Tbe buffer

Gels of different concentrations were prepared using 30% acrylamide and bis-acrylamide solution (Bio-Rad, 29:1) with 7 M urea in 0.5 × TBE buffer (Bio-Rad) and run on a PROTEAN II xi cell (Bio-Rad) or a Mini-PROTEAN Tetra cell (Bio-Rad). Samples were mixed 1:1 with TBE-urea sample buffer (Bio-Rad) and heated at 70 °C for 5 min before they were loaded into the wells. Gel concentrations were carefully chosen to ensure the proper separations between different topologies as well as the references. For imaging, gels were stained with GelRed (Biotium), and images were taken by Gel Doc XR+ (Bio-Rad) imaging system and processed by software Image Lab (v.4.0.1, Bio-Rad). For purification, gels (without staining) were visualized by UV shadowing against a fluorescent thin-layer chromatography plate and bands of interest were cut. The bands were then eluted using the crush-and-soak method and the eluent was purified and concentrated on 3 K Nanosep filters (Pall). The concentration of product was determined by measuring the OD₂₆₀. Optionally, the ssRNA knots and circles can be digested by RNase R (Epicentre) after the gel extraction to remove the unavoidable cleaved linear RNA during the purification. However, RNase R digestion is not useful for the hybrid BR.

RNase P cleavage was performed by mixing 300 nm MRPP1–MRPP2, 150 nm MRPP3, 10 units of RNase inhibitors (RNasin from Promega), and 400 nmin vitro transcribed pre-(mt) tRNA^Ile in a buffer of 30 mm Tris-HCl, pH 8, 40 mm NaCl, 4.5 mm MgCl₂, and 2 mm DTT to a total reaction volume of 8.25 μl. The reaction was performed at room temperature and stopped at set times by transferring 1 μl of the reaction mixture into 5 μl of 500 mm EDTA and heating to 95 °C. This sample was analyzed by denaturing PAGE on a 6% TBE-urea gel (Invitrogen) run in TBE buffer (Invitrogen). The gel was stained for 30 min with SYBR Gold (Invitrogen) diluted 1000-fold in TBE buffer from the manufacturer's stock and imaged using the Bio-Rad ChemiDoc imaging system.

For amplicons used in plasmid constructions, PCR was performed using the Q5 Polymerase (NEB #M0491S) following the manufacturer’s instructions. For transformant screenings (yeast and mycoplasma), PCR was performed with the Advantage2 polymerase mix (Takara #639201) following the manufacturer’s instructions. Multiplex PCR amplification was performed with the QIAGEN Multiplex PCR kit (QIAGEN #206143). The primer pairs used during simplex and multiplex PCR are all listed in Table S1.
Mccp agarose plugs and yeast agarose plugs were subjected to PFGE in a 1 % Bio-Rad Pulsed Field Certified Agarose (Bio-Rad #162–0137) gels in 0.5× TBE buffer (Bio-Rad #161–0733), with a CHEF-DR III PFGE system (Bio-Rad). Running conditions were set up as follows: switch angle 120 °, voltage gradient 6 V cm⁻¹, ramped switch time from 50 (initial time) to 90 s (final time), duration 22 h and temperature 14 °C. After electrophoresis, the gel was stained with SYBR Gold and visualization was performed using a Vilbert Lourmat E-BOX VX2 Complete Imaging system. The Bio-Rad Marker 0.225–2.2 Mb S. cerevisiae chromosomal DNA (Bio-Rad #170–3605) was used for DNA size estimations.

The PCR and PCR-RFLP products were separated in 1.5% and 2% agarose gels (peqGOLD, Peqlab Biotechnologie GmbH, Erlangen, Germany), respectively, in 1X Tris-borate-EDTA (TBE) buffer (Bio-Rad, Hercules, USA), at 90 V for 45 min to 1 h, stained with 1% aqueous solution of ethidium bromide (Merck, Darmstadt, Germany) and analyzed using GeneTools software (Syngene, Cambridge, UK).

Strain characterization was performed with a molecular serogroup-related PCR completed by the detection of flaA [9 (link),10 (link)].
Briefly, DNA was extracted as described before and reactions were carried out in a Gene Amp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). PCR products were run in 2% (w/v) agarose gel in 1× tris/borate/EDTA (TBE) buffer (Biorad, Hercules, CA, USA) and visualized by SYBR™ Safe DNA Gel Stain (Thermo Fisher Scientific, Waltham, MA, USA).

Multiplex PCR for the identification of the main L. monocytogenes serogroups (1/2a-3a, 1/2b-3b, 1/2c-3c, 4b-4d-4e) was performed as described by Doumith et al. [32 (link)]. The PCR was performed on a cycler Mastercycler^® pro (Eppendorf, Hamburg, Germany) using: 1.5 ×  PCR buffer (Promega, Madison, WI, USA), 2 mM MgCl₂ (Promega), 1.25 mM dNTPs (Promega), 0.5 μM of each primer (Oligo.pl, Warszawa, Poland), 1 U GoTaq DNA polymerase (Promega), ultrapure water (Sigma Aldrich, Saint Louis, MO, USA), and the previously isolated genomic DNA. The amplicons were electrophoretically separated in 1.5% agarose gel (Sigma Aldrich) stained with Midori Green (NIPPON Genetics EUROPE GmbH, Düren, Germany) in 1 × TBE buffer (BioRad, Hercules, CA, USA), in the presence of a DNA size standard (GeneRulerTM1000 bp DNA Ladder) (Fermentas, Waltham, MA, USA) (90 V, 1 h).
For each PCR reaction, the four selected L. monocytogenes strains examined by Wałecka-Zacharska et al. [33 (link)] were used as positive control strains for serogroup identification. The negative control in each reaction was a sample without DNA.