1. Cut some mycelial plugs (like 5) from a 10 -14 days old PDA culture.
2. Place the agar plugs in 250 mL Erlenmeyer flask with 40 ml of Fries medium.
3. Add 5.2 microliters of HYGRO to each flask. Get HYGRO ( from stock 383 ug/ml) from fresh stock found in Fridge #1.
4. Place the flasks in a shaker for 48 hours at 30 C. Shake at 250rpm . For mutants it may take as many as 4-5 days.
5. Obtain a new 250 mL Erlenmeyer flask and add 40 mL Fries medium. Again add 5.2 microliters of Hygro to each flask.
6. Transfer culture to the new 250 mL Erlenmeyer flask by filtering the culture through a funnel with miracloth. The mycelium and spores will be filtered through and the agar plugs will be thrown away. If the mycelium has grown around the agar plugs, then vortex the bottle to loosen the mycelium.
7. Place flasks in a shaker for 48 hours at 30 C. Shake at 250 rpm.
8. Filter the culture using a Filter flask, ceramic funnel, whatman paper (size= ceramic funnel), and vacuum pressure.
9. Collect the tissue in preweighed 14 mL Falcon tubes. Collect between 1.4-1.7 grams of tissue in each tube.
10. Parafilm the tubes and poke three holes in the parafilm. Leave samples in -80C ( Freezer #1) for at least 30 minutes.
11. Take the samples to the Lypholizer (located in centrifuge room) and let them stay overnight.
12. Obtain glass rods and use DNA away to disinfect the rods. This avoids contamination of the samples.
13. Crush the tissue using the glass rod,making sure to lose as little tissue as possible.
14. Place caps on tubes and store in -80C (Freezer #1).
******* If you are using the wild type and need more tissue, use 1L Erlenmeyer flasks with 100 mL of Fries medium. It is not necessary to add Hygro to the flasks.