Extraction of Genomic DNA from Plant Leaf Tissue Infected with Pathogenic Bacteria

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
Wizard Genomic DNA Purification Kit	Promega	A1120	Source of Nuclei Lysis, RNase, Protein Precipitation, and DNA Rehydration solutions
Liquid nitrogen	Air Products	N/A
Mastercycler pro with control panel	Eppendorf	6321000019
1250 W Microwave	Panasonic	N/A
5424 table top centrifuge	Eppendorf	05-403-93
Isotemp 215 digital water bath	Fisher scientific	15-462-15Q
Metal spatula	Sigma-Aldrich	Z283274
Mortar and pestle	Sigma-Aldrich	Z247464
LSE Vortex Mixer	Corning	6775
2-propanol	Sigma-Aldrich	I9516
Sterile 10 μL tips	TipOne	1161-3730
Sterile 200 μL tips	TipOne	1163-1730
Sterile 1250 μL tips	TipOne	1161-1750
Variable Volume Pipettor Kit	VWR	75788-460
Ethidium bromide	Sigma-Aldrich	E1510
Agarose	IBI Scientific	IB70041
EDTA	Thermo Fisher Scientific	17892
Acetic acid	Fisher Chemical	A38-212
Tris base	Sigma-Aldrich	10708976001
μcuvette	Eppendorf	6138000018
Biospectrometer basic	Eppendorf	6135000009