The 59 E. coli isolates were characterized by DNA macrorestriction analysis using PFGE in accordance with PulseNet International [23] . Briefly, bacterial isolates were inoculated onto Tryptone Soy Agar plates (Biokar Diagnostics, Beauvais, France) and incubated at 37 • C for 14 ± 2 h. From these plates, bacterial cultures were embedded in SeaKem Gold agarose (Bio-Rad Laboratories, Milan, Italy), lysed, washed, and the DNA was in situ digested with 50 U of XbaI (Thermo Fisher Scientific, Fermentas; Waltham, MA, USA) at 37 • C for 3 h. Resolution of the generated DNA fragments was obtained with 1% (m/v) SeaKem Gold agarose gels in 0.5× Tris-Borate EDTA Buffer (Bio-Rad Laboratories, Milan, Italy) at 14 • C and 6 V/cm with time ramped for 6.7/35.3 s over 18 h, using a CHEF DRII System (Bio-Rad Laboratories, Milan, Italy); pattern images were acquired in the Gel Doc™ EZ System (Bio-Rad Laboratories, Milan, Italy). The TIF images were normalized by aligning the peaks of the XbaI DNA fragments of the size standard strain Salmonella enterica serovar Braenderup H9812 DNA, which was loaded onto two lanes in each gel.
Seakem gold agarose
Manufactured by Bio-Rad
Sourced in Italy
SeaKem Gold agarose is a high-quality agarose product designed for use in a variety of electrophoresis applications. It provides consistent performance and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Chef dr 3 system, by Bio-Rad (2 mentions)
Seakem gold agarose, by Lonza (2 mentions)
S1 nuclease, by Takara Bio (2 mentions)
Fpquest software, by Bio-Rad (1 mentions)
Xbai, by Roche (1 mentions)
Chef dr 3 apparatus, by Bio-Rad (1 mentions)
Pcr dig probe synthesis kit, by Roche (1 mentions)
Dig luminescent detection kit, by Roche (1 mentions)
Hybond n nylon membrane, by GE Healthcare (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Dig high prime dna labeling and detection starter kit 1, by Roche (1 mentions)
Chef mapper pfge system, by Bio-Rad (1 mentions)
Chef dr 2 system, by Bio-Rad (1 mentions)
Xbai, by Thermo Fisher Scientific (1 mentions)
Gel doc ez system, by Bio-Rad (1 mentions)
5 protocols using seakem gold agarose
1
E. coli Isolates Genotyping by PFGE
2
Pulsed-Field Gel Electrophoresis for Bacterial Strain Typing
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PFGE was performed as described previously [41 (link)], with slight modifications. Briefly, the isolates were cultured in TSB supplemented with 1 μg/ml cefotaxime at 37°C overnight. After centrifugation, PFGE plugs were prepared by mixing culture pellets with 1% SeaKem Gold Agarose (Lonza, Basel, Switzerland). The plugs were digested with 30 U XbaI at 37°C overnight. The plugs were electrophoresed with a CHEF-DR III system (Biorad, Hercules, CA) in a 1% SeaKem Gold Agarose gel in 0.5× TBE buffer at 14°C and 6 V/cm for 16 h. Switching times were ramped from 2.2 to 54.2 s. Dendrographic analysis of the DNA fragments was performed using FPQuest software (Bio-rad). The Salmonella serotype Braenderup H9812 strain provided by Dr. Umeda, Osaka Institute of Public Health, was used as a DNA size standard.
