For DNA break analysis cells were washed with PBS, trypsinized and resuspended at 8.33 × 106 cells/ml cell density in incubation buffer (0.25 mM EDTA, 20 mM NaCl, 10 mM Tris, pH 7.5). This suspension (120 μl) was mixed 1:1 with 1% low-melting point agarose (Sigma) to obtain two agarose inserts each of them containing 0.5 × 106 cells. Afterwards, cells in plugs were lysed (0.25 mM EDTA, 20 mM NaCl, 10 mM Tris, pH 7.5, 1% N-laurylsarcosyl, 1 mg/ml proteinase K) for 48 h at 50°C, washed three times with TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) and run in 1% agarose gel (chromosomal grade; Bio-Rad) in a CHEF DR III PFGE apparatus (Bio-Rad; 120 angle; 60–240 s switch time; 4 V/cm) at 14°C for 20 h. Finally, gels were stained with SYBR® Safe and analysed using LAS-4000 system (Fujifilm).
Chef dr 3 pfge apparatus
Manufactured by Bio-Rad
Sourced in United States
The CHEF DR III PFGE apparatus is a laboratory instrument designed for performing pulsed-field gel electrophoresis (PFGE). PFGE is a technique used for separating and analyzing large DNA molecules. The CHEF DR III apparatus provides the necessary functions and controls to conduct PFGE experiments, including programmable voltage, pulse time, and temperature settings.
Automatically generated - may contain errors
Lab products found in correlation
Low melting point agarose, by Merck Group (1 mentions)
Las 4000 system, by Fujifilm (1 mentions)
Xbai enzyme, by Thermo Fisher Scientific (1 mentions)
Gel doc camera system, by Bio-Rad (1 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Seakem gold, by Lonza (1 mentions)
Amersham hybond n nylon membrane, by GE Healthcare (1 mentions)
Gel doc xr system, by Bio-Rad (1 mentions)
4 protocols using chef dr 3 pfge apparatus
1
Pulsed-field Gel Electrophoresis for DNA Break Analysis
2
Bacterial Fingerprinting by PFGE Analysis
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All bacterial isolates were fingerprinted by the PFGE method, using the PulseNet protocol developed by Centers for Diseases Control and Prevention [61 ]. Chromosomal DNA was subjected to restriction analysis with application of XbaI enzyme (Thermo Fisher Scientific, USA). PFGE analysis was conducted with CHEF DR III PFGE apparatus (Bio-Rad, USA). DNA separation was performed with the following parameters: 1% agarose gel (Prona Agarose) on 0.5 M Tris–Borate–EDTA buffer at 14°C for 19 h at 6.0 V/cm (200 V). Pulse time was ranging of 2.2–63.8 s. The gels were stained with SYBR® Safe - DNA Gel Stain (Thermo Fisher Scientific, Germany) and band patterns were visualized under UV light and photographed using a Gel Doc camera system (Bio-Rad, USA). Molecular Weight Marker ProMega-Markers® Lambda Ladders was used for analysis (Promega, USA). PFGE patterns were analyzed via visual assessment and the dendrograms were generated with UPGMA method using on-line software http://insilico.ehu.es/ .
3
PFGE Analysis of Chromosomal DNA
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Chromosomal DNA in agarose gel plugs was prepared as described previously (36 (link)) and digested with SfiI (TOYOBO) or NotI (TOYOBO). 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. The separated DNA was transferred onto Amersham Hybond-N + nylon membranes (GE Healthcare) under alkaline conditions and analyzed by Southern hybridization as described below.
4
Molecular Profiling of E. albertii Strains
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DNA extracts of E. albertii strains were digested with XbaI and fragments separated on a 1% agarose gel using a CHEF-DR III PFGE apparatus (Bio-Rad, USA). The pulse times were ramped from 2•2 to 54•2 s over 18 h, according to the protocol for E. coli O157:H7 from PulseNet, USA (http://www.cdc.gov/pul-senet/pathogens/index.html). Gel images were captured with a Gel Doc™ XR+ system (Bio-Rad, USA), converted to TIFF files, and analysed by BioNumerics v. 4•0 (Applied Maths, Belgium). An unweighted pair-group method with arithmetic mean dendrogram was constructed using BioNumerics software.
The nucleotide sequences determined in this study have been submitted to GenBank. Their accession numbers are KP197062-KP197093 for 16S rRNA genes; KP015856-KP016011 and KP064411-KP064472 for the seven housekeeping genes; KP197094-KP197126 for the eae genes; and KP197127-KP197158 for the cdtB genes.
The nucleotide sequences determined in this study have been submitted to GenBank. Their accession numbers are KP197062-KP197093 for 16S rRNA genes; KP015856-KP016011 and KP064411-KP064472 for the seven housekeeping genes; KP197094-KP197126 for the eae genes; and KP197127-KP197158 for the cdtB genes.
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