I have been having PCR trouble for two weeks now. It’s driving me crazy. One day, the assay worked, the next it didn’t. I’ve been repeating them for weeks, sure it was the dNTPs, since we weren’t using our usually brand, and that was the only reagent I’d changed.
The good news is that it wasn’t my technique. The bad news is that the machine is broken.
The way quantitative PCR works is you add a fluorescent probe to the normal PCR reaction. The probe binds to the middle of your DNA strand and is broken into bits when the taq enzyme replicates the DNA strand. Breaking up the probe separates the fluorescent reporter molecule from the quenching molecule, and the assay emits fluorescence detected by the special (read expensive) PCR machine.
Well, this doesn’t work when the lamp is broken. Not burned out, broken. So we have to get it fixed.
But this has been driving me to distraction for a fortnight, and affecting everything else I did. I’ve messed up genotyping, I’ve locked my keys in the truck at the gas station, I burnt the crust for my lime tart. It’s all been bad, just because I was obsessed with figuring out what I was doing wrong. And equally obsessed with why I could never seem to do anything right.
When Noodles (our postdoc) told me he thought the light was broken, I ran upstairs to ask the only other recent user if her plates had been working. And the tone of voice when she said no and the expression of relief when I told her that we thought the machine was broken told me she felt the same way I did.
It’s a hard thing to remember that sometimes you aren’t doing anything wrong when an experiment doesn’t work. The immediate instinct is that you have screwed up. When you can’t figure out how you are screwing up it’s extremely distressing. And when you realize you aren’t the problem it’s such an incredible relief it’s like falling up.