Genotyping MA crabs

I have all my crabs so I’m going to get cracking genotyping! Carolyn suspects there won’t be much genotype variation, and I agree with her. I expect most crabs to be CC, with maybe a smattering of CT since all of these crabs are from the southern, warm-adapted lineage. However, it doesn’t hurt to have the information in case there’s a bigger mix of genotypes than expected.

Methods

Chelex Extraction:

1. Use RNAse AWAY to clean bench space, pipets, tip boxes, etc.
2. Obtain samples in ethanol from the -80ºC freezer.
3. Obtain two sets of either an 8-strip PCR tube set or a 96-well plate. Label one set with sample ID, and another with sample ID and “S” (ex. 3 and 3S). Label an eppendorf tube “Chelex.”
4. Preheat the thermal cycler (protocol CHELEX) or set up a thermal block to 100ºC
5. Prepare a 10% Chelex solution (ex. 0.1 g Chelex beads in 1 mL of DEPC/nuclease-free water). Vortex thoroughly (10-15 seconds) and spin down briefly (5 secconds)
6. Add 70 µL of Chelex solution to each tube. Vortex Chelex solution for 10-15 seconds in between each sample tube since the Chelex beads settle quickly
7. Obtain and set up a flaming station and two pairs of tweezers. Ethanol and flame tweezers, then place on a clean kim wipe.
8. For each sample, use one tweezer to remove the leg joint from the sample tube with ethanol, and another pair of tweezers to remove the tissue from the leg. Be sure to avoid any exoskeletal pieces, as the chiton in the shell can inhibit the Chelex reaction. Place the tissue on a clean kim wipe and press to dab ethanol from the sample (which can also impede the Chelex reaction). After placing the blotted tissue in the sample tube, ethanol, flame, and toss the kim wipe. Use RNAse AWAY to clean bench.
9. Repeat step 7, ensuring a clean kim wipe is used to blot each sample after cleaning the bench with RNAse AWAY.
10. If doing extractions in a plate, seal wells with caps.
11. Vortex samples for 10-15 seconds, and spin samples briefly (5-10 seconds).
12. Place samples in the thermal cycler and run the CHELEX protocol, or on a heat block for 20 minutes at 100ºC.
13. If doing extraction sin a plate, quickly spin down samples, remove caps, and add 50 µL DEPC water to each sample to assist with evaporation issues. Recover plate with a foil seal.
14. Vortex samples for 10-15 seconds, then spin down for 2 minutes. Let tubes sit an additional minute or two, if possible, for Chelex beads to settle and make pipetting easier.
15. Pipet ~50 µL supernatant into new, labelled tubes or a plate (labelled “S”). Avoid the Chelex beads when pipetting.
16. Place the supernatant with DNA in the dirty -20ºC freezer until ready for PCR.

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.
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 and place on ice.
3. Add 0.5 µL enzyme to each PCR reaction. Either pipet up and down to mix or VERY gently and briefly vortex
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 PCR + restriction digest product, restriction digest product from a known CT crab, 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.
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 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 6 µL of PCR 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 35 minutes.
10. After running gel, remove gel tray and image.

2024-06-18

Notes

• Samples: 1, 3, 5, 19, 21, 25, 38, 40, 41, 74, 75, 76, 91, 92, 94, 109, 110, 112, 113, 6, 27, 44
• There wasn’t enough a lot of chitin-free tissue in sample 94
• When pipetting samples 1, 3, and a few others I can’t remember, there was a good amount of tissue in the Chelex tubes that was not spinning down. There may be some almost degraded tissue in the supernatant tubes
• PCR Master Mix calculations
  • GoTaq: 12.5 µL x 26 = 325 µL (6/3)
  • SMC F: 2.5 µL x 26 = 65 µL (mix of 6/4 and 6/18)
  • SMC R: 2.5 µL x 26 = 65 µL (mix of 6/4 and 6/18)
  • NF H₂: 5.5 µL x 26 = 143 µL (6/4)
• Used restriction digest product from sample 72A created 6/4/24 as a known CT reference

Results

Figure 1. Gel image for restriction digest product

Well, a good amount of my samples don’t have bands? My guess is that there isn’t enough extraction product, so I’ll need to Qubit and/or re-extract those. I also have two samples, 21 and 41, that have an ambiguous band pattern! They both look like they’re CC, but there’s a decent amount of clearer banding above the 301 + 389 double band that indicates a CC. I’ll rerun the restriction digest product for these two samples to get confirmation. As expected, the majority of my crabs I could genotype from this run are CC!

2024-06-20

Instead of redoing samples without clear genotypes, I decided to truck forward until all samples had been genotyped once.

