Maintaining subcultures:

Please check on the plates once a week, and subculture them once a month. I’ve attached a spreadsheet listing the plates that need to maintained- all of which are currently kept in a single incubator (top-right). Subcultures are plated onto quarter-strength PDA (7.6g PDA, 15g agar). Please copy the number from the initial isolate plate onto the subculture plate, also on the subculture plate add a letter indicating which subsequent subculture it is (eg 5230= initial isolate plate, 5230A= first subculture, 5230B= second subculture, and so on). Subcultures should be stored in a 10 deg C incubator.

Creating culture slants:

Please innoculate mini culture slants with fungi from plates. See the spreadsheet to see what plates correspond with what tube numbers. Culture slants are created using cryo tubes filled with 1 mL of PDA hard (36g PDA with 15g agar). Isolates are inoculated in the media and allowed to grow for a few days in the tube with a loose cap. Once the fungus has sufficiently colonized the surface of the media, the tubes are filled with 1mL of 20% glycerol and stored at -20C. Cryo tubes should be labeled on the top and side with the vial number. After you do this, could you update the website with this protocol and please add any additional info you feel is necessary.

PCR and sequencing:

All DNA extracts are kept in blue racks in the freezer. Primers which need to diluted are kept in shared boxes in the freezer labeled PRIMERS. Please make your own box with primer dilutions (or use your own from Jason’s lab). Exosap, extaq, etc. are in a box labeled SHARED.  Ladder and dye are in racks or sample boxes on the door of the fridge. I will provide a spreadsheet that details DNA extracts to undergo PCR, and the mastermix contents for the 25 uL reactions.

Asian ambrosia fungi

I’ll be sending back culture slants of fungi from Asia, which might need to be subcultured at DPI. Jiri or I will let you know what the needs are for this as they arise.

Adam,
Thank you in advance for your help with Latasha. Here is a summary of what we will do with Latasha on her first visit:

If she has beetles, we will identify them, and grind them up to extract symbionts. Here is how. We will plate them and wait a week.

If she doesn’t have beetles, or only species that are not easy to tell a story about, we will use our fungi.
– Raffaelea subfusca from Craig
– Ambrosiella hartigii from Martin Sigut
– Fusarium sp. from Martin Sigut
– Raffaelea lauricola from Marc
– Diplodia from oaks as a free-living control (or some better species?)

If these fungi are in decently clean cultures, we can subculture them directly into a competitive assay. If they need some cleanup, we will first subculture them alone.

Competitive assay: We will record single-direction reactions: each field has a record of the reactions of the fungus in the upper row. We also need reactions of each to itself, and a growth rate without a competitor. Can we do two replicates of each assay?

Better ideas welcome!
How should we score it? How about Strong inhibition, medium, weak, no effect? Or estimated percentage of growth decrease compared to the no-competitor control??
Thank you!