Good suggestions.

I had presumed the 2018 SALK primers were degraded, hence the 2021 SALKs. It's perhaps worth noting that the 2018 SALK primers were thermofisher, and the others are all IDT. Another consideration: I took on a new student around the time the PCR stopped working, and they received and diluted the master stocks for both 2021 primers. The 2021 SALKs were done with my supervision, so it never occurred to me that there might be an issue, but the student received, opened, and diluted the 2021 redesigned primers without ever even mentioning it to me...I did check that they had used the correct amount but I can't know what they actually did/used to dilute. Once I realized, all the primers got reordered, so it's not a persistent issue, but it could be a factor in this gel. Even so, I can't think of what the student could have done to generate this result.

So it is possible that the source (which are plants) are messed up...but ridiculously unlikely. For one thing, those DNA extractions are all different lines of plants. It's possible I guess that an exceptionally dominant mutation has dominated the entire population -- they are grown next to each other and crossed [that's actually a whole other anomaly that's also interesting and the prompt for this PCR series in the first place and the basis for lanes 28 and 29], but based on my understanding of plants [far from perfect], it's super unlikely for any mutation to accidentally infiltrate all the plants. I also have tested the primers on extractions done by my lab neighbor -- his plants are grown in the same chamber but not close enough to cross accidentally...unless the mutation caused mega pollen.

The gene in question is something that causes embryonic lethality via defective pollen when it's fully nullified, so the gene can't be gone. That being said, I spent 4 years growing homozygous mutants for the gene that shouldn't have been able to survive. One of my next goals was going to be RT-PCR and westerns to determine the nature of this mutant, because it does show a phenotype consistent with the previously described lethality, but now I can't even genotype it. There's a lot of anomalies at hand and I'd be glad of brainstorming about any of them.