Notes

• Samples: 8, 10, 11, 13, 28, 29, 30, 45, 47, 48, 79, 80, 81, 97, 98, 99, 114, 115, 116, 118
• I was conscious about adding slightly less tissue to my Chelex because I may have added too much with the previous batch! So silly how adding too much input material can actually lead to less DNA
• For samples 10, 13, 81, and 97, I had a hard time actually getting sample. These are bigger crabs, so I took less of the leg than I normally do. The tissue I was able to pull out was stringy and black. Not sure if this is necrotic tissue, chitin-covered tissue, or just what the tissue looks like in this part of the leg joint. The supernatant for these samples was also a light orange.
• PCR Master Mix calculations
  • GoTaq: 12.5 µL x 24 = 300 µL (6/3)
  • SMC F: 2.5 µL x 24 = 60 µL (mix of 6/4 and 6/18)
  • SMC R: 2.5 µL x 24 = 60 µL (mix of 6/4 and 6/18)
  • NF H₂: 5.5 µL x 24 = 132 µL (6/4)
• Used restriction digest product from sample 72A created 6/4/24 as a known CT reference
• Taught Heidi how to pour a gel and had Heidi and Catlin load samples on the gel

Results

Figure 2. Gel image for restriction digest product

This gel is really smearing! I think it’s because the students were (rightfully so) loading the gel a bit slowly in order to ensure they didn’t break the wells. I’ll definitely need to rerun this gel. In the future, I should use small gels for training instead of larger ones!

When I showed the gel to Carolyn, she agreed that the samples with bands are hard to interpret because there isn’t a lot of genotyping variation in these samples. She suggested adding a known CC and known TT interspersed with the samples so there’s some contrast, instead of adding a known CT since those are much easier to spot.

2024-06-21

I did two things today: rerun yesterday’s samples on a gel for 35 minutes, 40 minutes, 45 minutes, and 60 minutes. At each point, I imaged the gel than returned it to the gel rig to run longer. The goal of this exercise was to help me determine if there’s a specific running time that leads to clearer bands without running those bands off the gel.

The second thing I did was extract and PCR a new set of samples. If I have time to do the restriction digest and gel over the weekend I will, but if not I’ll return to these samples on Monday.

Notes

• Gel timing test
  • Samples: 8, 10, 11, 13, 28, 29, 30, 45, 47, 48, 79, 80, 81, 97, 98, 99, 114, 115, 116, 118
  • Used restriction digest product from sample 75A and 15A created 6/4/24 as a known CT reference
• New extractions and PCR
  • Samples: 14, 16, 17, 31, 32, 34, 49, 50, 51, 82, 83, 84, 100, 101, 103, 119, 120, 121
  • Had Catlin and Heidi practice Chelex extractions with samples 17, 31, 32, and 34. I did the rest of them
  • Samples 83, 84, 101, and 101 had that stringly black-colored tissue and orange supernatant. Since it didn’t impact the restriction digest when I saw that in samples yesterday, I’m less worried about it.
  • Redid PCR with samples 75A and 15A extracted on 6/4/24 since there seems to be some variation between digest runs
  • PCR Master Mix calculations
    • GoTaq: 12.5 µL x 26 = 325 µL (6/3)
    • SMC F: 2.5 µL x 26 = 65 µL (6/18 and 6/21)
    • SMC R: 2.5 µL x 26 = 65 µL (6/18 and 6/21)
    • NF H₂: 5.5 µL x 26 = 143 µL (6/4)

Results

Figures 3-5. Gel image for restriction digest product ran for 35, 40, or 45 minutes

45 minutes on a gel is the clear winner in terms of band separation! I couldn’t even attempt 60 since the yellow TriTrak dye was at the bottom of the gel after 45 minutes, and I didn’t want to risk the DNA running off the gel either.

Based on the 45 minute gel, I was able to genotype more samples than with the first iteration (and more samples than my initial set of extractions on 6/18). Weirdly, my restriction digest product from 15 was absent. There’s a chance I may have accidentally pulled product from 151, which was a known CT with no gel band from my restriction digest testing.

Carolyn and I also noticed that my product from 75A seemed a lot cleaner and tighter than the CC product around it on the gel. I thought this could be because 75A was digested for two hours, while my newer samples were only digested for one hour. While Carolyn agreed this could be a possibility, she thought a more likely explanation is that there is some variability in restriction digest runs. Instead of running previously-made restriction digest product on a gel, she suggested I take DNA from a known CC and TT, then redo PCR and the restriction digest with it to reduce any inter-digest variability.

2024-06-22

I extracted some new samples! Based on Carolyn’s suggestion, I re-extracted samples 75 and 15 to reduce any inter-digest variability. I didn’t have time to run my restriction digest until 6/24. I ran a gel on 6/26, and then ended up rerunning it on 6/30.

Notes

• Samples: 14, 16, 17, 31, 32, 34, 49, 50, 15, 51, 82, 83, 84, 100, 101, 103, 119, 120, 121
• Made new restriction digest product for 75 and 15
• PCR Master Mix calculations
  • GoTaq: 12.5 µL x 26 = 325 µL
  • SMC F: 2.5 µL x 26 = 65 µL
  • SMC R: 2.5 µL x 26 = 65 µL
  • NF H₂: 5.5 µL x 26 = 143 µL

Results

Figures 1-2. Gels for restriction digest product on 6/26 and 6/30

I FORGOT TO ADD A LADDER WHEN I RAN MY GEL ON 6/26. So I reran the gel using 8 µL of restriction digest product on 6/30 (and remembered to add the ladder). The gel looks a lot better with 8 µL of product, and I’ll probably add 10 µL in the next run. There are still a few samples that don’t have any restriction digest product, so I’ll need to add those to my list of samples to rerun.

Going forward

1. Run the MA experiment!
2. Continue genotyping MA crabs
3. Qubit and re-extract crabs with no gel bands
4. Develop lipid assay protocol
5. Develop heart rate protocol