Protocol 6: Selecting for Spizellomyces punctatus transformants
Sarah M Prostak, Edgar M Medina, Erik Kalinka, Lillian Fritz-Laylin
Abstract
Once the co-culture IM plates have grown for four days, it is time to select Sp transformants. Selecting these transformants requires harvesting the cells from the co-culture plates, dislodging and removing any remaining Agro from the culture, and plating Sp onto K1 selection media. In our laboratory, we use 300 mg/L Hygromycin B (Gibco #10687010) as our selection marker. Additionally, we add 50 mg/L Carbenicillin (Carb) and 50 mg/l Tetracycline (Tet) to our K1 plates to kill Agrobacterium. All steps here, with the exception of centrifugation steps, must be carried out in a sterile environment, ideally in the sterile area around an open flame.
Attachments
Steps
Steps
For each plasmid transformed, prepare a 50 mL conical with 30mL of DS solution.
From the appropriate conical for the plate to be harvested, remove 1mL of DS.
Distribute the DS among the four quadrants of the corresponding IM plate.
Incubate at Room temperature for 0h 5m 0s.
Using forceps, sterilize both sides of a single edge razor blade with the flame.
Tilt the rehydrated IM plate and gently scrape the surface of the agar and collect the growth into the DS pooled at the bottom.
Rotate the plate and scrape along the agar until most of the opaque areas on the plate are gone.
Use a 1000 μL pipette tip to scrap any remaining growth from the razor blade and resuspend the growth to the appropriate conical tube filled with DS.
Aspirate all of the liquid from the scraped plate and slowly dispense the liquid back into the appropriate conical tube.
From the top of the density gradient, remove 1mL of DS.
Wash the surface of the scraped IM plate several times with this fresh DS.
Return the volume back to the conical.
Invert the conical 3 times.
Vortex the conical for 1-2 seconds to dislodge any remaining Agro from Sp cells.
Repeat steps 2-14 for each plasmid transformed (i.e, for each IM plate you have)
Centrifuge the conical(s) for 2000rcf.
Check for the presence of a pinkish pellet, this is the chytrid Sp.
Gently pour off the supernatant with the high side of the pellet facing up.
Gently resuspend the pellet into 500µL of fresh DS.
Pipette 200µL of the resuspended Sp onto a K1 plate with selection antibiotics.
Spread the cells using 4-5 sterile glass beads.
Once the plate is dry, remove the glass beads.
Seal and incubate plates in a humidity chamber at Room temperature for 4 days or until colonies appear on the plate.
