McClean: Registering objectives on the Nikon

Overview

To use the PFS (perfect focus system) motorized nosepiece the objective information needs to be correctly registered on the microscope. Currently the objectives that we use most often (100x Plan Apo VC oil; 40x Plan Apo dry) are correctly registered. The 40x is registered to position 4 (and 1) and the 100x is registered to position 3 (and 2,5,6).

If you add a new objective that is compatible with the PFS, you will need to follow the procedure below to register it.

If the PFS is not working, it may be that the objectives are not registered correctly. Check the front display of the microscope to make sure that it displays information on the objective you are using (PA40x or PV100x). If it does not it is likely that the objectives are not registered or are not registered to the position you are using on the objective turret. To fix this, follow the directions below to register the objective you want to use to the appropriate turret position.

Protocol

• Let streaks grow 2 days 30°C.
• Inoculate overnight YPD cultures of the mating type testers.
• Dilute the overnights 1:10 with sterile media or water.
• Spread ~200 μl lawn on YPD plates, one for each mating type.
• Incubate 30°C for 30 minutes.
• Pin your strains of interest to the lawns, or use a toothpick to make small, well-separated patches on the lawn. Make sure to flame the frogger between each plate, or use a fresh toothpick for each plate. Do NOT pin onto tester plates thate are still wet. This will just cause your patches to run all over the plate.
• Incubate 30°C overnight.
• Score whether or not the patch has a halo of space around it. If it does, that means that the lawn strain responded to the pheromone emitted by the patch, and thus that they are of opposite mating type. So, if a halo formed around the patched strain on the a tester plate, the strain itself is alpha. The nice thing about having both the a and the alpha tester plates is that you can double-check your scoring by making sure there is a halo on only one tester plate.

References

Adapted from Maitreya Dunham's protocol (Dunham Lab Protocols) which was adapted from Katja Schwartz's protocol from the Botstein lab (Botstein Lab Protocols)

The strains were made in the Thorner lab. See Julius et al (1983) Cell, 32, 839-52 for documentation of DBY7730. DBY7442's exact genotype is unknown. The sst mutations make them super-sensitive to pheromone. When exposed to pheromone of the opposite mating type, the cells arrest.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

• Megan N McClean 11:02, 3 November 2011 (EDT) We have been having some trouble lately with the yMM422 strain. Strains of the opposite mating type have not been forming scorable halos. We may want to try fresh stock from the Botstein lab's -80°C stock. Alternatively, it seems to work 'sometimes' and since none of us are that careful about how many cells we seed or what growth phase they are in when we do so, perhaps we should play around with that and then be consistent in the future.

Contact

• Megan N McClean 14:01, 03 November 11(EDT)

or instead, discuss this protocol.