Cold Acclimation Green Crab Experiment Part 37

Continuing with Catlin’s crabs

Back in the lab after my self-imposed timeout to write (and some annoying allergies-turned-sinus issues)! Let’s see how quickly we can breeze through Catlin’s remaining samples.

Methods

DNEasy Extraction:

  1. Set the benchtop incubator to 56°C.
  2. Wipe down the bench surface, pipettes, and racks with RNase Away.
  3. Take out and label one 1.5 mL tube for each sample.
  4. Add 180 µL Buffer ATL to each sample tube.
  5. One at a time, cut a small amount of tissue from each sample (~1mm x 1mm) and place in the corresponding labeled 1.5mL tube. Make sure to ethanol and flame tools in between samples. If the specimen is stored in ethanol, make sure to blot off all ethanol before adding tissue to the labeled tube with Buffer ATL.
  6. Add 20 µL Proteinase K to each sample. Mix samples by vortexing and quick-spin tube(s).
  7. Place tube(s) in incubator at 56°C until tissue is completely lysed. Ideally, the incubation will be overnight. If opting for an incubation that isn’t overnight, set an initial timer for 90 minutes and vortex tube(s). If tissue appears to not be fully dissolved, continue to incubate samples and vortex every 15 minutes until tissue has dissolved.
  8. After incubation and complete lysis, vortex sample(s) for 15 seconds and quick-spin tube(s).
  9. Add 200 µL Buffer AL to each sample. Mix thoroughly by vortexing and quick-spin tube(s).
  10. Incubate sample(s) at 56°C for 10 minutes.
  11. While samples are incubating, take out and label one new 1.5 mL tube for each sample.
  12. Add 200 µL 200 Proof Ethanol to each sample. Mix thoroughly by vortexing and quick-spin tube(s).
  13. Pipet the entire sample volume (600 µL) into a labeled DNeasy Mini Spin column placed in a 2 mL collection tube (supplied). 14. Centrifuge for 1 minute at ≥6000 xg (8000 rpm). Discard the flow-through and collection tube.
  14. Place the spin column in a new 2 mL collection tube (supplied). Add 500µl Buffer AW1 to each sample. Centrifuge for 1 minute at ≥6000 xg (8000 rpm). Discard the flow-through and collection tube.
  15. Place the spin column in a new 2 mL collection tube (supplied). Add 500 µL Buffer AW2 to each sample and centrifuge for 3 minutes at 20,000 xg (14,000 rpm). Discard the flow-through and collection tube.
  16. Transfer each spin column to the correct labeled 1.5 mL tube (not supplied).
  17. Elute the DNA by adding 60 µL Buffer AE directly to the center of each spin column membrane. Dispense Buffer AE with pipette tip as close to the membrane as possible, without touching the membrane with the tip. It is strongly recommended to use a new tip for each sample.
  18. Incubate for 1 minute at room temperature (15°C - 25°C). Centrifuge for 1 minute at ≥ 6000 xg (8000 rpm).
  19. Take the eluted 60 µL and add it back to the membrane. Incubate at room temperature for 1 minute then centrifuge for 1 minute at ≥6000 xg (8000 rpm).

PCR:

  1. IF STARTING HERE: Use RNAse AWAY to clean bench space, pipets, tip boxes, etc.
  2. Label either an 8-strip PCR tube or a 96-well plate with sample ID and “P” (ex. 3P) and a 1.5 mL eppendorf tube “PCR MM.” Be sure to label an extra tube for the PCR blank. Label two tubes for a known CC and a known TT crab.
  3. Calculate the amount of reagents needed for PCR master mix for samples, a PCR blank, and two extra reactions.
    • GoTaq: 12.5 µL/sample
    • F primer: 2.5 µL/sample
    • R primer: 2.5 µL/sample
    • DEPC H2O: 5.5 µL/sample
  4. Preheat the thermocycler with the SMC_60RD PCR protocol
  5. Get ice and thaw PCR reagents and DNA at room temperature. Briefly vortex, spin down, and move onto ice as soon as reagents thaw.
  6. IF NEEDED, make new F and R primer aliquots. Take the 100 µM stock bottles (blue lids) and dilute into a new, labelled 1.5 mL eppendorf tube in a 1:10 dilution (ex. 10 µL primer stock, 90 µL NF water).
  7. Make master mix based on calculations in step 3 in the labelled PCR MM tube. Vortex, spin down, and keep on ice.
  8. Aliquot 23 µL of PCR MM from step 6 in each tube for the samples, extraction blank, and PCR blank.
  9. Once the DNA thaws, briefly vortex DNA and spin down. Place on an ice block tube holder.
  10. Add 2 µL of DNA into each sample tube. Add 2 µL DEPC water for the PCR blank.
  11. Seal wells completely. Use tube/well caps, foil plate seal, or Microseal A film to seal plates. Use roller to push down seal and use the plate sealer tool to ensure all wells are completely sealed.
  12. Vortex the sealed tubes and briefly spin down.
  13. Place in the thermocycler, close lid, and run the SMC_60RD PCR protocol.
  14. When protocol is complete, either place product in the 4ºC or proceed to the restriction digest.

