Chelex solution

DNA was extracted from each mosquito (whole body) using 5% Chelex solution (BioRad), according to the manufacturer's recommendations and eluted in 200 μl of double-distilled water.
Ten microsatellite loci were genotyped (see Additional file
1). Eight of the primer pairs were originally described by Conn et al.[25 (link)]. Due to amplification problems, the primers of locus ADC107 were redesigned. Furthermore, primers for two additional loci (ADMP2 and ADMP9) were obtained from the GenBank sequences reported by Calvo et al.[26 (link)], both from 3’UTR of unknown transcripts. Each locus was amplified by PCR using fluorescently labelled (FAM, NED, or HEX; Applied Biosystems) reverse primers. PCR reactions were conducted in a 20-μl PCR mix containing 1× PCR GoTaq® Flexi Buffer (Promega), 1.5 mM MgCl₂, 200 μM dNTPs, 0.5 μM of each primer, 0.5 U of Taq polymerase and 1 μl of DNA template. Cycling conditions included an initial denaturation at 94°C for 5 min, followed by 30 cycles at 94°C for 30 sec, annealing (52-57°C, Additional file
2) for 30 sec, elongation at 72°C for 35 sec; and a final extension step of 5–10 min at 72°C. Amplified fragments were separated by capillary electrophoresis in an automatic sequencer (ABI3730, Applied Biosystems) and sizes scored using the software GeneMarker (SoftGenetics) and GeneMapper (Applied Biosystems).

After sampling, we stored adult males individually in 96% ethanol for preservation. Prior to DNA extraction, we evaporated the ethanol, and then transferred the mites into 1.5 ml tubes containing 50 μl of 5% Chelex solution (Bio-Rad Laboratories, Hercules, CA, USA) and 3–4 zirconium beads. We homogenized samples for 20 s using a Precellys24 tissue homogenizer (Bertin Corp, Rockville, MD, USA), and added 2.5 μl of proteinase K (20 mg ml⁻¹) to each sample, followed by incubation at 56 °C for 60 min, then denaturation of the proteinase at 95 °C for 8 min. We centrifuged samples at 14 000 rpm for 2 min, and stored them at -20 °C until amplification. We collected individual inviable eggs using an ethanol- and flame-sterilized pin, and crushed them in 10 μl of 5 × Phire buffer (Thermo Fisher Scientific, Waltham, MA, USA) in a PCR tube. Samples were centrifuged at 4000 rpm for 2 min and then stored at −20 °C. Prior to amplification, we pipetted the buffer-egg mix up and down to facilitate mixing of buffer and sample. To generate control samples for the PCR process, we followed the above processes without transferring a sample into the reaction medium.

All recombineering experiments were performed using pSIM6 as described previously^{40 (link),41 (link)}. Briefly, the recombineering cassette was electroporated in an induced E. coli + pSIM6 strain. Then, cells were left to recover overnight at room temperature before plating on selective medium. Single isolated colonies were stabbed in a new plate before homogenization in 100 µL of 5% w/v Chelex solution (Bio-Rad). Afterwards, the Chelex mixture was heated to 56 °C for 25 min and 100 °C for 10 min in a PCR machine for DNA extraction. For PCR screening, 1 µL of freshly extracted DNA was added to the PCR mix. Positive clones were sub-cultured in 5 mL of selective LB broth overnight and frozen in 25% glycerol for storage. This protocol was applied to the deletion of the pilS in TP114 and the deletion of dapA in the chromosome of EcN, which are further described in the Supplementary methods section.

Genomic DNA was extracted from a small piece of cooked skeletal muscle tissue with a 10% Chelex solution (Bio-Rad, Hercules, CA, USA; Walsh, Metzger & Higuchi, 1991 (link)). Two gene regions were amplified separately with PCR: 350 bp of mitochondrial cytochrome b (cytb) and approximately 412 bp of 18S ribosomal RNA (18S) using L14841 and H15149 (Kocher et al., 1989 (link)) and 18R399 (Struck, Hessling & Purschke, 2002 (link)) and Small Subunit F (Englisch & Koenemann, 2001 (link)) primers, respectively. These primer pairs are identical to those used in the restriction digestion protocol used here for cytb (Tagliavini, Harrison & Gandolfi, 1995 ) and 18S (Frankowski & Bastrop, 2010 (link)). PCR reaction volumes were 18 µL and included 9 µL of 2x MyTaq Ready Mix (Bioline, Inc.), 6.4 µL of H₂O, 0.3 µL of each primer (10 mM), and 2 µL DNA (approximately 50 ng/µL). The thermal cycling profile started with an initial denaturation at 94 °C for 3 min followed by 33 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for cytb and 52 °C for 18S for 30 s, with a slow ramp of 1 °C/s to an extension temperature at 72 °C for 3.5 min. The last cycle was followed by a single extension incubation at 72 °C for 5 min. All samples were successfully amplified. Amplification success was visualized on 1.5% agarose gel.

A loopful of 48 h culture of C. pseudotuberculosis was suspended in 6% Chelex-solution (Bio-Rad Laboratories, Hercules, CA, USA) and then heated to 65°C for 30 min and to 100°C for 8 min; afterwards, it was centrifuged. The supernatant was collected in a new tube and stored at −20°C until use. Fragments of the selected housekeeping genes were amplified by PCR in an Applied Biosystems 2720 Thermal Cycler (Thermo Fisher Scientific, Carlsbad, CA, USA). The 25 μl amplification reaction mixture contained 1x Dream Taq Buffer, 200 nM dNTP-mix, 1 μM of each primer, 1 U Dream Taq (Thermo Fisher Scientific, Carlsbad, CA, USA), template DNA, and water. The PCR program included 3 min of initial denaturation at 95°C and then 30 cycles at 95°C for 30 s, at 60°C for 30, and at 72°C for 30 s and 7 min of the final elongation. PCR products were purified for direct sequencing using the Geneaid PCR purification Kit (Geneaid Biotech, New Taipei, Taiwan). Templates were sequenced with the PCR primers using the BigDye Terminator Ready Reaction Mix v3.1. Nucleotide sequences were run on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Genomic DNA extraction was carried out on the enterotoxigenic C. perfringens AHL 311 control strain and on the C. perfringens reference strain ATCC 13124 using the InstaGene matrix protocol with a 10% Chelex solution (Bio-Rad, Mississauga, ON, Canada) in water [19 (link)]. Briefly, the control and reference strains were subcultured on sheep blood agar plates (Oxoid, Toronto, ON, Canada) under anaerobic conditions at 37 °C for 24 h. Ten to 15 colonies were recovered from each agar plate, and resuspended in 1 mL of sterile distilled water. The bacterial suspension was vortexed and centrifuged at 13,000 g for 3 min. The supernatant was removed and the pellet was resuspended in 200 μL of a 10% Chelex solution. The microcentrifuge tubes were treated at 56 °C for 30 min on a hot plate, and were subsequently placed in boiling water at 100 °C for 10 min. After boiling, the bacterial suspension was vortexed and centrifuged at 13,000× g for 3 min. The resulting supernatant was collected and stored at −20 °C.