Cold Acclimation Green Crab Experiment Part 33

Re-genotyping MA crabs and finishing WA crabs

I’m continuing the genotyping work! I’m working with Jasmine to re-genotype the MA crabs, and I’ll move forward with the WA crabs myself.

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 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. 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.

2025-02-18

Jasmine came back into today! We finished up the samples we started previously.

Notes

  • Gel for: 14, 28, 112, 40, 74, 91, 41, 197, 196
    • 197: CT
    • 196: TT
  • Made 1.5% agarose gel mix in separate jar

Results

Image

Figure 1. Restriction digest gel image

Jasmine did a great job with her first gel! All the samples we did were confirmed to be CC. Samples 28 and 40 on the computer were faint and looked like CC, but it’ll be best to redo them for bright bands.

2025-02-20

Today I walked through DNEasy extractions with Jasmine using MA samples. She did an awesome job doing the extractions, and was already a bit more confident doing the PCR with minimal supervision! I also prepped samples from WA to finish tomorrow.

Notes

  • DNEasy extractions and PCR for MA samples: 29, 44, 75, 92, 45, 76, 97
    • Incubated for ~ 2 hours total to achieve total lysis
    • 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
    • We have NF H2 aliquots again!
    • Carolyn placed these samples in the 4ºC when the cycle finished
  • Started DNEasy extractions for WA samples: 145, 151, 155, 162, 166, 172, 177, 183, 186, 191, 198, 202, 205, 211, 216, 225, 227, 230, 236, 241, 244, 252, 257, 262
    • Completed the first 12 samples before Jasmine arrived at 10:30 a.m. Finished the last 12 samples and the extraction blank starting at 12:30 p.m. Kept the tools I was using aside so they wouldn’t be used for Jasmine’s samples.

2025-02-21

Notes

  • Finished DNEasy, PCR, digest, and gel for WA samples: 145, 151, 155, 162, 166, 172, 177, 183, 186, 191, 198, 202, 205, 211, 216, 225, 227, 230, 236, 241, 244, 252, 257, 262
    • Accidentally did AW1 twice
    • 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 and R primer aliquots
      • Initially miscalculated MM for 25 samples! When I added the amount for two samples extra, I was short some. The EB has less MM than the other samples but I think in the grand scheme of things that is fine.
      • May have mixed 10 µL of 262 into 257 when loading the gels…I’ll figure out what happened when I look at the results.

Results

Image

Figure 1. Restriction digest product gel

Lots of faint samples this time around! But the genotypes I have are clean. I need to run my gels for less than 75 minutes. I checked around the 65 minute mark and the gel was about to go off the edge! Next time I’ll check after 60 minutes.

Since I think I mixed 262 into 257 and they were both CT, I am suspicious. I’ll redo those samples along with my faint buddies.

2025-02-24

Notes

  • Started DNEasy extractions for WA samples: 147, 149, 163, 165, 169, 170, 178, 184, 185, 193, 104, 200, 206, 208, 214, 226, 229, 237, 239, 242, 243, 253, 255, 258, 260, 261
    • When doing the extractions, I think I may have blotted 261 in the same corner as 260. I created a new clean sample for 261

2025-02-25

Notes

  • Finished DNEasy extractions for WA samples: 147, 149, 163, 165, 169, 170, 178, 184, 185, 193, 104, 200, 206, 208, 214, 226, 229, 237, 239, 242, 243, 253, 255, 258, 260, 261
  • PCR and restriction digest for WA samples: 147, 163, 169, 170, 178, 184, 185, 193, 194, 200, 206, 214, 226, 237, 242, 243, 253, 255, 258, 260, 261, 149, 208, 229
    • 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

2025-02-26

Notes

  • Gel for WA samples: 147, 163, 169, 170, 178, 184, 185, 193, 194, 200, 206, 214, 226, 237, 242, 243, 253, 255, 258, 260, 261, 149, 208, 229
    • When loading the gel, I noticed that at sample 255 the number of used dye aliquots didn’t match the number of open tubes and used gel wells. I sorted it out so that sample 260 is good, but I am not sure about samples 243, 253, 255, or 258. I will need to redo those
    • Ran gel for 60 minutes instead of 75
  • PCR and restriction digest samples
    • WA: 165, 239, 154, 156, 172, 183, 186, 198, 202, 257, 262, 236, 227, 215
    • MA: 28, 40, 99, 38, 109, 16, 116. Used WA samples 197 and 196 as knowns
    • 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
    • When loading the PCR, I accidentally put sample 40 in 196B’s tube! So I will only have 197A, 196A, and 197B as knowns for the top row in my gel

Results

Image

Figure 2. Gel for WA restriction digest product

I truly do not know why the samples in the top row of the gel look so janky while the bottom row looks nice! I even tried running the gel for an extra 7 minutes to see if I could get some separation, but no dice. I’ll have to redo those samples (and maybe put them in the bottom row…).

