June 18, 2008

I Can Haz PCR Produkt

In rather happier and more personal news, I finally got PCR product out of Bolitotherus cornutus, my first successful attempt at doing so.

The gel shown here demonstrates that I successfully amplified DNA from my samples. This was extracted from the pronota of two B. cornutus, one male and one female. Not all the amplifications worked, though. The lanes labeled 1, 5, 9 and 13 are a segment of the large ribosomal subunit rDNA and those labeles 2, 6, 10 and 14 are the small subunit sequences. Lanes labeled 8 or below are using a 1:100 dilution and those labeled 9 or higher a 1:500 DNA dilution. The lower dilution clearly works better; I get at least two of my four products.

Two other sequences, the wingless gene and cytochrome oxidase 1 (COI) didn't amplify at all. That's probably because there are only a small number of copies of these present in the total genome, unlike the much-duplicated ribosomal sequences. I'll try a lower dilution, maybe just 1:20, and have another go at those later.

Mind you, I haven't actually sequenced these yet. This only shows that I amplified ribosomal DNA from something. While the primers I used are supposed to be beetle-specific, there's still a possibility that I'm looking at DNA that came from something that lives inside the beetle. I won't know that until I get back sequences and try aligning them with known ones.

That's the less likely scenario, though. This is just a small step forward for me, but I'm feeling pretty good about it at the moment. As for today, I expect to be spending more time in the field than in the lab. I'll get to cleaning and readying my products either late today or on tomorrow.

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