Pulsed field gel electrophoresis (S1-PFGE) was performed to determine the number and size of plasmids carried by strain 24835 as described previously. Briefly, agarose plugs containing whole-cell DNA of strain 24835 were treated with 8 U of S1 nuclease (Fermentas, Thermo Scientific, Waltham, MA, USA) and the reaction was stopped by adding 0.5 M EDTA (pH = 8). PFGE was conducted with a 1% SeaKem Gold agarose gel (Lonza, Basal, Switzerland) using a CHEF DRII system (Bio-Rad, Hercules, CA, USA) at 14°C, with a 6-V/cm current and run times of 12 h at switch time of 5–40 s followed by 8 h at switch time of 3–8 s. MidRange I PFG Marker (NEB, Ipswich, MA, USA) was used for size estimation.
Midrange 1 pfg marker
Manufactured by New England Biolabs
Sourced in United States
The MidRange I PFG Marker is a DNA size standard used for pulsed-field gel electrophoresis (PFGE) analysis. It contains DNA fragments ranging from 8 to 48 kilobase pairs, allowing for the effective separation and visualization of large DNA molecules.
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6 protocols using midrange 1 pfg marker
1
Plasmid Profiling by S1-PFGE
2
Phage DNA Isolation and PFGE Analysis
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The procedures described by Chang et al. [35 (link)] were used for isolating phage DNA and restriction enzyme digestion. Pulsed-field gel electrophoresis (PFGE) was performed as described previously [36 (link)], using the CHEF-DR III System (Bio-Rad Laboratories, Hercules, CA, USA) at 9 °C in 0.5 × Tris-borate-EDTA buffer, pH 8.0 at 6 V/cm with pulse ramps from 3.5 to 4 s for 19.5 h. The Midrange IPFG Marker (New England Biolabs, Ipswich, MA, USA) was used as the molecular size standard.
3
PFGE Analysis of Phage DNA Size
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PFGE was performed for phage DNA size determination as described previously [24 (link)] using a CHEF-DR III system (Bio-Rad Laboratories, Hercules, CA, USA) at 5 V/cm with pulse ramps from 1 to 6 s for 20 h for undigested phage DNA at 14 °C in 0.5 × Tris-borate-EDTA buffer. A Midrange I PFG Marker (New England Biolabs, Ipswich, MA, USA) was used as the molecular size standard.
4
Phage DNA Isolation and PFGE Analysis
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We followed the procedures described by Chang et al.51 (link) to isolate phage DNA and perform restriction enzyme digestion. PFGE was performed as previously described52 (link), and the CHEF-DR III System (Bio-Rad Laboratories, Hercules, CA, USA) was used under the following conditions: 9 °C in 0.5× Tris–borate–EDTA buffer (pH 8.0) at 6 V/cm with pulse ramps from 3.5 to 4 s for 19.5 h. A Midrange IPFG Marker (New England Biolabs, Ipswich, MA, USA) was used as the molecular size standard.
5
S1-PFGE for Plasmid Profiling
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S1-PFGE was performed to determine the number and size of plasmids carried by strain WCHEC13-8 as described previously35 (link). Briefly, agarose plugs containing whole-cell DNA of strain WCHEC13-8 were treated with 8 U of S1 nuclease (Fermentas, Thermo Scientific; Waltham, MA, US) and the reaction was stopped by adding 0.5 M EDTA (pH 8). PFGE was conducted with a 1% SeaKem Gold agarose gel (Lonza, Basal, Switzerland) using a CHEF DRII system (Bio-Rad, Hercules, CA, US) at 14°C, with a 6-V/cm current and run times of 12 h at switch time of 5 to 40 s followed by 8 h at switch time of 3 to 8 s. MidRange I PFG Marker (NEB, Ipswich, MA, US) was used for size estimation.
6
Determining Distal 4qA/4qB Variants
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The high-quality genomic DNA of the family numbers was extracted from peripheral lymphocytes, which were embedded in agarose plugs (InCert Agarose, USA). Then, 5 μg of DNA plugs was digested with the restriction enzyme EcoRI (or EcoRI/HindIII; New England Biolabs, USA) and EcoRI/BlnI (Takara, Japan). To determine the distal 4qA or 4qB variants, the corresponding DNA plugs were digested with HindIII. Then, the DNA digestions were separated on 1.2% agarose gel (Agarose III™, BBI, USA) for 39 h, according to the standard procedure for pulsed-field gel electrophoresis (PFGE). After PFGE, the DNA was transferred to a Nytran + membrane (GE Healthcare, USA) and hybridized with probe P13E-11, 4qA, or 4qB, as previously described.[10 (link)11 (link)] The position of the DRs was determined using an appropriate molecular size standard (MidRange I PFG Marker, New England Biolabs, USA). The D4Z4 units were calculated, as follows: D4Z4 unit = (D4Z4 length in EcoRI digestion [kb] − 5)/3.3.
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