Cold Acclimation Green Crab Experiment Part 32

Re-genotyping MA crabs

I’ve decided to rerun all MA CC samples to give myself peace of mind. If they are CC, great! If not, it’s better I know now.

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 30 minutes and vortex tube(s) every 10 minutes. If tissue appears to not be fully dissolved after 30 minutes, 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 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. Obtain a piece of parafilm and pipet 1.5 µL of TriTrak dye for each sample, extraction blank, and PCR blank. On the parafilm, the dye will bead up. Place the dots far enough apart to avoid contamination.
  8. Take 10 µL of DNA, 6 µL of PCR product, or 20 µL of restriction digest product and mix with a 1 µL dot of TriTrak dye by pipetting up and down at least 10 times. Then, pipet up 6 µL and load gel. Repeat for each sample until halfway through samples. Then, add 3 µL of diluted ladder + dye mix, and continue with remaining samples.
  9. Run gel for 75 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.

2025-02-05

Notes

  • PCR, RD, and gel for: 99, 38, 35, 109, 100, 30, 94, 16, 116
    • 35: CT
    • 100: TT
  • 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 F and R primers
  • When making primer dilutions and adding NF H2 to the PCR MM, I realized I used a 20 µL pipet tip by accident! I switched to the correct pipet tips and added the remaining quantities of water needed
  • Completely out of NF H2 aliquots. Had to use DEPC H2 for the PCR MM
  • Made a 1.3% gel. Previously I tried to make a 1.5% gel but not all the agarose dissolved in the gel. Figured I would start with 1.3% and see what happens.
  • Started DNEasy extractions for: 14, 28, 112, 40, 74, 91, 41

Results

Image

Figure 1. Restriction digest product gel

So…….this is weird. At first I thought that my CT looked great and my TT didn’t show up! but my 35 looks like a CC and the 100 looks like a TT?. 35 and 100 are definitely CT and TT…don’t really know what happened here. This is certainly better separation I’ve had with any previous testing gel from MA.

These samples are frustrating so I’m going to put them aside for now. I’m also going to use a known CT and TT from the WA crabs since I have higher confidence in those genotypes. These MA bands are also not as clean as the WA samples, so I may try and increase the gel to a 1.5% agarose gel (aka what I intended previously but may not have hit)

2025-02-06

Jasmine started today! We got through PCR and RD with our sample set, and will finish up with a gel next week.

Notes

  • Finished DNEasy extractions for: 14, 28, 112, 40, 74, 91, 41
    • 112 was yellow after the overnight incubation
  • PCR and RD for: 14, 28, 112, 40, 74, 91, 41, 197, 196
    • 197: CT
    • 196: TT
    • I used WA samples since my known MA samples were giving me grief
  • PCR Master Mix calculations
    • GoTaq: 12.5 µL x 15 = 187.5 µL
    • F: 2.5 µL x 15 = 37.5 µL
    • R: 2.5 µL x 15 = 37.5 µL
    • NF H2: 5.5 µL x 15 = 82.5 µL
  • Completely out of NF H2 aliquots. Had to use DEPC H2 for the PCR MM

Going forward

  1. Redo all MA CC samples
  2. Genotype WA samples
  3. Revise MA TTR analysis
  4. Revise WA TTR analysis
  5. Genotype Catlin’s samples
  6. Determine methods for comparing population responses
  7. Scaffold manuscript
  8. Troubleshoot lipid assay protocol
  9. Conduct lipid assay for crabs of interest
Written on February 5, 2025