3
Pulsed-field gel electrophoresis of Salmonella
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Agarose gel plugs were prepared by culturing isolates at 37°C for 18 h in trypto-soya broth (Nissui Phamaceutical) and SeaKem Gold agarose (Lonza). Plugs were treated with 1 mg/ml proteinase K (Sigma-Aldrich), digested with 18 U of S1 nuclease (Takara Bio), and electrophoresed on a CHEF-DRIII apparatus (Bio-Rad Laboratories) in 1% SeaKem Gold agarose at 14°C and 6 V/cm for 17 h with switching times of from 2.2 to 54.2 s. The Salmonella enterica serovar Braenderup strain H9812 digested with Xba I (Roche Diagnostics) was used as a marker for PFGE. DNA fragments were visualized by ethidium bromide staining and then transferred to Hybond N+ nylon membranes (GE Healthcare) using a capillary transfer system. Hybridization was carried out with a digoxigenin (DIG)-labeled blaKHM-1 probe prepared from DNA extracted from isolate OIPH-N069 using a KHM-F/KHM-R primer pair [8 ] and a PCR DIG probe synthesis kit (Roche Diagnostics). Hybridization signals were detected with a DIG luminescent detection kit (Roche Diagnostics) and an Amersham Imager 600 (GE Healthcare).
4
Phylogenetic Analysis of M. abscessus Isolates via PFGE
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The phylogenetic distance among M. abscessus isolates was analyzed by PFGE with modifications of the methods as described previously [11 (link)]. In brief, M. abscessus isolates were cultured to the logarithmic phase at 37°C in the 7H9 medium and were collected and resuspended in cell suspension buffer (CSB) to an OD600 of 1.6. Cell suspensions were mixed in a 1 : 1 ratio with 1% SeaKem Gold agarose (containing 1% SDS) and pipetted into disposable plug moulds (Bio-Rad). The plugs were lysed with 20 mg/ml lysozyme at 37°C for 3 h. The lysis solution was replaced with cell lysis buffer (CLB) [50 mM Tris/HCl (pH 8.0), 50 mM EDTA, and 0.1 mg/ml proteinase K] and incubated at 56°C overnight with shaking (160 rounds per minute). The plugs were washed once with ddH2O in a shaking water bath at 56°C for 15 min and then five times with Tris (10 mM)/EDTA (1 mM) buffer (pH 8.0) for 15 min each.
The plugs containing the genomic DNA were digested with DraI for 4 h at 37°C. Digested DNA was then separated on a 1% agarose gel using a CHEF-DRIII system (Bio-Rad Laboratories, Hercules, CA) in 0.5°×°TBE buffer [1 M Tris/HCl (pH 8.0), 0.9 M boric acid, and 10 mM Na2EDTA] with 200 μM thiourea. Pulse time was ramped from 5 to 35 s for 19 h at 14°C and a constant voltage of 6 V/cm. Similarity between PFGE patterns was analyzed by computerized band analysis with Bionumerics software version 3.3.
The plugs containing the genomic DNA were digested with DraI for 4 h at 37°C. Digested DNA was then separated on a 1% agarose gel using a CHEF-DRIII system (Bio-Rad Laboratories, Hercules, CA) in 0.5°×°TBE buffer [1 M Tris/HCl (pH 8.0), 0.9 M boric acid, and 10 mM Na2EDTA] with 200 μM thiourea. Pulse time was ramped from 5 to 35 s for 19 h at 14°C and a constant voltage of 6 V/cm. Similarity between PFGE patterns was analyzed by computerized band analysis with Bionumerics software version 3.3.
5
Plasmid Size Determination of mcr-10 and blaNDM-1
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S1-PFGE and Southern hybridization were conducted to determine the sizes of mcr-10 and blaNDM-1-carrying plasmids in strain 5549 and the transconjugants. The genomic DNA was digested with S1 nuclease (Takara Biotechnology, Dalian, China) and produced in Lonza Bioscience SeaKem® Gold Agarose, then electrophoresis by the CHEF-Mapper PFGE system (Bio-Rad) under the conditions of 14 °C, 6 V/cm, running for 16 h, 120° pulse angle and pulse times from 2.16 s to 63.8 s. Plasmid DNA fragments were hybridized with digoxigenin-labeled blaNDM-1 or mcr-10 specific probes after being transferred to nylon membranes and detected using DIG High Prime DNA Labeling and Detection Starter KitI (Roche, Mannheim, Germany).
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