Today, I finished all Oly larval measurements. The ROI sets are currently on Emu (in the Roberts lab). I will upload them to Github either at the end of this week or when my laptop is repaired.
I also helped dissect oysters for Ronnit’s experiment. We shucked three groups of oysters with each group containing diploids and triploids. They were either dessicated at room temperature or under heat. We took gill, mantle, and abductor tissue for qPCR and collected remaining tissue (excluding the gonad and visceral mass) in a ‘whole body’ sample. All samples are being stored in the first -80 freezer. The whole body tissues are on the final shelf while the other samples are on the 3rd shelf- not in a drawer.
Plan for another protocol test
Time to try this again. At lab meeting, we decided to try to get at least 1 ng/µL of DNA because that’s apparently all I would need for a Qiagen assay. I can’t use the Genome Sciences Thermomixer anymore, so I’m improvising. Steven and Sam suggested that I use a heating block to incubate the tubes at the proper temperature. I would keep the tubes on the heating block for a few minutes, then vortex at maximum speed for a few minutes. I would keep alternating until I kept the tubes on the heating block for at least an hour. I might need to extend the incubation time a bit longer to ensure the proteinkinase K digestion happens properly.
Based on my previous trial, I determined that the Tissue Tearor and two minute vortexing with glass beads provided the most consistently high DNA yield. To recap:
- Tissue Tearor at setting 2 for 20 s (Note: Laura said she used the Tissue Tearor at setting 15 in this lab notebook. She must have used one different than what’s in FSH 213, because there was no setting 15 on the one I used).
- Glass beads + vortexing for 2 minutes
I’m going to use three tissue samples, split between two tubes (each tube corresponds with each method). I don’t want my tube labels to overlap since I’m going to run these samples and the samples from the previous trial on the Bioanalyzer together. Here are what my tube labels will be:
I’m also not going to eliminate RNA from the samples. I would like to do both bisulfite sequencing and RNA-Seq with each sample, so I want to see if the protocol still works.
Kaitlyn’s helping me out tomorrow, so hopefully this will go smoothly! I’d really like to successfully extract DNA that can then be used for bisulfite sequencing :]
from the responsible grad student https://ift.tt/2CbOb6r
Yesterday (8/28), we set up a 24 hr exposure with triploid and diploid C. Gigas oysters to an elevated temperature + desiccation stress. In total, we had 40 oysters (20 diploid, 20 triploid). 10 control animals were kept for both the diploid and triploid animals in mesh bags in the aquarium in regular seawater. We had to place a foam cover over the mesh so that sea stars wouldn’t get in and potentially interfere with the controls. The other 20 animals (10 diploid, 10 triploid) were transferred to an incubator at 27 degrees Celsius where they were also kept in mesh bags. Exposure began at roughly 12:00 PM. Additionally, we decided to take 2 animals from both the control and the desiccation + elevated temperature exposure at 24 hrs to expose to a 1 hr acute heat shock at 45 degrees Celsius (for a total of 8 oysters exposed to acute heat shock). Thus, our sample size is N = 8 for the diploid/triploid controls and the diploid/triploid desiccation + elevated temperature treatment group.
Originally, we were planning to do a factorial stress response experiment, examining the effects of hypoosmotic stress (low salinity), desiccation + elevated temperature, and the two stressors combined. However, we ultimately decided to go with a simpler experimental design that would be more robust and could give us a baseline stress response to work with. I’m still interested in investigating interactive effects between multiple stressors, especially in triploids, so once we have this baseline single-stressor trial completed, we can definitely think about doing a factorial design!
Today (8/29), we’ll be sampling the oysters at the 24 hr mark, collecting mantle, adductor muscle, and gill tissue for follow-up RT-qPCR. At around noon, we’ll take 2 oysters each from the diploid/triploid control and treated groups to do an acute heat shock at 45 degrees Celsius for 1 hour.