PGFE was applied for typing the Lb. plantarum T571 strain and Lb. monocytogenes strains, according to Doulgeraki et al. (2010) (link) and Kostaki et al. (2012) (link), respectively. All isolates were digested with the restriction enzyme ApaI (10U) (New England Biolabs, Ipswich, MA, United States) according to the manufacturer’s recommendations for 16 h. Restriction fragments were separated in 1% PFGE grade agarose gel (Bio-Rad, Hercules, CA, United States) in 0.5-mM Tris–borate buffer on CHEF-DRIII (Bio-Rad, Hercules, CA, United States) equipment with running parameters as described at Papadopoulou et al. (2018) (link). The obtained restriction profiles were compared with the PFGE fingerprints of the inoculated Lb. monocytogenes strains and Lb. plantarum T571 strain.
Pfge grade agarose gel
Manufactured by Bio-Rad
Sourced in United States
PFGE grade agarose gel is a specialized type of agarose used in pulsed-field gel electrophoresis (PFGE) applications. It is designed to provide optimal resolution and separation of large DNA fragments, typically in the range of 50 kilobase pairs to 10 megabase pairs. The agarose gel is tailored for PFGE techniques, which utilize alternating electric fields to effectively separate high-molecular-weight DNA molecules.
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3 protocols using pfge grade agarose gel
1
PFGE-based typing of Lactobacillus strains
2
Clonality Analysis of EAEC Strains
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Thirty-one EAEC strains carrying the virulence plasmid genes were selected as representative strains and were typed using PFGE to investigate their clonal relationships according to a protocol described by Zhao et al. (18 (link)). The resulting fragments from PFGE were resolved by performing contour-clamped homogeneous electric field electrophoresis with CHEF Mapper system (Bio-Rad, USA) in an autoalgorithm mode and 1% PFGE-grade agarose gel (Bio-Rad) in 0.5 × TBE (44.5 mM Tris-HCl, 44.5 mM boric acid, and 1.0 mM EDTA [pH 8.0]) at 6 V/cm for 18 hours at 14°C. The gels were stained with ethidium bromide (30 mg/L) and were digitized for computer-aided analysis.
Chromosomal DNA (225-2,200 kb; Bio-Rad) of Saccharomyces cerevisiae was used as a DNA marker. The DNA fragments were digested with XbaI and were separated on 1% agarose gel. Images of PFGE patterns were clustered using GelCompar II software (Applied Maths, Belgium). Similarity percentage was determined using Dice coefficient. Strains were considered to be clonally related if their Dice coefficient of correlation was ≥80% (19 (link), 20 (link)).
Chromosomal DNA (225-2,200 kb; Bio-Rad) of Saccharomyces cerevisiae was used as a DNA marker. The DNA fragments were digested with XbaI and were separated on 1% agarose gel. Images of PFGE patterns were clustered using GelCompar II software (Applied Maths, Belgium). Similarity percentage was determined using Dice coefficient. Strains were considered to be clonally related if their Dice coefficient of correlation was ≥80% (19 (link), 20 (link)).
3
PFGE Analysis of S. aureus Isolates
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The isolated S. aureus strains from lung tissues and inoculated test and control S. aureus strains were compared using PFGE analysis. For this purpose, agarose plugs were prepared as previously described by Durmaz et al. [40 ]. Bacterial DNA in the plug was digested by 30 U Smal (Thermo, Waltham, MA, USA) at 30 °C for 16 h, and the plugs were loaded into 150 mL 1% (w/v) PFGE grade agarose gel (Bio-Rad, Hercules, CA, USA). PFGE gel was run in 0.5× TBE buffer at 14 °C for 22 h, 5–40 s pulse time, 120°, and 6 V/cm current using CHEF-DR II PFGE system (Bio-Rad). After electrophoresis, PFGE gel was stained by 2.5× EZ-Vision DNA dye (VWR Amresco), and the bands were visualised in the Gel Doc™ EZ gel documentation system (Bio-Rad). The obtained band patterns were analysed by BioNumerics Version 7.6 (AppliedMaths, Sint-Martens-Latem, Belgium).
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