McClean: Tetrad Dissection
This protocol describes dissection of yeast tetrads. In our lab, we primarily use tetrad dissection for constructing strains for genetic and biochemical experiments.
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 richly 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 looks like under the dissecting scope, etc.
- 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
- "Dry" YPD dissection plates
- Everyone has a particular way that they like their plates for tetrad dissection. You are basically aiming for dry and level. To make dissection plates, add 25 ml YPD with a plastic strippette to plates on a very level surface. Once solid, invert. Let dry at room temperature for ~3 days. Bag. These plates are best after aging for a while.
Stock Solution 1
- This is a very simple solution, so we only need a one line description of how to make it.
Stock Solution 2
This is a more involved solution, so we will describe how to make it in several steps:
- Step 1
- Step 2
- Step 3
- Step 1
- Step 2
- Step 3
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- *Megan N McClean When I can't age my dissection plates on my bench for a few days, I will stick them in the 30°C or 37°C warm room the morning of the day I dissect to dry them out a little bit. It is absolutely infuriating, if not impossible, to try dissection on plates that are wet.
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- *Megan N McClean Different tetrad dissection protocols call for using different enzymes to digest the ascus wall. Our protocol uses a β-glucuronidase from Sigma (G7770) which is a mixture of enzymes derived from Helix pomatia (the Roman snail). Zymolyase, another commonly used enzyme, consists mostly of β-1,3-glucan laminaripentaohydrolase. It hydrolyzes glucose polymers at the β-1,3-glucan linkages releasing laminaripentaose as the principal product. β-glucuronidase catalyzes hydrolysis of β-D-glucuronic acid residues from the non-reducing end of mucopolysaccharides (also referred to as glycosaminoglycans).
Adapted from Maitreya Dunham's protocol (http://dunham.gs.washington.edu/sporulationdissection.htm) and the Botstein lab protocol (http://www.princeton.edu/genomics/botstein/protocols/Sporulation_and_Tetrad_Dissection.pdf)
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.