Monthly Archives: December 2006

Protoplast Preparation

Here is a protocol for preparation of protoplasts. I first used it for C. graminicola while I was a postdoc in Lisa Vaillancourt’s lab at the University of Kentucky, but we’ve made a few modifications to it since then.

FCG LAB, 2005

1. Harvest conidia from 2 or more plates that are 3-4 weeks old, center inoculated. Expect about 1×108 conidia per plate.
2. Wash conidia once with sterile water. Resuspend the last time in Fries medium.
3. Count with hemacytometer.
4. Incubate in Fries medium for 48 hours with shaking at 30C. I use a 100 mL volume culture in a 250 mL Erlenmyer flask and shake at 150 to 175 rpm. Apparently if the culture receives too much aeration the culture will be induced to vegetative growth rather than the production of oval conidia.
5. Filter through Nytex membrane.
6. Count with a hemacytometer. Expect about 20X to 25X the number of starting falcate conidia.
7. Pellet in 50 mL centrifuge tubes in a bench-top centrifuge, 3000 rpm, 5 min at room temp. Resuspend in enzyme solution to a conidium concentration of 1.5×108/mL.
8. Incubate at 30C with slow shaking for 4-5 hours.
9. Pellet in a bench-top centrifuge, 3000 rpm, 5 min, 4C. Resuspend in 40 mL STC, ice cold. Keep the protoplasts on ice for the rest of the procedure. Count with a hemacytometer.
10. Pellet as in the previous step. Resuspend in STC medium to a concentration of 108/mL.
11. Divide into aliquots and freeze at -80C. I usually use 500 uL aliquots, which is enough for 5 transformations.

Enzyme solution
Glucanex*, 100 mg/mL dissolved in 0.7M NaCl.
Filter through a 0.2 uM filter before use. The solution will have a high solids content so only 6-7 mL of this solution can be passed through a 0.2 uM syringe filter.

*in the United States:
Novo Nordisk Biochem NA
State Road 1003 Box 576
Franklington NC 27525
Phone 800-879-6686
(note – this is no longer available directly from Novo Nordisk. Lysing Enzyme from Sigma (Catalog # L1412) is reported to be the same product.

STC 50 Ml

1.2 M Sorbitol (10.9 g)
10mM Tris (500 uL of a 1 M stock)
50mM CaCl2 (2.5 mL of a 1 M stock)
Adjust to pH 7.5