July 03, 2008

Gel of the Day: Ambitious but Only Slightly Successful

I was feeling a bit ambitious today; my new COI primers arrived. They didn't work, but I did try them. Amplifying COI is turning out to be a bit tricky. I'm going to have to do some digging on this problem.

The mystery COIs from last time are still more or less a mystery... but they are at least Coleoptera and they are cytochrome oxidases. That much is good. There's nothing with better than 86% identity to them on GenBank, though, including what is supposed to be a Penthe species; I have my doubts as to whether that sequence is actually from that genus.

Anyhow, today's gel:

007 is my putative Pentaphyllus. I tried to amplify lsu and ssu from it. Didn't work. Neither did the same genes from Penthe pimelia (004). 009 and 010 are a pair of Bolitotherus cornutus; I got lsu and ssu from them but no COI. Why? I don't know. For some reason, I can't get any ribosomal sequences out of 007 and 004, only COI... and I can't get COI out of B. cornutus, even though they should all have the same conserved sequences. 015 is a new addition, Diaperis maculatus, a teneb from the subfamily Diaperinae. I only got the ribosomal sequences out of that one, too. Why? I don't know.

I'm neither entirely happy nor entirely unhappy with these products. I at least get to add some more data to my set, and that's something, anyhow. I'll clean and sequence these on Monday and chuck 'em into my alignment. I do wish I had gotten the Penthe and Pentaphyllus rDNA, though. It would have cleared up a couple of questions I still have about these two insects. I may be able to do another extraction from Penthe; it's a big bug and there's still enough left of my one specimen to try one more time. My Pentaphyllus stock is used up, though. They're tiny insects, 3-4 mm long, and I can only use heads and pronota for extraction to avoid contamination by gut symbionts. It's a bit like trying to suck the juice out of a pinhead.

Think I'll go have a nice Scotch now.

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