Premise

Pilot calcium assay

I found a positive correlation between developmental stage and hemolymph calcium levels in female broodstock

Full run Calcium Assay

To investigate the effect of pH on hemolymph calcium levels and the correlation between reproductive stage and hemolymph calcium, I performed a colorimetric calcium assay on temporal hemolymph samples from geoduck broodstock reared under constant low pH (6.8)

https://docs.google.com/spreadsheets/d/10AC6N0UNAnLc6LoQY8UDl8lZkm2ZCZcVk63-SMPvopE/edit?usp=sharing

Hemolymph samples from animals with gonad histology:

  • Ambient: 11 animals with 3 time points and 10 animals with 1 time point
  • low pH: 10 animals with 3 time points and 12 with 1 time point

Total samples: 85

Run samples and standards in triplicate:

  • 96 well plate:
    • 24 wells for standards (8 standards)
    • 72 wells for samples (24 samples/plate)
  • need 85/24 = 3.5 plates

Assay set up

1: Prepare 1x Assay Buffer

  • 5mL of concentrated buffer + 45mL HPLC grade H2O
    • combined the 5mL concentrated buffer from each of the two kits and 90 mL nanopure H2O in a flask

2. Prepare standards

  • prepared 2x the volume the protocol described so:
    • A: 200uL assay buffer
    • B: 5uL Ca standard + 195uL assay buffer
    • C: 10uL Ca standard + 190uL assay buffer
    • D: 20uL Ca standard + 180uL assay buffer
    • E: 40uL Ca standard + 160uL assay buffer
    • F: 60uL Ca standard + 140uL assay buffer
    • G: 80uL Ca standard + 120uL assay buffer
    • H: 100uL Ca standard + 100uL assay buffer
  • added prepared standards to dilution plate wells 89-96 (column 12)

3. Prepare 1:4 dilutions of hemolymph

  • this took about 3 hours

  • locate samples
  • scrape out some frozen lymph into labeled eppy tubes using a new pipette tip for each sample
  • aliquot out 10uL of each into a 96-well PCR plate
  • add 30uL 1x Assay Buffer on top and pipette up and down to mix (careful not to touch the tips to anything! and just hover)
    • sample 01/23-037 only had 5uL so this was a 1:7 dilution rather than a 1:4 diluiton like the rest

4. Load assay plates

5. Prepare Working detection reagent

  • 37mL R1 + 37mL R2

6. Add reagent to wells

  • added 200uL to all wells using a multichannel pipette
  • sealed plates with plastic film provided in kits
  • incubated 5 minutes while I walked over to meet Emma at GS around 1:45pm

7. Read plates