Grace’s Notebook: Crab Meeting

Today we had Crab Meeting #5. We discussed the new plan for using the lyophilizer (freeze drier) and Tri-Reagent protocol for extracting RNA. I recorded the meeting and will edit and publish tomorrow as a new DecaPod episode. I also went over to FSH building with Sam and he showed me how the lyophilizer works. On Monday, I will put the samples in and retrieve them on Tuesday, at which point they will be ready for Sam to extract RNA from and for me to run the lyophilized pooled sample on the Qubit.

Crab Meeting #5 and Lyophilizer Plan

Pam is back from her second (surprise) survey trip to the Bering Sea! We all met and discussed the pooled sample we have over at NWGC, as well as the new plan of testing out what protocols and methods will work best.

Plan:

  1. Lyophilize Monday-Tuesday the following samples:
    • 8 pelleted crab hemolymph (from day 26)
    • 8 RNAlater crab hemolymph supernatant (from day 26)
    • 1 pooled sample of the 25 samples from Sam’s Qiagen RNeasy extraction that had “out of range” Qubit results
  2. Sam –> extract RNA from lyophilized 16 samples using Tri-Reagent protocol
    Me –> re-quanitify RNA of the pooled sample using Qubit

We also have some extra tubes in the boxes that were mistakes, but still have some hemolymph in them (from two crabs) and some extra “test” samples that we can use to try out protocols on. I will find these and make sure we use them to test things out with.

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Ronit’s Notebook: Link to Spreadsheet and Labelling System Notes

Attached below is a link to the Excel spreadsheet for the C. Gigas exposure from 8/28-8/29. The column on the left indicates the oyster’s label (see labelling system below) and the subsequent column records the weight of the oyster. All oysters were weighed prior to shucking on 8/29.

Spreadsheet: https://docs.google.com/spreadsheets/d/e/2PACX-1vTHPB2YUKnaZ_-OKJR2fWATWSlcDFADULn_1pjz1_2BYh9gSpRW-VHKwpdCFmlvxfiR1C8g7DsAoxHx/pubhtml?gid=0&single=true

Labelling system: 

D01-D08: Diploid oysters exposed to control conditions (water in aquarium)

D09-D10: Diploid oysters exposed to control conditions (water in aquarium); subsequently exposed to 1 hr acute heat shock at 45 degrees Celsius.

D11-D18: Diploid oysters exposed to desiccation + elevated temperature (27 degrees Celsius) for 24 hrs

D19-D20: Diploid oysters exposed to desiccation + elevated temperature (27 degrees Celsius) for 24 hrs; subsequently exposed to 1 hr acute heat shock at 45 degrees Celsius.

T01-T08: Triploid oysters exposed to control conditions (water in aquarium)

T09-T10: Triploid oysters exposed to control conditions (water in aquarium); subsequently exposed to 1 hr acute heat shock at 45 degrees Celsius.

T11-T18: Triploid oysters exposed to desiccation + elevated temperature (27 degrees Celsius) for 24 hrs

T19-T20: Triploid oysters exposed to desiccation + elevated temperature (27 degrees Celsius) for 24 hrs; subsequently exposed to 1 hr acute heat shock at 45 degrees Celsius.