Genotyping MA crabs (back in troubleshooting land)

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. 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 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 10 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 40 minutes.
10. After running gel, remove gel tray and image.

2024-07-15

I’m trying two different troubleshooting approaches today:

1. Re-extract samples with no bands in initial runs (some of these samples were used in the dilution series)
2. Run a PCR with samples I extracted 2024-07-13, but add BSA to the PCR to reduce the effect of any PCR inhibitors

Notes

• Samples for re-extractions, PCR, and digest: 1, 3, 6, 8, 11, 16, 17, 21, 25
  • Used 75 and 15 from extractions Catlin and Heidi did as known CC and TT crabs. Not sure why my most recent PCR attempts are being weird with these two samples, but those extractions were from a time when things were going much better
• Samples for PCR + BSA and digest
  • 60, 61, 62, 88, 89, 90, 108, 128, 122
  • Used 80 as a known CT since my known CC (75) and TT (15) from last year’s experiment are being weird. I have tons of known CC, but I don’t have any known TT from this year!
• Used brand new GoTaq aliquot, made new F and R primers, and new NF H₂ aliquot
• PCR Master Mix calculations (without BSA)
  • GoTaq: 12.5 µL x 15 = 187.5 µL
  • F: 2.5 µL x 15 = 37.5 µL
  • R: 2.5 µL x 15 = 70 µL
  • NF H₂: 5.5 µL x 15 = 82.5 µL
• PCR Master Mix calculations (with BSA)
  • In a 50 µL total reaction, Sarah adds 1 µL BSA. Since my reactions are 25 µL total, I will add 0.5 µL BSA and reduce the amount of water added by 0.5 µL.
  • GoTaq: 12.5 µL x 15 = 187.5 µL
  • F: 2.5 µL x 15 = 37.5 µL
  • R: 2.5 µL x 15 = 70 µL
  • NF H₂: 5.0 µL x 15 = 75 µL
  • BSA: 0.5 µL x 15 = 7.5 µL

Results

Figure 1. Gel for re-extractions (top) and BSA addition test (bottom)

STILL NO AMPLIFICATION I AM LOSING MY MIND. WHY, OH WHY, CRUEL WORLD.

2024-07-16

Today to confirm exactly how I’m losing my mind, I took some of Carolyn’s DNA (that had bands!!) and ran it through PCR and the restriction digest. Carolyn used a Qiagen column kit for extractions which takes longer, but yields much cleaner DNA. If Carolyn’s extractions don’t work, then we know there’s an issue with the thermocycler or reagents. If Carolyn’s extractions work, then there’s an issue with the Chelex or with the MA crabs specifically.

Notes

• Samples for PCR and digest: Carolyn 1, 2, 3, 4, 5, 6, 7, 8
• PCR Master Mix calculations (without BSA)
  • GoTaq: 12.5 µL x 11 = 137.5 µL
  • F: 2.5 µL x 11 = 27.5 µL
  • R: 2.5 µL x 11 = 27.5 µL
  • NF H₂: 5.5 µL x 11 = 60.5 µL

Results

Figure 2. Gel for Carolyn’s extractions

Of course, her samples had bands. So that means there’s specifically an issue with the Chelex extractions. I really don’t know why all of a sudden our Chelex protocol isn’t working. Maybe there’s something specific in the MA crabs inhibiting the Chelex or the PCR afterwards, since I developed the protocol specifically with WA crabs?

2024-07-17

The last thing I can think of to do right now is to extract some of the crabs from this year’s WA experiments with Chelex to determine if there’s a specific issue with the MA crabs. A Hail Mary, if you will.

Notes

• Samples for PCR and digest: 145, 146, 160, 161, 175, 190, 205, 206, 220, 235, 250
• Sample 206 had orange-colored supernatant
• Sample 175 evaporated during the Chelex incubation. I added 70 µL of DEPC water to the tube before vortexing and centrifuging. While centrifuging, the water escaped the tube! So there is likely something not working with that tube closure mechanism. I scraped the sample all together.
• 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

Results

Figure 3. Gel for WA crabs

Some faint bands, but really nothing to write home about. F. Me.

Carolyn suggested a few things:

1. Ask the department to see if anyone else has Chelex beads I can use to determine if something went wrong with ours
2. Look up Chelex issues online
3. Try using Chelex with WA samples from last year that I know worked (aka any of them)
4. Just move forward genotyping a few crabs per tank with DNEasy

I started looking up Chelex issues online. I found something on the BioRad website to suggest that Chelex should be stored at ambient temperature, and the lab air conditioning is currently not working. Unsure just how related that is, but oh well.

I emailed the department to see if anyone had some Chelex we could use! Mindy offered some, so I have a different Chelex to try when I return to troubleshooting.

Going forward

1. Finish genotyping MA crabs
2. Genotype WA crabs
3. Analyze TTR data
4. Develop lipid assay protocol
5. Develop heart rate protocol
6. Conduct lipid assay with pilot crabs
7. Conduct lipid assay with remaining crabs