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Yeast Spotting Assay To set up a yeast spotting assay, you'll need:

Single colonies or patches of your yeast strains, YPD plates (or other media), Toothpicks, ddH2O, 1.5 ml Eppendorf tubes, and possibly the use of the Nanodrop.

0. You want DRY plates for this. choose the older plates of those aailable, dry if neccessary.

1. Scoop about 10ul by eye of yeast from your colony or patch with a sterile toothpick. ALTERNATIVELY: start from a fresh YEPD overnight culture.

2. Propeller this into 200ul of ddH20 (Baxter). If you will be comparing multiple strains quantitatively, take 100ul of this 200ul, and put it into 900ul of baxter water to check its OD600 on the Nanodrop. See Measuring OD600 on the Nanodrop. ALTERNATIVELY: start with 200ul of a 1:10 dilution of your overnight instead of 200ul of resuspended yeast from a plate.

3. Take 2ul of this initial suspension into 198 ul (1:100 dilution) of ddH2O. If you are comparing multiple strains quantitatively, adjust the 2ul so that you add the same amount of each strain, normalized by OD600. If this last sentence doesn't make sense to you come ask Mark, Dan, Scott, or Brett; Mark and Scott have a spreadsheet template for this calculation as well.

4. From this initial 1:100 dilution, make five more 1:10 dilutions, 20ul into 180 ul ddH20. You will have 7 tubes: your initial suspension, and a series of 6 1:10 dilutions. Set the initial suspension aside.

5. Using either Dan's spotting assay template (ask Dan for it), or any 96-well plate, as a template for the correct spacing, add 3.5ul from each tube to create a series of six spots ranging across your dilutions. Move down one row and repeat for any replicates, other strains, etc. You can fit 6 rows of 6 spots onto a normal plate. N.B.: Do not put a and alpha strains onto the same plate.

6. The spots can run until they are dry. Do not move them or be prepared to move them VERY GENTLY. If your spots run together you will have to start over. If you aren't sure how gentle I'm takin bout, make a demo YPD plate with water spots to convince yourself how carefully you'd have to move them to not run the spots.

7. Put the plates into the 30C Incubator, and take pictures of them using the Plate Camera once every 24 hours for the next three days, or longer for slow-growing strains or conditions.

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