Returning to ACTUAL genotyping

I finally have a protocol that works! I’m going to finish up the MA samples before moving onto the WA ones. Here’s the overall protocol:

• DNEasy extraction with overnight incubation
• PCR
• Restriction digest
• Gel at 90 V for 60 minutes

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.

2024-11-25

I returned to the samples I left for incubation on Friday to finish up the extraction, PCR, and restriction digest.

Notes

• Samples for extraction: 1, 6, 8, 11, 21, 25, 27, 100, 110, 113, 114
• Noticed that sample 1 had a much smaller volume and was kind of yellow after the multi-day incubation
• Forgot to incubate the first elution for 1 minute before centrifuging. I incubated the second elution for 2 minutes before centrifuging.
• Used 20 µL of restriction digest product for the gel
• 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 11 = 82.5 µL
• Ran the gel an extra 10 minutes for a total of 70 minutes
• Set up next set of samples for overnight extraction: 17, 18, 55, 31, 32, 34, 49, 50, 51, 82, 83 84, 85, 86, 103, 104, 105, 119, 120, 121, 122, 123, 125

Results

Figure 1. Restriction digest gel

Well…that’s weird. When I took the gel out at 60 minutes, I noticed that it was really fuzzy! I thought if I ran it out for an extra 10 minutes that it will help with the fuzziness. Clearly (lol) it did not. Even the ladder looks fuzzy! Carolyn was stumped when I showed it to her. The PCR and digest worked, but somehow the gel isn’t showing up well! She suggested I run the gel again but replace the TAE buffer to see if that helps.

2024-11-26

Today I redid the PCR and restriction digest so I can get a clearer gel, and I finished up the DNA extraction I started yesterday.

Notes

• Samples for DNEasy extraction: 17, 18, 55, 31, 32, 34, 49, 50, 51, 82, 83 84, 85, 86, 103, 104, 105, 119, 120, 121, 122, 123, 125
• I noticed that sample 103 was really yellow when the overnight incubation was done
• Samples for PCR and digest: 1, 6, 8, 11, 21, 25, 27, 100, 110, 113, 114
• 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 11 = 77 µL
• I didn’t run the extraction blank from the samples since it came up blank in the really blurry gel!

Results

Figure 2. Restriction digest gel

Figure 3. Photo of computer screen which shows clearer double banding for fainter samples.

So, I clearly forgot to add a ladder…..but what an EXCELLENT gel to forget a ladder for! Almost every single crab is a CT, with the exception of a TT crab. The fainter samples are hard to see on the printed gel image, but are clear enough to genotype on the computer. I think I have more genotypic diversity in this population than Carolyn expected, which will be really interesting when looking at genotype- and population-specific effects on righting response.

Next week, I’ll finish up the MA samples and start on the WA samples.

Going forward

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