Karissa Milbury
posted 21 days ago
A.K.A. Point Mutation 🔬

Dilution spot assay for yeast

This is a quick run-through of the protocol to make dilution spot plates for S. cerevisiae. This assay is great for identifying broad fitness defects but isn't quantitative.

Note that temperatures for temperature sensitive strains will need adjustment from the standard 30°C. Even if you aren't aware of any temperature-related defects, it's easy to check and nice to add -- I like to make several replicates of each plate and grow them in parallel at multiple temperatures.

You can also plate each set of strains to multiple media types. For my purposes, looking for drug sensitivity, I often plate from a base media (like YPD or synthetic complete) as well as media + drug. In these cases when using a replicator I stamp to the base media plate first, then drug plates, as I'm a little paranoid about carry-over (e.g. don't want to pick up residual drug on the replicator and then put it back into the culture). This can also be addressed by washing between each stamp.

You can also use this assay to identify and isolate suppressor colonies, i.e. drug-resistant cells caused by reversion or secondary mutations, that can be further analyzed by backcrossing or your downstream analysis of choice.

  1. Grow yeast overnight in 5 ml liquid medium (e.g. YPD) at 30°C.
  2. In the morning, check the OD600, then dilute each culture to 0.1 OD in YPD. Allow to grow until they return to log phase (3-4 hrs at 30°C).
  3. Meanwhile, ensure that media plates that you'll be spotting to are dry; older plates seem better for this. Wet plates will cause the spots to run into each other. Consider marking a spot on the edge of the plate as the corner where you'll align the first sample of the first strain, so you can make sure everything is oriented properly when you take pictures later.
  4. Measure OD again and dilute cultures to 0.1 OD in 200µL YPD into the first column of a 96-well plate. You can dilute to higher concentration if desired, but be consistent across samples.
  5. Pipette the dilution series: start with your 0.1 OD cultures, then dilute 1/10, then 1/10 from that, etc into each subsequent column until you have minimum 4 dilutions of each culture. The max you'll be able to fit on a standard 10cm plate is 6 dilutions for 8 samples.
  6. Drop dilutions onto appropriate plates using one of two methods: A) A multipipette. Create a grid of descending cell concentrations. Drop size depends on dryness of plates and medium used: usually between 2 and 5 µl. Make sure the tips are well aligned. B) Alternatively (preferably) use a pin tool (also called a replicator or "frogger": https://www.fishersci.com/shop/products/boekel-replicators-2/054509) to stamp from the 96-well to the plate. Prior to stamping, wash the pins by dunking in water, then 100% EtOH, and then passing over a flame. Let cool for 5-10 seconds before stamping so you don't roast your cells.
  7. Once dilutions are plated, leave the plates face-up on the bench until liquid dots have dried. This may happen quickly or take hours, depending on plate wetness and lab humidity. If you absolutely must get them into an incubator before this is complete, you can CAREFULLY move them in there and leave face-up, but be sure to flip them once dry.
  8. Incubate plates at appropriate temperature for your experiment.
  9. Take photographs at different days of incubation, as differences between strains may become more or less apparent as the spots grow to completion. We often used a standard computer scanner; dark backgrounds work well, and it looks best if you can block out as much of the ambient light around the scanner as possible. If the scanner top can't come down properly with plates on it, put a box over it. Also note that warm plates will quickly fog up the scanner surface, so consider letting the plates rest at room temp for a little while before scanning.
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