September 25, 2007

Where Are the PCR Pixies When You Need Them?

As you might have noticed, I still haven't posted the results of sequencing that Vietnamese Lentinula sp. ITS region. I blame the PCR pixies for their not showing up to save the day. Let me explain.

I ran the reactions and attempted to have the sequences taken, but when I got my results back they were an utter mess. Apparently there was no cysteine in the gene product. Well, there was something there, but my sequence showed conflict after conflict after conflict. I had only one indel, so that wasn't the problem, and I did have a couple of loopy-looking peaks due to not getting all of the ethanol out of my mix after cleaning, but those things are easily enough resolved by looking at complementary sequences. The problem is that at least a third of my c peaks have been squeezed into non-existence because, I am told, the polymer we have is too thick. I don't understand why that should be, since it's swapped out at least once a week when the sequencer isn't in use. Sure enough, though, five other people's sequences came back with exactly the same problem as I'm having.

So we haven't much choice but to ship plates off to somewhere far, far away to get our sequences done in such a way that we don't all have to spend the next week sitting in front of a Sequencher edit screen. Thus, we're all pooling our samples on a few plates to be shipped out tomorrow afternoon.

To add to the problem, coordination of this effort wasn't exactly stellar. I finally sent an email around this morning asking everyone to reserve space on the plates, and thus it wasn't until 3:30 that I found out that we needed to ship the things out tomorrow. Because of my schedule, I don't have any time during the day to run the necessary PCR, so I didn't get started on them until nearly 4:00 and by the time I'd whipped up my little microtubes of reaction mixes it was 4:45, and so I can't pull the things off the block until about 8:15 tonight... so I have to go back in and do that.

I don't get it. Look, the helpful magic faeries have been known to go out of their way for everyone from princesses to shoemakers, haven't they? You would think that in this day and age there would be helpful PCR pixies on call to come in at night and do little things like put reaction tubes on ice and whip up buffers and master mixes and maybe design a primer or two... but no. Despite my willingness to leave out as many pipette tips as they could ever want as payment, I am told that the PCR pixies don't include our lab on their nightly route. I must say that I'm rather offended by this fact. Here we are doing all sorts of interesting and important and wondrous molecular biology and reconstructing fungal phylogenies and not a single PCR pixie will deign to remove my mixes from Bob the Friendly PCR Machine (yes, we have pet names for our PCR machines; another one is named Helga).

Stupid PCR pixies. Next time I see one, it's going for a swim in a big tub of liquid nitrogen.

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