To distinguish the Inc types of possible plasmids, 2 or 3 colonies of fresh cultures of all K. oxytoca isolates were resuspended in 100 μl of nuclease-free water, heated to 95°C for 10 min, and centrifuged at 14,000 × g for 5 min. The supernatant was carefully removed and used for PCR. The PCR mixture contained 2× OneTaq master mix (New England Biolabs) and 0.2 μM each primer (Table 4 ) (39 (link)– (link)41 (link)).
The PCR conditions were set at 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 68°C for 1 min. The final extension was performed at 68°C for 5 min. The PCR products were analyzed using agarose gel electrophoresis on a 1% agarose-TBE gel and were stained with Midori green (Biozym Scientific GmbH).
The PCR conditions were set at 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 68°C for 1 min. The final extension was performed at 68°C for 5 min. The PCR products were analyzed using agarose gel electrophoresis on a 1% agarose-TBE gel and were stained with Midori green (Biozym Scientific GmbH).
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