Sometimes, a perfectly good gel gets funkified in the process of handling. I think that's what happened with this gel, which was otherwise indicative of a near-perfect PCR:
The specimens in this case were three adult and one larval Bolitotherus cornutus which I collected at the MSA foray in Pennsylvania on August 10. The last eight lanes, 25-32, are from the larva. I haven't successfully sequenced a larva before; maybe my dissection technique is improving. The cox1 bands (31) look a bit weak, but the product should do for sequencing.
If you're familiar with electrophoresis gels or have at least been peeking at previous gels of the day, you're probably asking about now, "Mike, what the heck happened to lanes 21, 22 and 27?"
Well, I'll tell you. In order to take this photo using a UV light, I have to remove the gel from the casting tray, put it in a weigh boat and take it to a dark room upstairs from my lab. In the process of transferring the gel from tray to boat to camera, it gets a little scrape over an edge or two. This time, for whatever reason, that caused the bands in three lanes to get smeared. Nonetheless, there are bands there and since they are duplicates from two different dilutions (as far as specimen and gene go, 21=17, 22=18 and 27=31), it's not a problem.
Also, every fourth well is a control; there shouldn't be anything there. There seems to be a very faint band in lane 4, but it's nothing to worry about. I may just try and sequence it to see what the contamination is, since the position of that band is unlike that of any of the legitimate bands on this gel. I suspect that the culprit is a dirty microtube; if my primers, buffers, etc. had been contaminated, I would expect to see that phantom band in all of the control lanes.
The reason I'm running another batch of B. cornutus is that these are the first specimens I've collected from a site outside of Massachusetts. It's worth looking at them just to see if there are any polymorphisms present in introns in the sequences I'm analyzing.
I also read a very interesting paper today:
Leschen RAB and TR Buckley. 2007. Multistate characters and diet shifts: evolution of Erotylidae (Coleoptera). Systematic Biology 56(1):97-112. DOI: 10.1080/10635150701211844.It's very much relevant to my own research. In this paper, Leschen and Buckley analyze the evolution of Erotylidae, a family of beetles only distantly related to the family I'm studying, and link their hyperdiversity to a shift to a diet of macrofungi. It sounds rather familiar, no? It's a very interesting read if you're into this sort of thing. It's based on morphology, not molecular biology, and finds a good link between diet and the external reproductive structure (gonocoxa and ovipositor) of Coleopteran females. Mycologists (at least those with an interest in basidiomycetes) and coleopterists alike will undoubtedly find something of interest in the paper. Enjoy.