McClean: Frogging Tetrads
This is a protocol for scoring tetrads (for mating type, auxotrophies, drug resistance, etc) by "frogging" them from the "frog-pond" (a 96-well plate containing your spores in individual wells) onto different selective plates.
- Sterile "Frog Ponds" (96-well Costar #3370 Flat-Bottom polystyrene plates)
- Sterile toothpicks
- Sterile water and reservoir
- Multichannel pipetter and sterile reservoir or repeat pipetter
- Frogger and Petri dish half full of EtOH (95%) (This MUST be GLASS!!!)
- Overnight cultures of yMM421 and yMM422 for testing mating type
- Fill the "frog-pond" with sterile ddH2O or YPD. Use YPD if you want to freeze down this plate as a backup at -80°C, otherwise water is fine. There are two convenient ways to fill the pond:
- Fill a sterile plastic reservoir with water or YPD. We keep these in the "cryostorage" drawer and the main stock is in Room 115 on the top shelf. Use a multichannel pipetter with 8 pipet tips to fill the 96-well plate or “frog pond” with 150µl sterile ddH2O or YPD in each well. Repeat with additional plates if necessary.
- Transfer 50ml of YPD or water into a 50ml conical. Use the Eppendorf repeat pipettor ("pipette" drawer) and the appropriate disposable tip (on top shelf in the main room) to dispense 150 μL of sterile H2O or YPD into each well.
- Using a sterile toothpick, select one spore from a tetrad and inoculate a well. Inoculate each well with its own spore. Record the pattern in which the tetrads were inoculated.
- Sterilize the frogger by dipping the prongs into EtOH. KEEP the EtOH AWAY from the flame!!! After a quick shake insert the prongs into a Bunsen burner flame. Be CAREFUL not to burn yourself. Do NOT allow flaming EtOH to drop into your dish of EtOH. KEEP THEM SEPARATED!! Allow the frogger to cool slightly (1 minute). If you do not allow the frogger to cool enough, you will kill your cells and the frogging won't work well.
- Align the prongs of the frogger with the wells and then place the prongs into the wells. Wiggle the frogger within the well to ensure that the culture in each well is uniformly mixed. Stamp frogger firmly but gently onto an agar plate. Repeat this step to stamp additional plates.
- To test auxotrophies: Stamp the cells onto each dropout media (e.g. SC-URA, SC-LEU, SC-HIS, etc) of interest to determine genotype. Allow to grow 2-3 days at 30°C and compare extent of growth. Carefully monitor the plates during their growth. It is a good idea to always stamp onto a YPD plate last to make sure that everything grows up on the YPD plate (indicating that you have transferred a sufficient number of cells to all of the SC- plates).
- To test mating type: Prepare plates seeded with mating type tester as described in: Yeast Mating Halo Assay. Stamp the spores onto the plate seeded with yMM421. Sterilize the frogger to ensure accurate mating type determination. Then dip the frogger back into the frog ponds and stamp onto the plate seeded with yMM422. Allow cells to grow at 30°C and monitor halo formation.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
Adapted from the Goode Lab protocol: The Goode Lab at Brandeis University
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.