This article details a method for genotyping a pathogenic bacterium, Yersinia ruckeri, using a sterile technique to ensure genetic homogeneity. The process involves isolating a single colony, lysing the cells, and extracting genomic DNA for further analysis.
Begin with colonies of a pathogenic bacterium grown on a nutrient-rich agar medium.
Choose a single colony, which ensures a genetically homogeneous population of bacterial cells and reduces variability during genotyping.
Using a sterile inoculation loop, transfer the selected colony into ultrapure water to minimize the risk of introducing contaminants into the sample.
Vortex the suspension to break the colony and disperse the bacterial cells uniformly.
Next, heat the suspension to disrupt the bacterial cell envelope, which consists of the outer membrane, peptidoglycan layer, and inner membrane.
This causes the bacterial cells to lyse and release intracellular contents, including genomic DNA, into the water.
Centrifuge the suspension at high speed to separate cellular debris from the genomic DNA.
Transfer the supernatant to a fresh tube for the downstream genotyping application.
To begin, use sterile inoculation loops to sow out Yersinia ruckeri pure cultures on any suitable agar type on Petri dishes. Incubate at 22 degrees Celsius for one to two days or 15 degrees Celsius for three to four days. After that, using inoculation loops, pick a single representative colony from each Petri dish and transfer to 1.5-milliliter centrifuge tubes, containing 50 microliters of ultra purified water to resuspend.
Vortex briefly and incubate for seven minutes on a heating block at 100 degrees Celsius. Then, centrifuge at 16,000 g for three minutes and use a pipette to carefully transfer the supernatant template DNA into empty 1.5-milliliter centrifuge tubes. Proceed to the next step or store the DNA at minus 20 degrees Celsius.
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