Today we started off troubleshooting our gels from late last night (thanks Sarah and Ruth!) This is one of the most helpful processes for me. For example, how unexpected results from PCR could be from…
- Using too many cycles so that the reagents are used up and the amplified product degrades
- Incorrect temperatures
- Primer dimers and how to tell the difference (make your product bigger!)
- Nonspecific amplification
We started off with a bunch of PCR. The others started making DIG probes using the primers that had worked for the target pathogens in seastars and corals yesterday and the PCR DIG Probe Synthesis Kit: Roche cat. no 11 636 090 910. Their notes should have more detail on how the abalone RLO protocol was modified.
I re-ran the CAR-1 (coral-associated rickettsia) primers with a different protocol – the same touchdown PCR I described yesterday but it’s nested. This means I ran it once, then I took 1 uL of product from each of the 5 sample tubes from this first run and used that as the template for a second run.
I also ran the Ehrlichia primers with an annealing temperature of 56.5 (otherwise our standard PCR steps and reagents). Yesterday it ran best at 55 and we got this improved protocol from Lisa.
Sarah photographed the gel. Overall, the Ehrlichia primers did not amplify anything while the CAR-1 primers amplified a clear, bright band in all the coral samples and not the negative control. However, that band was not around 170 bp as we expected based on Casas et al. so we’re not sure what these primers are amplifying. Looking forward to more troubleshooting discussion tomorrow!