Stationary phase cells were embedded in agarose plugs and prepared using standard procedures [91 (link)]. CHEF gel analysis of whole yeast chromosomes was performed using a BioRad CHEF-DR II Pulsed Field Electrophoresis System. Whole chromosomes were resolved in 0.8% LE agarose gels with a switch time ramped from 300–900 seconds at 100 volts for 68 hours in 0.5X TBE at 14°C. BamHI digested chromosomal DNA fragments were resolved in 1.0% LE agarose gels with a switch time ramped from 47–175 seconds at 165 volts for 62 hours in 0.5X TBE at 14°C. Gels were stained with ethidium bromide and photographed. Southern blotting and hybridization were performed using standard procedures.
Chef dr 2 pulsed field electrophoresis system
Manufactured by Bio-Rad
Sourced in Belgium, Sweden
The CHEF-DR II Pulsed Field Electrophoresis System is a laboratory equipment designed for the separation and analysis of large DNA molecules. It utilizes pulsed-field gel electrophoresis (PFGE) technology to effectively separate DNA fragments ranging from 10 kilobases to 10 megabases in size.
Automatically generated - may contain errors
Lab products found in correlation
Pulsed field certified agarose, by Bio-Rad (1 mentions)
Smai restriction enzyme, by New England Biolabs (1 mentions)
Chef dna size standard lambda ladder, by Bio-Rad (1 mentions)
Gel doc camera system, by Bio-Rad (1 mentions)
Typhoon fla 9000 gel imaging scanner, by GE Healthcare (1 mentions)
Certified megabase agarose gel, by Bio-Rad (1 mentions)
7 protocols using chef dr 2 pulsed field electrophoresis system
1
Separation of Yeast Chromosomes by CHEF
2
PFGE Analysis of Genomic DNA
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Pulsed-field gel electrophoresis (PFGE) was carried out using the CHEF-DR II Pulsed Field Electrophoresis System (Bio-Rad). A 1% agarose gel was cast using Pulsed Field Certified Agarose (cat.no. 1620137, Bio-Rad) in fresh 0.5X TBE buffer. A CHEF DNA Size Marker, 0.2–2.2 Mb, S. cerevisiae Ladder (cat.no. 1703605, Bio-Rad) agarose plug was loaded in the first lane as a size reference. One µg total DNA with a volume of 40 µl was loaded in each well. The PFGE ran for 40 h at 14 °C, at 5 V/cm, initial SW: 47 s, and final SW: 170 s. The gel was post-stained in a 3X GelRed solution for 30 min.
3
Yeast Chromosomal DNA Extraction for PFGE
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Collected yeast cells were washed once with water and then fixed with 70% ethanol for 1 h at room temperature. To remove cell walls, cells were resuspended in LiSorb buffer (100 mM lithium acetate, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM sorbitol) and treated with zymolyase at 37°C for 1 h. Then cells were resuspended in TE buffer (10 mM Tris, 10 mM EDTA, pH 8.0). An equal volume of 1% melted agarose (after cooling down to 50°C) was added into cells. After agarose was solidified, cells embedded in agarose blocks were subject to digestion with lysis buffer (100 mM EDTA, 10 mM Tris, 1% sarkosyl, 100 μg/ml proteinase K, pH 8.0) overnight at 50°C. After that, the agarose blocks were washed with TE buffer twice and were ready for PFGE analysis. The CHEF-DR II pulsed-field electrophoresis system (Bio-Rad, Richmond, CA) was used. The running time was 20 h at 6 V/cm with a 60–120-s switch time ramp (14°C).
4
PFGE Typing for Clonal Analysis
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To determine the clonal relationship among isolates, PFGE typing was conducted using a previously described method11 (link) with some modifications. In brief, the samples were digested with the SmaI restriction enzyme (New England Biolabs) and separated by electrophoresis using the CHEF-DR® II Pulsed-Field Electrophoresis System (Bio-Rad Laboratories, Nazareth, Belgium) with the following parameters: duration, 20 h; temperature, 14°C; first shot duration, 3.5 s; end shot duration, 23.5 s; shot angle, 120°; and current, 6 V/cm2. The Pearson correlation coefficient and the unweighted pair group method with arithmetic mean were used for band and cluster analysis, respectively. Based on the Pearson correlation coefficient, strains with ≥95% similarity were accepted as the same clone and those with <95% similarity were considered as different clones.
5
Pulsed-Field Gel Electrophoresis (PFGE) of Y. enterocolitica O8
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PFGE was performed to compare the genetic characteristics of Y. enterocolitica O8 isolates. The PFGE was carried out as described by Iwata et al. [13 (link)]. Briefly, chromosomal DNAs were digested by the restriction enzyme NotI (TaKaRa, Kusatsu, Japan) for 3 hr at 37°C. The DNA fragments
were separated in 1.2% agarose NA (GE Healthcare, Bioscience AB, Uppsala, Sweden) on a CHEF-DRII Pulsed Field Electrophoresis System (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Electrophoresis was carried out for 24 hr at 14°C and 200 V with pulse times of 2 to 25 sec. A CHEF DNA Size Standard Lambda Ladder (Bio-Rad) was used as molecular size marker. The gels were
stained with AtlasSight DNA Stain (Bioatlas, Tartu, Estonia) and photographed using a Gel Doc camera system (Bio-Rad).
were separated in 1.2% agarose NA (GE Healthcare, Bioscience AB, Uppsala, Sweden) on a CHEF-DRII Pulsed Field Electrophoresis System (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Electrophoresis was carried out for 24 hr at 14°C and 200 V with pulse times of 2 to 25 sec. A CHEF DNA Size Standard Lambda Ladder (Bio-Rad) was used as molecular size marker. The gels were
stained with AtlasSight DNA Stain (Bioatlas, Tartu, Estonia) and photographed using a Gel Doc camera system (Bio-Rad).
6
Chromosomal DNA Separation by PFGE
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Preparation of chromosomal DNAs and their separation by PFGE were performed as previously described26 (link). Chromosomal DNAs were prepared in 0.8% low melting agarose gels (Nacalai tesque, 01161-12) and resolved using a CHEF-DRII pulsed-field electrophoresis system (Bio-Rad) under the following conditions. For broad-range PFGE, a 1600 s pulse time at 2 V cm−1 for 42 h followed by a 180 s pulse time at 2.4 V cm−1 for 4 h, at 4 °C in 1× TAE buffer using 0.55% Certified Megabase agarose gel (Bio-Rad, 161-3109)). For short-range PFGE, a 40–70 s pulse time at 4.2 V cm−1 for 24 h, at 4 °C in 0.5× TBE buffer using 0.55% Certified Megabase agarose gel. DNAs were stained with 0.2 µg mL−1 EtBr (Nacalai Tesque, 14631-94) and detected using a Typhoon FLA9000 gel imaging scanner (GE Healthcare). Gel images were processed using ImageJ software or Adobe Photoshop Elements (Adobe, San Jose, CA).
7
Chromosomal Analysis of Spheroplasts
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Spheroplasts were prepared from Sd. ludwigii cells, embedded in agarose plugs, lysed and processed for electrophoresis of intact chromosomes as previously described 39 , using the CHEF-DR II Pulsed Field Electrophoresis System (Bio-Rad).
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