Genotyping MA crabs (but now it’s hell)

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.

2024-07-09

Today I decided to rerun samples with faint or no gel bands with 10 µL of product. I had success with 8 µL of product and Carolyn’s gel with 10 µL of product looked pretty good!

Notes

• Samples: 11, 21, 28, 17, 51, 80, 115, 116, 8, 29, 44, 14, 16
  • I also wanted to run restriction digest product for samples 1, 3, 6, 25, 27, 41, 84, 110, 113, and 118 from June 18. However, I couldn’t find these samples in the fridge! I must have accidentally tossed them when I was cleaning out unneeded sample tubes.
  • There are also a few more samples I forgot to include in my gel plan. I tossed all restriction digest products after running this gel…so I have some work to potentially redo.
• I did not include RD product from 75 or 15 in the gel because the samples I had and ladder fit very nicely into a 14-well gel

Results

Figure 1. Gel run with 10 µL for previously-digested samples

10 µL really helps with visibility! There are some samples with no gel bands in the previous runs that have clear enough bands to read now. I still have a few I want to redo because the bands aren’t clear enough. 21 is really confusing to me, because the position of the bands suggests that it’s a CC crab, but it looks like it has two bands similar to a CT crab. When I redo 21, I want to run it in the middle of the gel next to a ladder or known CC/TT so I can more easily compare its genotype and avoid any warping of the gel image on the edges.

2024-07-10

I did the extraction, PCR, and restriction digest on 7/10, and ran the gel on 7/11

Notes

• Samples: 18, 55, 56, 35, 36, 59, 52, 53, 54, 85, 86, 87, 104, 105, 106, 122, 123, 125, 57, 58
• Sample 52 had black-colored tissue and the resulting supernatant had an orange tint
• Sample 122 all evaporated! There was no water left in the tube, so that became another blank
• I used Sara’s four corners method where I use a different corner of a kim wipe to blot four individual samples
• Made new restriction digest product for 75 and 15
• Accidentally placed samples on heat block without starting the incubation program so they likely digested longer than 1 hour.
• PCR Master Mix calculations
  • GoTaq: 12.5 µL x 28 = 350 µL
  • SMC F: 2.5 µL x 28 = 70 µL
  • SMC R: 2.5 µL x 28 = 70 µL
  • NF H₂: 5.5 µL x 28 = 154 µL
• Gel made by melting down previously poured gels. One of the gels had been sitting for a few hours on the bench before being melted down.

Results

Figure 2. Gel image for extracted samples

Welp…everything is faint? Since I used previously poured gels that were sitting for a bit, maybe I needed to add more Gel Red when I melted them back down. And I don’t know why samples 75 and 15 aren’t even showing up. Maybe the extraction was long enough ago that the DNA has broken down?

I quantified my samples on the Qubit and the yields seemed okay:

Table 1. Concentrations of samples

Sample	Concentration (ng/µL)
18	3.29
55	4.29
56	4.57
35	3.48
36	2.02
59	2.72
52	6.70
53	3.64
54	3.00
85	5.48
86	4.08
87	2.71
104	3.94
105	4.02
106	3.84
123	5.15
125	4.42
57	3.49

I’ll need to redo some part of this protocol. At least my extraction blank was clean and I know I can use Sara’s four corners method moving forward!

2024-07-11

Apart from running the gel for samples extracted 7/10, I wanted to Qubit samples with no or very faint gel bands in my previous runs to start troubleshooting. Based on my Qubit, Carolyn suggested I add more DNA to the PCR to get better amplification and hopefully, brighter gel bands.

