ApaLI restriction enzyme

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	Transconjugants containing one plasmid were extracted and analyzed by RFLP using ApaLI (TaKaRa, Dalian, China) digestion.

Protocol tips
Transconjugants containing one plasmid were extracted and analyzed by RFLP using ApaLI (TaKaRa, Dalian, China) digestion.

Publication protocol

Incompatibility (Inc) groups were assigned by PBRT. To analyze the location of the mcr-1 gene, S1 nuclease-PFGE and Southern blot analysis were performed. Briefly, whole-cell DNA of the donor strains and the transconjugants harbouring mcr-1 were extracted and embedded in agarose gel plugs. Subsequently, the agarose gel plugs were treated with S1 nuclease (TaKaRa, Dalian, China) and the DNA fragments were separated by PFGE. Southern blot hybridization was then performed with DNA probes specific for the mcr-1 gene, which was non-radioactively labeled with a DIG High Prime DNA labeling and detection kit (Roche Diagnostics, Mannheim, Germany). Transconjugants containing one plasmid were extracted and analyzed by RFLP using ApaLI (TaKaRa, Dalian, China) digestion. The genetic context surrounding the mcr-1 gene was investigated by PCR mapping and sequencing according to the plasmids sequences which had been submitted to GenBank. The primers used to determine the regions upstream and downstream of the mcr-1 gene are listed in Table 2. To assess the stability of mcr-1-positive plasmids, the original strains and transconjugants were cultured by in daily serial passages with the absence of colistin for two weeks.

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