Protocol 7: Picking colonies of transformed Spizellomyces punctatus (Sp)
Sarah M Prostak, Edgar M Medina, Erik Kalinka, Lillian Fritz-Laylin
Abstract
If transformation was successful, antibiotic-resistant Spizellomyces colonies should appear after 4-6 days of growth on selection media. These colonies should be rough, white, and opaque. When viewed under a microscope, there should be clustering of sporangia and some movement of zoospores. Picking these colonies and culturing them until there are enough cells for cryopreservation and to use in downstream molecular analysis is the last protocol in the entire transformation process. All steps here, with the exception of centrifugation steps, must be carried out in a sterile environment, either in a laminar flow hood or in the sterile area around an open flame.
Attachments
Steps
Steps
Divide one K1 plate with selection antimicrobials into four sections using a marker on the bottom of the plate.
Aliquot 50µL of DS into one microcentrifuge tube per colony to be picked.
Using an 18G needle, gently lift the colony of interest from the agar.
Resuspend the colony into the appropriate tube pre-filled with DS.
Pipette gently to mix and break up the pellet.
Pipette 50µL of the resuspended colony onto one quadrant of the K1 plate prepared in step 1.
Repeat steps 3-6 for each colony to be picked.
Let the cells grow for 2-3 days before rehydrating the quadrants with 100µL of DS and transferring each colony to its own quadrant of a new K1 selection plate.
After 2-4 rounds of colony expansion there is enough inoculum to transfer each isolated colony into its own plate.
Continuing subculturing each colony until enough culture exists to freeze and perform downstream analyses.
