A reason for decontamination station

Today I worked with Vanessa to assess a possible reason for why I was still at decontamination station: SMC primer contamination. Carolyn suggested I run a few DNA samples with the C. maenas COI primers. If my PCR comes out uncontaminated, then that would mean that the SMC primers are contaminated or amplifying contaminats. If the PCR comes out contaminated, then it means that there is some other floating green crab contaminant in the lab.

I walked Vanessa through the PCR protocol for six samples and an extraction blank from 11/8, and a COI PCR blank. After the PCR, we ran a gel with these samples, as well as 11/13 and 1/22 SMC blanks. Since my previous PCR was contaminated, I wanted to see if these blanks were contaminated. I suspected that they were and that we just didn’t run the gel out for long enough to see the bands separate. We ran the gel for 40 minutes today to get clear separation.

Figure 1. Gel for samples 35, 160, 163, 11, 185, 70, ladder, extraction blank, COI PCR blank, 11/13 SMC PCR blanks (2), and 1/22 SMC PCR blanks (2)

First off, I accidentally used an undiluted ladder, hence the giant smear in the middle. Oops. BUT what the gel clearly showed is that the COI PCR blank is NOT contaminated, while all of the SMC primer blanks are. This means that there’s an issue with the SMC primers! Carolyn ordered a new set of SMC primer stock, so we’ll see if that helps.

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. Update methods and results of 2022 paper
7. Examine HOBO data from 2023 experiment
8. Demographic data analysis for 2023 paper
9. Update methods and results of 2023 paper