Culturing Xanthomonas
Murray Grant, Shannon Greer, Joana Vicente
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Abstract
This is the protocol for culturing strains of Xanthomonas species in preparation for extracting genomic DNA for sequencing.
Steps
Streak isolates onto King’s B plates. Incubate at 28°C for ~48 hours.
Pick a single colony and inoculate to 10ml King’s B liquid media in 30ml universal tubes. Incubate over night at 28°C and 220rpm.
Pellet 1.8ml of liquid culture in a 2ml Eppendorf tube by centrifuging at 5000g for 10mins. Pour off and discard the supernatant.
Pipette another 1.8ml culture into the tube and repeat step 3. This will pellet 4ml of culture in a 2ml Eppendorf tube.
Spin the tubes for 2mins at 5000g. Remove the supernatant by pipetting and discard.
Proceed to the extraction process or flash freeze the pellets in liquid nitrogen and store at -20C until use.
