation of ssDNA at the fork (median values of ssDNA regions at the junction) and frequency of fork reversal. Results from two independent experiments are displayed for untreated U2OS cells and for each genotoxic treatment. (B) HEK293T cells were EdU-labeled as indicated in Fig. S5 C and treated with sublethal doses of genotoxic drugs (0.5 mM HU, 200 nM MMC, or 25 nM CPT). Proteins and relative posttranslational modifications associated with replication forks were isolated by iPOND procedure and detected with the indicated antibodies. The thymidine (Thy; 10 µM) chase experiment is used to discriminate proteins associated with chromatin behind replicating forks. In the control (Ctrl) experiment, the click reaction is performed using DMSO instead of biotin azide. 1 µM CPT treatment is used as positive control to induce high replication stress and DSBs. (C) Immunofluorescence staining for U2OS cells grown on coverslips and treated with the indicated drugs for 1 h. Red staining, RAD51; green staining, EdU; blue, DAPI. Bar, 15 µM. (D) DNA fiber spreading. Statistical analysis of IdU replicated track length in U2OS cells, comparing not treated (NT) conditions with the indicated treatments. U2OS cells were transfected with siRNA against luciferase (siLuc) or RAD51 (siRAD51) 24 h before CldU or IdU labeling. At least 100 tracks were scored per sample. Horizontal lines represent the median value, and boxes and whiskers indicate 10–90th percentiles. Statistical analysis: one-way ANOVA; ns, not significant; ***, P ≤ 0.001. (E) PFGE analysis for DNA breakage detection in untreated U2OS cells and upon 1-h treatment with indicated doses of genotoxic treatments. U2OS cells were transfected with siRNA against luciferase or RAD51 24 h before treatments. 1 µM camptothecin (CPT) treatment is used as a positive control for DSB formation. The graph shows quantitative DSB induction from three independent experiments and includes average value and standard deviations (error bars). Statistical analysis: two-way ANOVA; ns, not significant; *, P ≤ 0.05.

Article Snippet: Electrophoresis was performed for 21 h at 14°C in 0.9% (wt/vol) Pulse Field Certified Agarose (Bio-Rad Laboratories) containing Tris-borate/EDTA buffer in a PFGE apparatus (CHEF DR III; Bio-Rad Laboratories), according to the following protocol (block I: 9 h, 120° included angle, 5.5 V/cm, 30 to 18-s switch; block II: 6 h, 117° included angle, 4.5 V/cm, 18 to 9-s switch; block III: 6 h, 112° included angle, 4.0 V/cm, 9 to 5-s switch).

Techniques: Labeling, Isolation, Control, Positive Control, Immunofluorescence, Staining, Transfection, Luciferase

Journal: FEMS Microbiology Letters

Article Title: Giant linear plasmids of β-lactam antibiotic producing Streptomyces

doi: 10.1111/j.1574-6968.1995.tb07749.x

Figure Lengend Snippet: Fig. 1. CHEF gel electrophoresis of DNA preparations prepared by in situ lysis of @-la&m producing Streptomyces spp. Lanes 1-7: (1) S. canleya; (2) S. chuligerus; (3) S. grisew; (4) S. jumonjinensis; (5) S. lipmannii; (6) S. iividans 1326; (7) h ladder molecular mass marker (New England Biolabs). Numbers to the side of the figures indicate molecular mass marker positions (size in kb), C indicates zone of compression.

Article Snippet: Pulsed field gel electrophoresis GLPs were separated using a Bio-Rad CHEF DR II apparatus [13] capable of resolving DNA fragments up to at least 2 Mb in size.

Techniques: Nucleic Acid Electrophoresis, In Situ, Lysis, Marker

Journal: FEMS Microbiology Letters

Article Title: Giant linear plasmids of β-lactam antibiotic producing Streptomyces

doi: 10.1111/j.1574-6968.1995.tb07749.x

Figure Lengend Snippet: Fig. 2. Sucrose gradient fractionation of linear plasmid DNA from a proteinase K high molecular mass DNA preparation of S. clauuligerus. S. clauuligerus DNA was fractionated and electrophoresed in a CHEF gel as described in the materials and methods, and stained with ethidium bromide. Lanes 1 and 20 are A ladder/A Hind111 molecular mass markers, lanes 2 to 19 correspond to sucrose gradient fractions, with lane 2 being the top of the gradient, lane 19 the bottom. Numbers to the side of the figures indicate molecular mass marker positions (sizes in kb.)

Article Snippet: Pulsed field gel electrophoresis GLPs were separated using a Bio-Rad CHEF DR II apparatus [13] capable of resolving DNA fragments up to at least 2 Mb in size.

Techniques: Fractionation, Plasmid Preparation, Staining, Marker

Journal: FEMS Microbiology Letters

Article Title: Giant linear plasmids of β-lactam antibiotic producing Streptomyces

doi: 10.1111/j.1574-6968.1995.tb07749.x

Figure Lengend Snippet: Fig. 3. Southern transfer and hybridization analysis of total cellular DNA in situ preparations of 8. clauuligerus, S. gri.seus, S. jumonjinensis, and 5. liuidans 1326 with linear plasmid probes. A: Ethidium bromide stained CHEF gel mn under the same conditions as in Fig. 1, with the DNA samples: (1) A concatamer ladder molecular mass markers; (2) 5. clauuligerus; (3) S. griseus; (4) S. jumonjinensis; and (5) 5. liuidans 1326. Panels B to H represent autoradiographs of the gel shown in panel A after hybridizations with random primer labeled probes: (B) pSCL1; (0 pSCL2; (D) pSCL3; (El pSGL1; (F) pSJL3; (G) pSJL4; and_(H) SLP2. Scale and lane contents remain constant between panels. Numbers to the side of the figures indicate molecular mass marker positions (size in kb), C indicates zone of compression.

Article Snippet: Pulsed field gel electrophoresis GLPs were separated using a Bio-Rad CHEF DR II apparatus [13] capable of resolving DNA fragments up to at least 2 Mb in size.

Techniques: Hybridization, In Situ, Plasmid Preparation, Staining, Labeling, Marker