Tetrad Digestion and Dissection

— Ryla Cantergiani 2020/03/30 16:43

This protocol describes dissection of yeast tetrads. In our lab, we primarily use tetrad dissection for constructing strains for genetic and biochemical experiments.

• β-glucuronidase (Sigma G7770, Stored in 4°C refrigerator. Comes as an aqueous solution in ~1.0 M ammonium sulfate with 3 mM sodium azide as preservative.)
• Sporulated yeast culture
• Sterile Water
• YPD dissection plates - Draw a line on the left side of the plate ~2-3cm from the edge.
  • Everyone has a particular way that they like their plates for tetrad dissection. You are basically aiming for dry and level.
• Dissection scope

The spores are contained in a specialized cell wall called an ascus. In order to be able to separate the spores, the ascus is digested with the enzyme β-glucuronidase. Ideally, the ascus is digested just enough that the spores can be separated but not so much that the spores fall apart entirely.

1. Spin down 1 mL of sporulated culture at 3000rpm for 2 minutes. Don't forget to balance the centrifuge.
2. Remove the supernatant and resuspend cells in 200 μL sterile water by pipetting up and down.
3. Mix 17 μL washed culture with 3 μL β-glucuronidase (can vary by strain). Set up three tubes like this – you will stop them at different time points to check digestion.
4. Let sit at room temperature 15, 20, and 25 minutes. Stop the digestions as in step 5. (The ideal timing for digestion varies by strain, culture, and ambient conditions – if you are having trouble with your dissections, you may need to try less or more time.)
5. Gently add 200 μL water. Adding water too aggressively will break the tetrads apart and you will have no tetrads to choose from during dissection. The goal is to suspend the cells without breaking up the tetrads. Tap the tube gently to mix.
6. Plate 50 μL by placing drops along the line drawn on the plate. Tilt the plate so that the liquid drops spread over the marked off part of the plate. This spreads the tetrads for easy selection. Let the digested culture fully absorb into the plate.

1. Find a tetrad
2. Using the tetrad dissection sheet, go to the first set of coordinates. Break apart the tetrad, leaving one cell. Follow the coordinates, leaving one cell at each.
3. Repeat for twenty tetrads

The sporulation cultures are good for weeks, though the spore viability may start to decrease.

Before digestion, tetrads are held tightly in a tetrahedron and it is often difficult ot see all four spores at once. After digestion they relax into a diamond shape.

How to distinguish a tetrad from two budded cells adjacent to each other?

• Generally, the spores of a tetrad are smaller than a budded cell.
• Tetrads, if moved gently, will remain intact while two adjacent budded cells will not.
• The four spores in a tetrad are often very similar in size, while a budded cell usually has a large mother cell and a smaller bud.

An accomplished yeast biologist can dissect a plate of spores in 20 minutes; for a beginner, 2 hours is not unusual. Tetrad dissection is a learnable skill. Your initial attempts will likely be frustrating. If you persevere, you will be rewarded by your new ability to wield one of the most powerful tools in the yeast geneticist's toolbox. When you first learn to do tetrad dissection, make sure to ask for help from someone in the lab who is experienced at doing it! It really helps to have someone with a practiced eye point out what a well-digested culture looks like, what a tetrad like under the dissecting scope, etc.

You cannot restart a tetrad plate. If for some reason you cannot complete your plate, place the plate in the 30°C incubator to grow up. Start a new plate.

β-glucuronidase (Sigma G7770, Stored in 4°C refrigerator. Comes as an aqueous solution in ~1.0 M ammonium sulfate with 3 mM sodium azide as preservative.