Cold Acclimation Green Crab Experiment Part 34

Finishing WA crabs and starting on Catlin’s crabs

Home stretch? I hope?

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.

2025-03-03

I lied to myself last week. I had 15 samples I needed to re-PCR today! These samples were too faint in their original run.

Notes

  • PCR and restriction digest for WA samples: 163, 169, 170, 178, 184, 185, 193, 194, 200, 206, 214, 226, 239, 253
    • PCR Master Mix calculations
      • GoTaq: 12.5 µL x 16 = 200 µL
      • F: 2.5 µL x 16 = 40 µL
      • R: 2.5 µL x 16 = 40 µL
      • NF H2: 5.5 µL x 16 = 88 µL

Results

Image

Figure 1. Gel image for samples

I could genotype most of these! I’m pretty sure 163, 169, and 200 are faint hets, but 239 and 253 have slightly funkier bottom bands. I will redo these samples along with some from my initial runs without the 1.5% gel next.

2025-03-04

I was waiting on new primers from IDT today since I’m out of my F primer, so I had Jasmine work on extractions for Catlin’s project today!

Notes

  • DNEasy for Catlin’s WA samples: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12
  • PCR for Catlin’s WA samples: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11
    • Realized we extracted one more sample than what would fit in a single row of a gel!
    • PCR Master Mix calculations
      • GoTaq: 12.5 µL x 14 = 175 µL
      • F: 2.5 µL x 14 = 35 µL
      • R: 2.5 µL x 14 = 35 µL
      • NF H2: 5.5 µL x 14 = 77 µL
    • Made new R primer

2025-03-06

Today I did took my remaining WA samples from PCR to gel. I also finished up the restriction digest and gel for the samples Jasmine started Tuesday.

Also, my IDT primers arrived! I’m running low on restriction enzyme so Carolyn put in an order.

Notes

  • PCR, restriction digest, and gel for WA samples: 163, 167, 169, 195, 200, 217, 223, 228, 229, 235, 239, 253
    • PCR Master Mix calculations
      • GoTaq: 12.5 µL x 14 = 175 µL
      • F: 2.5 µL x 14 = 35 µL
      • R: 2.5 µL x 14 = 35 µL
      • NF H2: 5.5 µL x 14 = 77 µL
  • Restriction digest and gel for Catlin’s WA samples: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11
  • Extractions for Catlin’s samples
    • WA: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30
    • ME: 32, 33, 34, 35, 36, 37
      • These samples are in a box and tubes labelled “MA.” According to Catlin’s spreadsheet, these should be ME samples. I confirmed this with him as well.

Results

Image

Figure 2. Restriction digest product gel for my samples (top) and Catlin’s samples (bottom)

All the samples I thought were hets, are hets! Sample 229 was a TT with a false bottom band in my previous gel, and it is showing up consistently with this pattern. Since the two samples around it are CT, it’s easier to compare a TT with false bottom band to a CT. There’s slightly more of a gap between the two true CT bands, and the bottom of the false band is less defined. Based on this, I think two of Catlin’s samples are TT. I will redo them with sample WA 1 since that band was too faint to genotype. I’m not sure why WA 1-5 have a wavy band design. I must have done something weird when I placed or took out the well comb.

NOW I HAVE ALL MY 2024 COLD CRAB RESULTS AND CAN START ACTUALLY WRITING. YAY!

Going forward

  1. Write methods section in manuscript
  2. Add MA results to manuscript
  3. Revise WA TTR analysis
  4. Add WA results to manuscript
  5. Add Catlin’s methods and results to manuscript
  6. Genotype Catlin’s samples
  7. Determine methods for comparing population responses
  8. Troubleshoot lipid assay protocol
  9. Conduct lipid assay for crabs of interest
Written on March 3, 2025