West Coast Green Crab Experiment Part 31

Moving past decontamination station (for now)

…I think we’re past decontamination station?

After two months of no extractions and no fume hoods kicking up random stuff into the lab, I decided to revisit my contamination issues. I used PCR reagents from November and got a brand new set of reagents. I made new SMC F and SMC R primers and made enough PCR MM for four samples for each batch of reagents (11/8, 11/9, 11/13, and 1/22). Side note: I think I didn’t pipet the correct amount of SMC F and SMC R into the new aliquots when I made them, because when I checked my pipet after adding SMC primers into my PCR MM, the pipet said 2 µL instead of 10 µL. So, I added another 8 µL of SMC F and SMC R into the new aliquots, and then 8 more µL of the aliquots into my PCR MM.

Screenshot 2024-01-22 at 4 18 27 PM

Figure 1. PCR blanks using reagents from 11/8, 11/9, 11/13, and 1/22

The gel imaged horribly but both Carolyn and I agree that the bands are likely the primer-dimer! So, things are okay?

One of the 11/9 bands looks funky to me, so I might toss those reagents (or run that blank on a gel another day). But the 11/8 and 11/13 bands look good! The 1/22 bands are REALLY bright, which leads me to believe that I may have added too much SMC F and R to the master mix. I’m going to see how much of the stock is left. I should have used 10 µL of the primers to make the PCR MM, so I can check to see if the stock quantity is different than 90 µL.

Going forward

  1. Work with Vanessa to finish extracting respirometry samples
  2. Work with Vanessa to continue with Chelex extractions, PCRs, and gels for TTR crabs
  3. Perform Qubit assay with any sample consistently not showing up on a gel
  4. Genotype remaining samples
  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
Written on January 22, 2024