West Coast Green Crab Experiment Part 36

Testing out new SMC primers

Today I used the new primer set for PCR, but with two modifications. One, I UV crosslinked EVERTYHING I could: pipet tip boxes, pipets (just the outside), open empty tubes, and tube racks. Two, I used some of my DNA as well as some DNA from Chelex extractions Sara completed the day before. Her samples were from Tillamook, OR in 2019, but she had already extracted DNA and brought it through the rest of her protocol so she had extra DNA from me. I took all of these DNA samples and their blanks through PCR then ran a gel.

Screenshot 2024-02-06 at 7 11 28 PM

Figure 1. Gel for PCR product from samples 35, 163, 11, my extraction blank, 2/6 PCR blank, 1/22 PCR blank, ladder, 2/5 PCR blank, 11E, 11F, 11G (Sara’s samples), and 11H (Sara’s blank)

MY 2/5 AND 2/6 PCR BLANKS ARE ACTUALLY BLANK!! There’s some smearing on those two, but when you look at 1/22 you can see a clearer separation between some blobs indicating that the 1/22 is likely contaminated. Another interesting thing is that my extraction blank is contaminated, but Sara’s is not. The likely cause of my contamination here is both the SMC primers, samples, and perhaps some general laboratory SMC product contamination that can be taken care of by extra cleaning and UV crosslinking.

What’s next? I return to Chelex land to try and re-extract the samples I’ve been working with. Then, I’m going to rerun PCR and gel. I’m going to run the gel for 30 minutes instead of 40. I’ve been running the gel longer than my original protocol because of some bad separation during a previous gel, but I think I’m going to return to the original time and check for band separation at an earlier timepoint to avoid smearing. Of course, I’ll be UV crosslinking everything possible and cleaning extra hard. Fingers crossed that these positive results are replicated!

But first I get to toss the previous primer sets AND that batch of DNA. We love a good clean-up!

Going forward

  1. Work with Vanessa to leave decontamination station
  2. Work with Vanessa to finish extracting respirometry samples
  3. Work with Vanessa to 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. Genotype remaining samples
  6. Examine HOBO data from 2023 experiment
  7. Determine best statistical approach for analyzing performance data
  8. Demographic data analysis for 2023 paper
  9. Update methods and results of 2023 paper
Written on February 6, 2024