These are my protocols for the D2O adaptation experiments. I’m making this post so I don’t need to write a post daily unless something out of the ordinary happens.

Making YPD:

1. Use autoclaved beakers and bottles for water handling and storage.
2. Measure water volume: D2O usually is supplied as a little over 90ml per bottle, DDW is about 100ml per bottle, and DI is readily available in whatever quantity needed.
3. Measure YPD powder and add 5g per 100ml of water.
4. Add a clean stir bar and stir (using hotplate/stirrer) until YPD is fully dissolved. For D2O and DDW stir at a low speed to minimize air mixture with solution.
5. Using a syringe and a filter, filter the YPD into the bottles. This step is necessary for D2O and DDW YPD to ensure minimization with H2O/D2O contamination. For DI YPD autoclaving is sufficient.
6. Label bottles and date.

Daily Prep:

1. For starter cultures, add 10ml of YPD to an autoclaved test tube. For daily measurements, add 9ml of YPD.
2. Inoculate yeast in liquid media. For daily measurements, inoculate 1ml of culture from the previous generation’s water type (ie add 1ml of 99% D2O yeast to 99% D2O YPD).
3. Add to incubator/shaker and incubate at 30C and 185RPM.

Nanodrop Measurement:

1. Aliquot a minimum of 400ul of sample into semi-micro cuvettes. Normally I measure at 0 hours and at 24 hours (or every 24h thereafter for prolonged experiments) for daily measurements and hourly starting with the 0h measurement for time trial experiments.
2. Blank the nanodrop with pure YPD and make sure to press the cuvette checkbox (I always forget this!).
3. Measure each sample.