Genomic DNA was prepared as described previously with some modifications.22 (link) Isolated colonies were harvested from Mueller-Hinton agar plates after overnight incubation at 37°C, and the suspension was adjusted to a concentration of 109 CFU/mL in cell suspension buffer (100 mM Tris-HCl, 100 mM EDTA, pH=8). After a short incubation of approximately 5–10 mins at 37°C, the bacterial suspension was mixed with an equal volume of 1% Gold Agarose (Lonza, USA) and allowed to solidify in a 100-µL plug mould. The DNA block was incubated overnight at 54°C in 1 mL of cell lysis buffer (50 mM Tris-HCl, 50 mM EDTA, 1% sarcosyl, 100 µg/mL proteinase K, pH=8). To eliminate the lysed bacterial material and inactivate proteinase K activity, the DNA blocks were washed four times at 50°C in 4 mL of Tris-EDTA buffer (100 mM Tris-HCl, 1 mM EDTA, pH=8). A slice of each plug was cut and incubated with SpeI (Takara, Japan). Restriction fragments of DNA were separated by pulsed-field gel electrophoresis (PFGE) with a CHEF MAPPER apparatus (Bio-Rad, USA) through 1% Gold Agarose. Electrophoresis was performed at 6 V/cm and 14°C. The run time was 18 h, with the pulse time ramping from 5 to 60 s. XbaI-digested DNA of Salmonella enterica serotype Braenderup H9812 was electrophoresed as the size marker.
Gold agarose
Manufactured by Bio-Rad
Sourced in United States
Gold Agarose is a laboratory product manufactured by Bio-Rad. It is a type of agarose gel used for electrophoresis applications in research and analytical settings.
Automatically generated - may contain errors
2 protocols using gold agarose
1
Genomic DNA Preparation and PFGE Analysis
2
Genomic DNA Extraction and PFGE
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Genomic DNA was prepared as described previously (12 (link)). Isolated colonies were harvested from Mueller–Hinton agar plates after overnight incubation at 37°C, and the suspension was adjusted to a concentration of 109 CFU/ml in cell suspension buffer (100 mM Tris–HCl, 100 mM EDTA, pH = 8). After a short incubation of ~5–10 min at 37°C, the bacterial suspension was mixed with an equal volume of 1% Gold Agarose (Lonza, Rockland, MD, USA) and allowed to solidify in a 100-μl plug mould. The DNA block was incubated overnight at 54°C in 1 ml of cell lysis buffer (50 mM Tris–HCl, 50 mM EDTA, 1% sarcosyl, 100 μg/ml proteinase K, pH = 8). To eliminate the lysed bacterial material and inactivate proteinase K activity, the DNA blocks were washed four times at 50°C in 4 ml of Tris–EDTA buffer (100 mM Tris–HCl, 1 mM EDTA, pH = 8). A slice of each plug was cut and incubated with XbaI (Takara, Shiga, Japan). Restriction fragments of DNA were separated by pulsed-field gel electrophoresis (PFGE) with a CHEF Mapper apparatus (Bio-Rad, Hercules, CA, USA) through 1% Gold Agarose. Electrophoresis was performed at 6 V/cm and 14°C. The run time was 20 h, with the pulse time ramping from 5 to 35 s. XbaI-digested DNA of Salmonella enterica serotype Braenderup H9812 was electrophoresed as the size marker.
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