chef dr iii pfge apparatus  (Bio-Rad)

Journal: Nucleic Acids Research

Article Title: Fission yeast Stn1 maintains stability of repetitive DNA at subtelomere and ribosomal DNA regions

doi: 10.1093/nar/gkab767

Figure Lengend Snippet: Rearrangements at rDNA and subtelomeres are incurred in stn1-1 . ( A ) Sensitivity to HU of wild-type, reb1 Δ, and stn1-1 reb1 Δ. Five-fold serial dilutions of the indicated strains were spotted onto YES plates containing 4 mM HU or no HU (control), and incubated for 4 days at 30°C. Two independent clones of stn1-1 reb1 Δ were examined. These separated images (top and bottom) were taken from a single photo of the respective plate. ( B ) Pol1-myc localization at rfp4 and gal1 + loci tested by ChIP. gal1 + was used as a control locus. Each symbol indicates independent experiments. Error bar indicates SEM ( n = 3). P -value was calculated by a paired two-tailed Student's t -test (* P < 0.05). Three biological replicates were tested. ( C ) Instability of rDNA repeats in prolonged cultures of stn1-1 . Independent clones were cultured in liquid YES for the indicated days at 25°C, and then genomic DNA was digested by Sfi I endonuclease and analyzed by PFGE followed by Southern hybridization with a radiolabelled rDNA probe (left panel). The position of Sfi I sites, telomeric repeats (blue boxes), rDNA repeats (shaded boxes) and centromeres (oval) are shown at the top right schematic diagram. Signal intensity was quantified along each lane (the quantified range is indicated by a line at the right side of the gel) and normalized to the maximum value for each lane. The density plot for every clone is shown at the right side. Gray and red colors represent Day1 and Day10, respectively. ( D ) The copy number of rDNA repeats relative to that of the ade6 + gene was determined by qPCR. Each dot represents the result from an individual colony ( n = 5). Horizontal lines indicate the median value. A Mann–Whitney test was used for statistical analysis (* P < 0.05; ns, not significance).

Article Snippet: The digested DNA was separated by PFGE with a CHEF-DR-III PFGE apparatus (BioRad) under the following conditions: 1% SEAKEM Gold (LONZA) agarose gel in 0.5× TBE; electrode angle 120°; voltage gradient 6.8 V/cm; initiating switching time 40 s; final switching time 80 s; run time 15 h; temperature 10°C.

Techniques: Incubation, Clone Assay, Two Tailed Test, Cell Culture, Hybridization, MANN-WHITNEY