This protocol was submitted by Matt Kaeberlein.
Required Reagants:
Sporulation Protocol:
Tetrad Dissection Protocol:
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.