Extraction, PCR, and gel for remaining crabs

Vanessa did a set of extractions, PCR, and gel for me while I was away on May 3! Today, I set out to extract more samples, as well as re-extract some with faint gel bands from my previous gel and from Vanessa’s gel.

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
   • SMC F primer: 2.5 µL/sample
   • SMC R primer: 2.5 µL/sample
   • DEPC H₂O: 5.5 µL/sample
4. Preheat the thermocycler with the TD65_48L 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 SMC 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 TD65_48L PCR protocol.
14. When protocol is complete, place PCR product in the 4ºC fridge until ready to load a gel.

Gel:

1. Obtain PCR product, 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 6 µL of diluted ladder + dye mix, and continue with remaining samples.
9. Run gel for 30 minutes.
10. After running gel, remove gel tray and image.

Notes

• Vanessa samples fron May 3: 13, 54, 67, 108, 129, 155, 170
• My samples from today: 15, 57, 72, 111, 130, 162, 172, 8, 47, 151, 170
  • 8, 47, and 151 were faint in this gel
  • 170 was faint in Vanessa’s gel (Figure 1)
• I used 3 µL of ladder for my gel
• PCR Master Mix calculations
  • GoTaq: 12.5 µL x 16 = 200 µL (from 4/22)
  • SMC F: 2.5 µL x 16 = 40 µL (mix of 4/22, 4/26, and 4/29)
  • SMC R: 2.5 µL x 16 = 40 µL (mix of 4/22, 4/26, and 4/29)
  • NF H₂: 5.5 µL x 14 = 88 µL (from 4/22)

Results

Figure 1. Gel image for PCR blank, extraction blank, 170, 155, 129, ladder, 108, 67, 54, and 13

Blanks look great and all samples except 170 have a bright enough gel band! There’s also an interest artifact Vanessa noted, likely contamination from the gel tray used for imaging. I’ll be sure to clean it thoroughly prior to imaging in the future.

Figure 2. Gel image for 15, 57, 72, 111, 130, 162, 172, ladder, 8, 47, 151, 170, extraction blank, and PCR blank

Interestingly my gel looks like it needed ~5 more minutes to fully separate the bands! In any case, I didn’t do that and my blanks look good, so there’s that. I will rerun 72, 151, and 170 on my next gel. When I was pipetting TriTrak dye, more than 1 µL was added to 72, so I think there could be an issue with DNA/dye ratio in that well. Samples 151 and 170 look like they could be bright enough, but because there isn’t clear band separation I will rerun just to be sure!

Going forward

1. Finish Chelex extractions, PCRs, and gels for remaining crabs
2. Genotype remaining samples
3. Examine HOBO data from 2023 experiment
4. Determine best statistical approach for analyzing performance data
5. Demographic data analysis for 2023 paper
6. Update methods and results of 2023 paper