June 30, 2008

Gel of the Day: ...and Then a Miracle Happens

If you're the kind of person who reads this blog then you have probably seen the cartoon with two physicists standing in front of a chalkboard. In the middle of a bunch of equations are the words "and then a miracle occurs" and one of the physicist is saying "I think you should be more explicit in step 2."

Today I had a similar thing happen with one of my gels:

The first two are DNA from taxa that didn't come out last time I tried. The last group of four is a positive control. I used stock DNA and the same primers for all. Now, here's the really weird part: lanes 4 and 8 shouldn't have a product in them at all. No bands should be there. They're the products of a cytochrome oxidase I primer that I discovered today should not work. The primer I was given is for the wrong beetle family; the one I'm studying is too divergent. I was just investigating the problem this morning, in fact, right after I put these samples in the thermocycler. I wound up designing two new Tenebrionidae-specific COI primers and sending an email off to two of my committee members telling them that I'd done so.

So, of course, the primers that have never worked before, and shouldn't have worked this time, are the only ones that produced bands in the first two taxa... but NOT in the positive control. The only band I got in that one is for the small subunit. I should have one for the large subunit as well. It worked last time using the same dilution and primers.

I would just like to say "Argh." Really, what else can I say?

Now, it may be that I used the wrong primers. I've been very out of sorts after not getting enough sleep last night and I could have screwed up. Still, the way I load my tubes, I should have gotten the same result in the positive control (#001) if that were the case. But I didn't.

Tomorrow, I'll clean the PCR products and get them ready for sequencing using the old COI primers. My bet is that I'll get back nothing. And then I'll just start all over again. Maybe someone tampered with the annealing temperature on the thermocycler or something.

I dunno.

I need a drink.

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