Lab day 2 - Homogenization & Sonication
Today’s lab work involved homogenizing the tissue in solution, then sonicating to lyse the cells.
Added 500 µl 50 mM NH4HCO3 + 6M urea solution that we made the day prior to each sample; homogenize tissue using pestle.
- I did not spin the sample down to remove supernatant, as I am only working with tissue. Should I have larvae, or other sample with shell, I’ll need to do that, as per the protocol.
- At this point I should have kept the samples on wet ice until sonication, but was following a protocol that did not outline that in detail. This needs to be updated in the protocol.
Walked over to the Genome Science Building with samples, dry ice, and wet ice. Working with Emma, made an ethanol dry-ice bath by getting a small plastic container and filling with ethanol to dip samples in one at a time. Slowly added small chunks of dry-ice until they are not dissolving very fast; kept adding dry ice throughout the sonication to maintain “bubbles” Sonicated each sample three times, but not in a row.
- First round: 5 seconds (should have been 10 seconds, but I had received conflicting direction).
- Second & Third round: 10 seconds
- After sonicating, dipped sample in ethanol + dry ice for 5s, and wedged the tube into wet ice until next sonication.
- Between samples, dipped the sonicating probe into 1) ethanol 2) nanopure, and 3) nanopure; rinsed again with ethanol, and dried with a kimwipe.
- After completed with sonication, kept samples on dry ice for ~1 hr, then moved to wet ice (realized that it needed to be thawed for the next step)
Brought samples back to our lab, and aliquoted 11ul of each sample to a new labeled tube for BCA assay. Saved remainder for mini-trypsin digestion. Stored all tubes at -80°C.
Next time, keep samples on wet ice throughout the day, rather than dry ice, to keep cold but not freeze