Chef dr 3 pulsed field electrophoresis system

Pulsed-field gel electrophoresis of yeast cells was performed as described by Litwin et al. [52 (link)]. Briefly, 3 × 10⁷ cells were embedded in low melting agarose and digested with lyticase for 2 h at 37 °C (10 mM Tris, pH 7.2, 50 mM EDTA, 1 mg/mL lyticase). Next, cells were washed and incubated overnight with proteinase K at 50 °C (100 mM EDTA, pH 8.0, 0.2% sodium deoxycholate, 1% sodium lauryl sarcosine, 1 mg/mL proteinase K). Yeast chromosomes were resolved in 1% agarose at 6 V/cm for 24 h with a 60–120s switch time ramp and 120° switch angle at 4 °C using a CHEF-DR III pulsed-field electrophoresis system (Bio-Rad, Hercules, CA, USA). For pulsed-field electrophoresis of human DNA, 10⁵ cells were embedded in low melting agarose and incubated with proteinase K for 24 h (0.5 M EDTA, 1% N-laurylsarcosyl, proteinase K 1 mg/mL). Next, agarose plugs were washed with TE buffer and the DNA was resolved in 1% agarose at 4 V/cm for 24 h with a 60–240 s switch time ramp and 120° switch angle at 14 °C. To visualize the DNA electrophoresis, gels were stained with ethidium bromide and analyzed using Bio-Rad ChemiDoc MP System and Image Lab software (Software 6.0.1, Hercules, CA, USA).

PFGE was performed on DS1, DS2, and RS to determine if the isolates were from the challenge strains or any extraneous Salmonella introduced during the study. At each timepoint, we randomly selected two isolates (from two different chickens) per group for analysis. PFGE was performed according to CDC’s PulseNet protocol.¹⁸ (link) DNA was digested with 50 U of XbaI restriction enzyme (New England Biolabs, Ipswich, MA, USA) for at least 2 h at 37°C. Salmonella Braenderup H9812 was used as a molecular reference marker. Electrophoresis was performed using CHEF-DR^® III Pulsed-Field Electrophoresis System (Bio-Rad Laboratories, Hercules, CA, USA) with the following conditions and reagents: 1% SeaKem Gold agarose (FMC BioProducts, Rockland, Maine, USA) in 0.5% Tris-borate EDTA buffer, temperature: 14°C; voltage: 6 V/cm; run time: 18 h with switch times ranging from 2.2 to 63.8 s. The gels were stained with ethidium bromide and the DNA bands were visualized under UV trans-illumination (Gel Doc™ 2000, Bio-Rad Laboratories, Hercules, CA, USA) and gel images were captured using the Quantity one 1-D analysis software (Bio-Rad Laboratories, Hercules, CA, USA). PFGE gels were consolidated and visualized using Bionumerics software V. 4.61 (Applied Maths NV, Belgium).

PFGE of NotI-digested chromosomal DNA was performed using a CHEF-DR III Pulsed-Field Electrophoresis System (BioRad) under the following conditions: 1% SeaKem Gold Agarose (Lonza) in 0.5× TBE; temperature, 10 °C; initial switch time, 40 s; final switch time, 80 s; run time, 18 h; voltage gradient, 6.8 V/cm; and angle, 120°.

1 × 10⁸ cells were resuspended in 50 µl of SEZ buffer (1 M Sorbitol, 50 mM EDTA, 0,5 mg/ml zymolase-100T) and 30 µl of agarose low gelling (Sigma-Aldrich) were added to a 0.75% final concentration. Cell suspension was transferred in one plug mold. Plugs were incubated at 37 °C during 2 h and 12 h for vegetative and quiescent cells, respectively. Plugs were rinsed 30 min in TE-1% SDS and incubated twice 12 h in Proteinase K buffer (0.5 M EDTA, 50 mM Tris-HCl, pH 9.5, 1 mg/ml Proteinase K). Plugs were rinsed three times in TE containing 1 mM PMSF for 1 h. Finally, plugs were incubated 3 h at 37 °C in 1× TAE containing 100 µg/ml of RNase. Plugs were loaded onto 0.8% agarose gel in 1× TAE buffer. Electrophoresis was performed in a CHEF DR III pulsed-field electrophoresis system (Biorad) in 1× TAE buffer with the following settings: 48 h at 2 V/cm with a 1.8 T switch time at an included angle of 106°. DNA was visualized by ethidium bromide staining (1 µg/ml) for 30 min.

To create an rDNA specific probe, the pNOY373 plasmid, a derivative of the high copy number plasmid YEp351 carrying rDNA with a promoter starting from –206 with a XhoI–NotI flanked enhancer, LEU2, 2µ, amp, was used. The pNOY373 plasmid was kindly provided by Prof. Masayasu Nomura (University of California, USA) (Wai et al. 2000 (link)). pNOY373 DNA containing the 18S rRNA coding region [1 µg] was labelled with digoxigenin-11-deoxyuridine 5′-triphosphate (dUTP) using the DIG Nick Translation Mix (Roche) according to manufacturer’s instruction. After PFGE separation (CHEF-DR^®III Pulsed Field Electrophoresis System, Biorad), yeast chromosomes were transferred onto a nylon membrane (Roche) by capillary transfer. Then, the membrane was hybridised to a digoxigenin (DIG)-labelled rDNA-specific probe and rDNA was detected with an alkaline phosphatase-conjugated anti-DIG antibody. The chemiluminescence signal was detected with the substrate for alkaline phosphatase (CDP-Star) and the G:BOX imaging system (Syngene, Cambridge, UK). rDNA and ERCs were quantified using GelQuantNET software (http://biochemlabsolutions.com/GelQuantNET.html) using the background correction option. The amount of genomic rDNA and multimer ERCs were calculated per mean amount of DNA.

PFGE was performed as previously described [53 (link)]. In brief, cells harvested from the indicated time points were embedded in agarose plugs, spheroplasted, and deproteinized. Plugs were loaded into 0.5X TBE gels and run on a Bio-Rad CHEF-DR III Pulsed Field Electrophoresis System for 12 hours to achieve chromosome separation. Gels were stained by ethidium bromide and Sytox (Molecular Probes) and then transferred onto Hybond-N+ membranes (GE Healthcare) using standard capillary transfer technique. Membranes were probed with anti-BrdU antibody (BD) and α-mouse secondary antibody (GE Healthcare). Membranes were scanned with Fujifilm LAS-3000 luminescent image analyzer, which has a linear dynamic range of 10⁴ to achieve reliable quantification. The percentage of gel entry for each chromosome was calculated by dividing the chromosomal band signal by the sum of chromosomal band signal and well signal, after background subtraction. The positions of each chromosome were derived from [54 ]. Southern blotting of Chr XII, Chr III, and rDNA were performed using specific probes hybridizing to each region, and primers used for probe amplification are available upon request.