Finishing MA genotyping

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 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 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.
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 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 60 minutes at 90 V.
10. After running gel, remove gel tray and image. If needed, run gel for longer in 5-10 minute intervals.

2024-12-02

Today I did PCR and restriction digest for the samples I finished extracting last week. I also started the extraction for the last batch of MA samples!

Notes

• Samples for PCR and restriction digest: 17, 18, 55, 31, 32, 34, 49, 50, 51, 82, 83 84, 85, 86, 103, 104, 105, 119, 120, 121, 122, 123, 125
• PCR Master Mix calculations
  • GoTaq: 12.5 µL x 26 = 325 µL
  • F: 2.5 µL x 26 = 65 µL
  • R: 2.5 µL x 26 = 65 µL
  • NF H₂: 5.5 µL x 26 = 143 µL
  • Made new F primer for today
  • Ran gel for 65 minutes
• Samples for DNEasy extraction: 56, 57, 58, 35, 36, 59, 60, 61, 52, 53, 54, 62, 87, 88, 89, 90, 101, 106, 108, 128
  • Initially I planned to extract 63 and 126 based on the entries in my genotyping spreadsheet. Turns out these samples don’t exist since the crabs died before the experiment started!
• When using the repeater pipet to add the ATL buffer, I noticed that it would suck up a lot of air, or something would block the pipet tip so it wouldn’t suck up the full volume needed. I made sure all samples had roughly the same amount of ATL buffer before adding in the tissue.

Results

Figure 1. Restriction digest gel

Figures 2-3. Restriction digest gel images on the computer

Once again, the printed image and the computer image are a bit different! I may have to finagle my settings a bit before printing. In any case, I’m able to genotype most of the samples, and the majority are CT with a few TT. My EB and PCR blanks are clean, so once again I think this is real and not some weird contamination thing. I initially thought this may be a population difference between Eel Pond and New Bedford Harbor samples, but I think that the local population is just more CT-heavy than Carolyn realized!

There are a few samples I’ll need to redo, but I can add them to tomorrow’s gel. Carolyn also mentioned grabbing a few samples from each gel run and running them together, just to QC across runs.

2024-12-03

Notes

• Samples for DNEasy extraction, PCR, and restriction digest: 56, 57, 58, 35, 36, 59, 60, 61, 52, 53, 54, 62, 87, 88, 89, 90, 101, 106, 108, 128
  • Sample 52 had a yellow tinge after incubation that was cleaned up through the various washes
• Added 160 mL 200 proof ethanol to new AW2 reagent to finish extractions
• Additional samples for PCR and restriction digest: 18, 82, 86, 103
• 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 for today in the same tubes
• Ran gel for 65 minutes

Results

Figure 4-5. Restriction digest gel printed and computer images

Well…this is one hard to read gel! Apart from the fact that 103 is a TT genotype, I can’t tell much. I’m going to redo the gel and empty out the TAE buffer from the gel rig and fill it with new buffer and hope that helps. I’ll also need to re-extract the really yellow sample, 52!

2024-12-05

I redid the PCR and digest using samples from Tuesday. I also started to extract Washington samples!

Notes

• Samples for PCR and digest: 56, 57, 58, 35, 36, 59, 60, 61, 52, 53, 54, 62, 87, 88, 89, 90, 101, 106, 108, 128, 18, 82, 86
• PCR Master Mix calculations
  • GoTaq: 12.5 µL x 24 = 300 µL
  • F: 2.5 µL x 24 = 60 µL
  • R: 2.5 µL x 24 = 60 µL
  • NF H₂: 5.5 µL x 24 = 132 µL
  • Made new R primer today
• Samples for DNEasy: 52, 146, 150, 160, 168, 179, 182, 190, 201, 209, 212
  • 52 is from MA, all other samples are from WA
  • I am out of tissue for sample 52. I used the little that was left, but I may need to use a different tissue type to complete future extractions. I kept the old extracted DNA to run Qubit on in the future
  • The WA samples were chosen since they were some of the 30 crabs alive at the end of the experiment. I want to genotype these first
  • I am doing a smaller amount of extractions today because I want to make sure that the protocol works for the WA samples! I’m not sure why they wouldn’t…but you never know

Results

Figures 6-7. Restriction digest gel printed and computer images

All of these crabs are hets?! These are all from Eel Pond. Looking at my genotype spreadsheet, all my CC crabs are in the New Bedford population, whereas Eel Pond is predominantly CT crabs. Once I have all my genotype, I’ll see if each population is in Hardy-Weinberg equilibrium. My guess is that Eel Pond is certainly not.

2024-12-06

A few things were done today:

• Finished DNEasy extraction I started with WA crabs
• Did Qubit with old and new extracted DNA for MA 52
• Ran PCR and gel for WA crabs, as well as MA crabs I wanted to double check the genotypes for

Notes

• Samples for Qubit: 52 (old) and 52 (new)
• Samples for PCR and digest: 52 (new), 146, 150, 160, 168, 179, 182, 190, 201, 209, 212, 52 (old)
• Samples for QC gel to double-check genotypes: 103, 82, 62, 31, 3, 100, 21, 19, 8, 118, 35, 113
• Made new AW1 buffer by adding 130 mL 200 proof ethanol to the provided reagent
• 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 today

Results

Table 1. Qubit dsDNA HS results for sample 52. S1 = 43.67, S2 = 25761.48

Sample	Extraction Date	Concentration (ng/µL)
52A	12/3/24	46.70
52B	12/6/24	5.53

Alright, looks like there is DNA in both the sample I extracted on Tuesday and the sample I extracted today. As expected, there’s more DNA in the sample I extracted on Tuesday since there was very little tissue in the extraction today. However, today’s sample was not yellow. It’s possible that there’s some weird contamination in the old extraction, even though DNEasy extractions are meant to be cleaner.

Figures 8-9. Restriction digest gel printed and computer images

Exciting that I have a genotype for sample 52 now! Must be that the yellow stuff was indicative of some PCR-inhibiting contaminant. The not so exciting news is that my 3 CC QCs (19, 3, 118) are showing up as CTs. Seeing how my extraction and PCR blanks are blank, and my TT QC is still showing up as a TT, I don’t think there’s a contamination issue. I did run that previous gel for 40 minutes instead of my now standard 65, so maybe the difference in gel protcol is leading to this? I’m going to rerun these three samples again next week to be very clear about what is happening. The more concerning thing is that 19 was genotyped in June, and I feel good about calling that a CC. If there is indeed a discrepancy in genotyping and running this sample again leads to a CT, I may need to re-genotype samples I’ve already finished.

Going forward

1. Troubleshoot QC gel discrepancies
2. Individual-level TTR data analysis for MA samples
3. Genotype WA samples
4. Individual-level TTR data analysis for WA samples
5. Clarify methods for average TTR analysis (reach out to Andy, Nic, or Megan?)
6. Determine methods for comparing population responses
7. Troubleshoot lipid assay protocol
8. Conduct lipid assay for crabs of interest