As far as the 237-260 fiasco goes, here are my thoughts:

  • Sample 237 has to be good
  • Sample 260 I know is good
  • The mix-up happened somewhere in the middle.
    • If I put 10 µL of 237 into 242 by accident, then added 10 µL of 242, then I would get a different banding result if 242 was a CT or TT. Since I only see CC bands, I think I am safe in thinking 242 is a CC. I will likely redo this anyways just for increased peace of mind.
    • If I put 10 µL of 242 into 243 by accident, then 243 could be a CT or TT. I will need to redo this one
    • 253 is blank, so that’s a redo
    • I saw the discrepancy when I got to 253 and noticed I took some 255 in my pipet, so I need to redo 255 and 258 since I was a little extra confused and didn’t resolve the issue until I got to 260.

Image

Figure 3. Gel for WA and MA restriction digest product

Yet another weird thing with the first samples loaded! Sara suggested I ensure the gel is drying level and to double check the connections of the electrical cables to the gel rig. I normally place the drying gel box on a paper towel in case there’s a spill, but perhaps this is leading to weird drying! I’ll change that for next time and see if it helps.

I thought I put 2 µL of sample 40 in 196B (which is why there are 2 40s). But 196A looks suspicious too. Since I’m going to do those samples again, I guess I’ll have to check then. Sample 257 looks like it could be a TT or a CT, so that will need to be checked too.

2025-02-27

Notes

  • Started DNEasy extractions for MA samples: 47, 48, 79, 81, 98, 99
    • No more sample 99 left after I pulled very little left from the leg.
    • Incubated samples for two hours
    • Sample 81 was yellow after incubation
  • PCR and restriction digest for samples
    • MA: 47, 48, 79, 81, 98, 99, 94, 28, 40, 30. Used WA 197 and 196 as knowns
    • WA: 147, 163, 165, 239, 243, 243, 255, 257, 258, 261
    • 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 and R primer. I’m OUT of F primer!

2025-02-28

Today I wrapped up the gel for the restriction digest product from yesterday! Jasmine wasn’t able to come in, but I finished up the restriction digest and gel with her samples from 2/20/2025.

Notes

  • Gel for samples
    • MA: 47, 48, 79, 81, 98, 99, 94, 28, 40, 30. Used WA 197 and 196 as knowns
    • WA: 147, 163, 165, 239, 243, 243, 255, 257, 258, 261
  • Restriction digest and gel for MA samples: 29, 44, 75, 92, 45, 76, 97. Used WA 197 and 196 as knowns
    • When I was loading this gel, I realized I could be more time efficient if I added 2 µL of TriTrak to the strip tubes, pipetted up and down to mix, then put it in the gel well. You can estimate ~2 µL based on the gradations on the pipet tip so you can do one sample all in one go! It saved me a lot of time.

Results

Image

Image

Figures 4-5. Gel and computer image for samples

All samples had bands and I was able to genotype! The bands for 163 and 239 aren’t clear for me to distinguish between CT and TT, so I’ll need to redo those. I also didn’t redo 253, which was blank in my gel from 2/26/2025. Three more samples and I’ll have all the WA genotypes! It may be worth going through my gel images just to identify CT samples where the CC band is considerably fainter. I think I have a good feel for when a sample is a CT vs. a weird TT, but it doesn’t hurt to look at several more and confirm my original suspicions.

Image

Figure 6. Gel for Jasmine’s samples

Jasmine’s extraction and PCR blanks are clean!! And all of these MA samples are CC…which means 1) I FINISHED ALL THE MA SAMPLES and 2) I don’t have to redo my analysis!!!!!!!!!!! What a great day to end the week (and month).

Going forward

  1. Write methods section in manuscript
  2. Add MA results to manuscript
  3. Add Catlin’s methods and results to manuscript
  4. Genotype WA samples
  5. Revise WA TTR analysis
  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 February 19, 2025