Starting chelex extractions for genotyping

Carolyn and I agree that it would be nice to present genotype data for PICES later this month! So, it’s time to do DNA extractions. I’m using Chelex extractions to get DNA quickly. Since we’re using Sanger Sequencing to genotype data at two loci, it’s not essential for me to have the highest quality DNA. What comes out of the Chelex extraction is good enough.

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 and flame the tweezers.
9. Repeat step 7, ensuring a clean kim wipe section is used to blot each sample.
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.
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. Make master mix based on calculations in step 3 in the labelled PCR MM tube. Vortex, spin down, and keep on ice.
7. Aliquot 23 µL of PCR MM from step 6 in each tube for the samples, extraction blank, and PCR blank.
8. Once the DNA thaws, briefly vortex DNA and spin down. Place on an ice block tube holder.
9. Add 2 µL of DNA into each sample tube. Add 2 µL DEPC water for the PCR blank.
10. 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.
11. Vortex the sealed tubes and briefly spin down.
12. Place in the thermocycler, close lid, and run TD65_48L PCR protocol.
13. 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.
6. Once gel is hardened, place in gel box with 1x TAE buffer.
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.

Results

Figure 1. Gel image for samples 3, 5, 10, 31, 32, 52, and 60. I picked seven respirometry crabs to test out the protocol.

First off, my PCR blank is contaminated! This means I need to redo the PCR with a new set of PCR reagents (MM, primer, NF water, etc.) to see if that helps the contamination issue. I also don’t have any band for sample 10. I think this is my pipetting error: I remember when I was mixing the PCR product with the TriTrak dye that I couldn’t tell if I actually pipetted up any PCR product.

Going forward

1. Redo PCR with these samples to see if that fixes contamination issue
2. Continue with Chelex extractions, PCRs, and gels
3. Treatment-wise TTR analysis
4. Treatment-wise respirometry analysis
5. Get existing genotype data from Julia
6. Incorporate genotype into TTR and respirometry analyses
7. Prepare talk for PICES
8. Update methods and results of 2023 paper