Thermal Lysis-Based Genomic DNA Extraction from a Pathogenic Bacterium

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 a