Notes

• Samples: 1, 3, 6, 8, 11, 16, 17, 21, 25, 27, 31, 32, 41, 51, 83, 110, 113, 114, 118
• PCR Master Mix calculations
  • GoTaq: 12.5 µL x 24 = 300 µL
  • SMC F: 2.5 µL x 24 = 60 µL
  • SMC R: 2.5 µL x 24 = 60 µL
  • NF H₂: 2.5 µL x 24 = 60 µL
• Added 20 µL of PCR Master Mix and 5 µL of sample to each tube

Results

Table 2. Sample concentrations

Sample	Concentration (ng/µL)
1	1.53
3	1.95
6	1.63
8	1.77
11	0.869
16	1.13
17	1.83
21	1.85
27	2.81
31	2.70
32	9.67
41	0.750
51	2.57
83	2.95
110	2.21
113	1.97
114	1.49
118	0.125

Looks like all of these samples have really low yields! Based on this, Carolyn suggested two potential approaches for getting brighter gel bands: increase or decrease the amount of DNA. There should be enough DNA to get decent amplification, but just in case I could start by adding more DNA to each PCR reaction. If I wanted to add 5 µL of DNA to each reaction but maintain a final volume of 25 µL, then I needed to add only 20 µL of PCR Master Mix. Instead of messing with the amount of GoTaq or primers in each reaction, I needed to reduce the amount of water in each reaction. Carolyn suggested using 2.5 µL of water per reaction instead of the usual 5.5 µL for my Master Mix.

Figure 3. Gel image for samples with faint gel bands in previous runs

NOTHING AMPLIFIED. This means that Carolyn’s initial suspicion is correct, and that there’s likely some sort of PCR inhibitor that should be diluted out. I’ll need to give that a shot!

2024-07-13

Today I’m following up on my previous gel with no amplification. I performed 1/10 and 1/100 dilutions for a handful of samples, then performed PCR, the restriction digest, and ran out the digest on a gel.

Notes

• Samples: 1, 113, 75, 15
• Dilutions
  • 1/10: 1 µL DNA and 9 µL NF H₂
  • 1/100: 1 µL DNA and 99 µL NF H₂
• PCR Master Mix calculations
  • GoTaq: 12.5 µL x 15 = 187.5 µL
  • SMC F: 2.5 µL x 15 = 37.5 µL
  • SMC R: 2.5 µL x 15 = 37.5 µL
  • NF H₂: 2.5 µL x 15 = 82.5 µL
• Extracted remaining samples
  • 60, 61, 62, 88, 89, 90, 108, 128, 122

Results

Figure 4. Gel for serial dilution test

STILL NO AMPLIFICATION. I shot Carolyn a quick message to see if I should try a 1/1000 dilution, or if I should just re-extract the samples that had bad bands.

2024-07-14

Alright, a lot to do today!

1. Try a dilution series with 1/1000 and 1/10000 dilutions
2. Run a PCR with the samples I just extracted and the dilution series
3. Figure out the next steps

Notes

• Samples for dilutions: 1, 113, 75, 15
  • Made a 1/1000 dilution using 1 µL of the 1/100 dilution and 9 µL of water
  • Made a 1/10000 dilution using 1 µL of the 1/1000 dilution and 9 µL of water
• Samples for PCR
  • 60, 61, 62, 88, 89, 90, 108, 128, 122
  • 1/100, 1/1000, and 1/10000 dilutions: 1, 113, 75, 15
  • 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!
• PCR Master Mix calculations
  • GoTaq: 12.5 µL x 28 = 350 µL
  • SMC F: 2.5 µL x 28 = 70 µL
  • SMC R: 2.5 µL x 28 = 70 µL
  • NF H₂: 2.5 µL x 28 = 154 µL

Results

Figure 5. Gel for remaining samples (top) and serial dilution test (bottom)

Welp, still no amplification! I’m especially concerned that sample 80, a sample I extracted before going to SEB and ran out on a gel earlier this week, has NO bands when it had really bright bands earlier. Truly every extraction/PCR/digest I’ve done this week has yielded a gel with faint or no bands. What is happening?! I’m truly at a loss and I feel like pulling all my hair out.

I need to genotype my WA samples before my experiment so I’ll prioritize working through that. Hopefully I won’t run into any weird issues with my WA samples?!

Going forward

1. Finish genotyping MA crabs
2. Qubit and re-extract crabs with no gel bands
3. Develop lipid assay protocol
4. Develop heart rate protocol