Thermal Lysis-Based Genomic DNA Extraction from a Pathogenic Bacterium

Abstract

Source: Gulla, S., et al. Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis. J. Vis. Exp. (2019).

This protocol extracts genomic DNA from Yersinia ruckeri by suspending isolated colonies in ultrapure water and applying heat to lyse the cells. Centrifugation separates the DNA-containing supernatant from cell debris, yielding template DNA suitable for PCR-based genotyping and epidemiological analysis.

Protocol

1. Bacterial Cultivation and Extraction of Genomic DNA

1. Sow out Y. ruckeri pure cultures on any suitable agar type (the authors used 5% bovine blood agar) and incubate at 22 °C for 1-2 days, or 15 °C for 3-4 days.
2. From each agar plate, pick a single representative colony with an inoculation loop and transfer to 1.5 mL centrifuge tubes containing 50 µL of ultrapurified water. Suspend, vortex briefly, and incubate for 7 min on a heating block at 100 °C.
3. Centrifuge at 16,000 x g for 3 min and use a pipette to carefully transfer the supernatant into an empty 1.5 mL centrifuge tube. Proceed to the next step using the supernatant as template DNA or store at -20 °C until such time.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
22°C/15°C incubator	As preferred.	NA
Centrifuge(s)	As preferred.	NA
Eppendorf Safe-Lock Tubes, 1.5 mL	Eppendorf	30120086	Centrifuge tubes used during DNA extraction.
Freezer	As preferred.	NA
Heating block	As preferred.	NA
Milli-Q water	NA	NA	Purified water used during PCR and capillary electrophoresis. Standard recipe; produced in-house.
Pure culture of Yersinia ruckeri	NA	NA	E.g. cryopreserved or fresh.
Vortexer	As preferred.	NA