July 20, 2008

Gel of the Day: Moderate Success

Sound the trumpets! I actually got some PCR products out of today's experiment with COI primers.

It looks from the results like both primer sets work, although my new Tenebrionidae-specific primers (PATten and JERten) amplifies a smaller fragment than the traditional Coleoptera COI primers. Also, it seems that the traditional primer set worked for one species but not for another in the same tribe (Diaperini), and the new primers worked for the other member of that tribe (both are Diaperini; the new primers worked on Pentaphyllus but not Diaperis and the old ones on Diaperis but not Pentaphyllus). Why? I dunno. I still have my suspicions about how monophyletic tribes, and even subfamilies, really are in this family, and maybe that's part of what I'm seeing in these results. I may know more once I try aligning these sequences and especially when I add this data together with the large and small subunit stuff.

Otherwise, both primer sets worked on everything except one miss by the new set on a single Bolitotherus cornutus, but it worked on the other specimens of the same species, so I really have no idea with that one.

Still, here's the gel. There are a couple of faint bands in the lower half that may be hard to see in the reduced photo, but they're in there. Hopefully I'll still be able to get sequences out of those:

I used several different dilutions (1:100, 1:10, 1:5) and got some products from all of them. It all comes down to using a lower annealing temperature for the COI stuff. From here on in, I should be able to get those sequences reliably.

Now I'm just hanging around and waiting for my sequencing reactions to finish up, which they should do in another 2.5 hours. Thankfully, that Del Tenney DVD I have is a double feature; I can watch Curse of the Living Corpse while I'm waiting. I'll be here late on a Sunday, but I should be ready to go when tomorrow's sequencer run starts up at 11:00 AM. Go, me.

If anybody wants me, I'll be in the conference room.

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