Extractions, PCR, and gel for respirometry + TTR crabs

I’m jumping back into extractions! I’m going to finish extracting DNA from respirometry crabs, and from the extra crab in each tank that was used for TTR over the course of the experiment. Some tanks, like T2, didn’t end up having these crabs since they needed to be ~ 20.00g for back-up respirometry crabs. Other crabs were sequenced by Julia for her experiment.

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 another 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 RNAse AWAY the tweezers and use RNAse AWAY to clean bench.
9. Repeat step 7, ensuring 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. 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 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 ladder + dye mix, and continue with remaining samples.
9. Run gel for 30 minutes.
10. After running gel, remove gel tray and image.

Notes

• Samples run: 15-035, 15-160, 15-163, 5-011, 25-185, 5-070, 15-098, 25-117, 5-126, 25-176, 25-168
• Samples 15-163, 25-117, 5-126, 25-176, and 25-168 may have had 4 µL DNA added instead of 2 µL because I couldn’t tell if anything actually made it into the tube.
• PCR Master Mix calculations
  • 12 samples + 1 PCR blank + 2 extra = 15 aliquots
  • 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: 5.5 µL x 15 = 82.5 µL
• After finishing my Chelex extractions, I immediately loaded the vortexed samples into PCR tubes.

Results

Figure 1. Gel image for 135, 160, 163, 11, 185, 70, 98, ladder, 117, 126, 176, 168, extraction blank, and PCR blank.

My blanks are REALLY contaminated :( I have no idea why I’m having issues again. Carolyn suggested I run a PCR tomorrow just using blanks. I’m going to create a new PCR MM with brand new reagents, but also use the PCR MM from today and run them. If the mix from today is contaminated while the new mix isn’t, that’ll be an easy solution. If both mixes are uncontaminated, then we have something else to figure out. There’s a slight possibility that the fume hood issues may be kicking up all kinds of things in lab and we’ll need to do a full-scale decontamination.

Carolyn also suggested running sample 160 on the Qubit to see if there’s any DNA to begin with. If there is DNA, there may be PCR inhibitors in the DNA. I could then do a serial dilution (1:10, 1:100, 1:1000) prior to PCR to see if I can get PCR product after diluting out the inhibitors.

Going forward

1. Identify contamination source and decontaminate
2. Finish extracting respirometry samples
3. Continue with Chelex extractions, PCRs, and gels for TTR crabs
4. Perform Qubit assay with any sample consistently not showing up on a gel
5. Update methods and results of 2022 paper
6. Examine HOBO data from 2023 experiment
7. Demographic data analysis for 2023 paper
8. Update methods and results of 2023 paper
9. Revisit Julia’s genotypes