Yeast Tetrad Analysis



This protocol was submitted by Matt Kaeberlein.

Required Reagants:

Sporulation Protocol:

  1. Grow diploid cells overnight at 30C on YPD or synthetic media. Sporulation seems to be more efficient when cells are pregrown on YPD, so if you don't need to select for a plasmid do this.
  2. Innoculate cells heavily (should be cloudy) into 5mL of 2% KOAc.
  3. Add any amino acids that your strain is auxotrophic for. I add ~20uL of a stock 1% solution for each amino acid.
  4. Incubate at room temperature overnight on a roller or with shaking.
  5. Transfer tubes to 30C and incubate overnight on a roller or with shaking.
  6. Verify microcopically that you have tetrads.

Tetrad Dissection Protocol:

  1. Spin down 200uL of the KOAc solution with spores.
  2. Wash 1X in 500uL of water
  3. Resuspend in 200uL of zymolyase solution.
  4. Incubate for 10-20 min in a 37C water bath.
  5. Add 1mL water and place on ice
  6. Aliquot 25-50uL of spores onto a YPD (or other media) plate. Some people put the spores on one side and dissect tetrads across the plate, other people put the spores down the middle of the plate and dissect tetrads to both sides. I prefer the former, but you can fit more tetrads per plate with the latter.
  7. Allow the solution to dry into the plate.
  8. Under a microscope, try to find nice 4-spore tetrads that you are able to pick up with the needle without also picking up surrounding cells. Move to a predefined position (see tip#4 below) on the plate and deposit the tetrad. Set the needle lightly on the tetrad and tap the side of the scope. If your zymolyase treatment worked, the tetrad should split into 4 individual spores.
  9. Using the micromanipulator place the 4 spores far enough away from each other that the colonies won't grow into each other.
  10. Once you've done enough tetrads, place the plate at 30C and go home!

Tips:

You can make your stock sporulation solution already supplemented with the required amino acids, that way you don't have to add them to individual tubes each time. For example, make 250mL of 2% KOAc. If you have 1% stock solutions of each amino acid, add 1mL each of ADE, HIS, LEU, TRP, LYS, and URA (these are the most common markers). Some people claim that doing this reduces sporulation efficiency, but in the strains I've used that isn't the case.

I don't actually use the stock zymolyase solution. After the wash, I resuspend the tetrads in 200uL of water. Then I dip the end of a toothpick into the zymolyase powder and transfer a little bit to the eppendorf tube. We use Zymolyase-100T, Athrobacter luteus (100,000 U/g) from ICN Biomedicals, Inc.

Don't try to dissect tetrads on a wet YPD plate. It's much easier if the plate is a few days old. If it's too wet, it's very difficult to pick up the spores with the needle.

Most scopes have a series of numbers along the X and Y axes of the stage. I use 5 unit gradations both horizontally between individual spores and vertically between individual tetrads. For example, the spores from tetrad #1 would end up positioned at (160,10), (155,10), (150,10), and (145,10). The sprores from tetrad #2 would end up at (160,15), (155,15), (150,15), and (145,15), and so on.

At first, dissecting tetrads can be VERY VERY VERY frustrating. Don't give up, it gets much easier. If it's easy to pick up the tetrads/spores, but your having a hard time getting them off of the needle - it's not you, it's the needle. If you're going to be doing a lot of tetrads, it's definitely worth making a good needle. I wish I could tell you what the trick is for making a good dissecting needle, but I don't know. You'll know when you've got one! If anyone out there does know the secret email me.

After your tetrads have formed colonies, you'll want to genotype them by replica plating to various selective media to test for markers. There are a lot of ways to organize the data. Here's the standard form I use, just fill in your own markers and genes.