Restriction Digest:

  1. Preheat the thermocycler with the SMCRD_IN incubation protocol
  2. Obtain Alul enzyme from the -20ºC. VERY gently vortex, spin down, and place on ice.
  3. Add 0.5 µL enzyme to each PCR reaction. Pipet up and down to mix
  4. Briefly spin down
  5. Place in thermocycler, close lid, and run the SMCRD_IN protocol
  6. When incubation is finished, either place product in the 4ºC or proceed to gel imaging.

Gel:

  1. Obtain product for gel, ladder, and TriTrak from the 4ºC fridge and move into gel room.
  2. Microwave pre-made 1.5% agarose gel mix in the microwave for 3 minutes in 1.5 minute intervals. Be sure to swirl the bottle to mix the gel liquid. If gel mix is needed, make a 1.3% gel.
  3. Allow the bottle with gel mix to cool for a few minutes. While cooling, tape off sides of the gel tray.
  4. After bottle has cooled enough to the touch but the gel mix is still in liquid form, pour the mix into the taped gel tray slowly to avoid bubbles. Use combs to make wells by slowly inserting them into the gel to avoid bubbles.
  5. Allow gel to harden for at least two hours.
  6. Once gel is hardened, place in gel box with 1x TAE buffer. If an extra gel was made, slide and place in a Ziploc bag with TAE buffer. Place that Ziploc bag in the same drawer as the pre-made gel mix, away from the light.
  7. For each sample, pipet 1.5 µL of TriTrak dye by eye (above the first gradation on the pipet tip and about a third of the way to the second gradation) directly into the sample tube. Mix up and down at least 10 times, then load 10 µL of restriction digest product into the well. Pipet a second 10 µL of restriction digest product into the well for a total of 20 µL restriction digest product in the well.
  8. Add 3 µL of diluted ladder into the designated well.
  9. Run gel for 65 minutes at 90 V.
  10. After running gel, remove gel tray and image. If needed, run gel for longer in 5-10 minute intervals. If the image isn’t as bright or contrasted as it is on the computer, adjust the settings on the thermal printer and print again.

2025-03-20

Notes

  • PCR, restriction digest, and gel: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36
    • PCR Master Mix calculations
      • GoTaq: 12.5 µL x 27 = 337.5 µL
      • F: 2.5 µL x 27 = 67.5 µL
      • R: 2.5 µL x 27 = 67.5 µL
      • NF H2: 5.5 µL x 27 = 148.5 µL
    • Made new R primer
  • Started DNEasy extractions: WA 152 (my last sample to genotype), 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58

Results

Image

Figure 1. Gel image for samples

I have a couple more potential TTs with false bottoms! I’m keeping track of them so I can redo them at the end.

2025-03-21

Notes

  • PCR, restriction digest, and gel: WA 152 (my last sample to genotype), WA 257 (redoing PCR), 37 (previously extracted), 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58
    • PCR Master Mix calculations
      • GoTaq: 12.5 µL x 27 = 337.5 µL
      • F: 2.5 µL x 27 = 67.5 µL
      • R: 2.5 µL x 27 = 67.5 µL
      • NF H2: 5.5 µL x 27 = 148.5 µL
    • Made new F primer with new IDT stock
    • Made new R primer
    • Used new GoTaq and new NF H2
  • When running the restriction digest, I forgot to press start on the thermocycler program! The samples sat at 37ºC for a little over an hour before I got to them.

Results

Image

Figure 2. Gel image for samples

I ended up with an abnormally blurry top row! I thought I loaded it fast enough, but maybe not. I ran the gel for an extra 5 minutes (70 minutes total), which I think helped clarify some of the smudge. There are samples that I’m pretty sure are CTs even though they’re blurry because there’s an “underline” where that CC band should be. I have a handful more potential TTs with false bottoms that will need to be re-run for clarity.

Going forward

  1. Genotype Catlin’s samples
  2. Add Catlin’s methods and results to manuscript
  3. Analyze 2024 HOBO data
  4. Analyze Julia’s HOBO data
  5. Analyze Catlin’s HOBO data
  6. Create an experimental setup figure
  7. Outline introduction
  8. Outline discussion
  9. Determine methods for comparing population responses
  10. Troubleshoot lipid assay protocol
  11. Conduct lipid assay for crabs of interest
Written on March 20, 2025