I haven't posted much, because change has been gradual. I'm advancing like an ice skater over a cattlegrid: slowly, carefully, hazardously.
I spend most of my days in the molecular lab, except for when I really can't stomach it, and then I write my thesis or read papers or do one of the other things I have to do to advance my PhD.
I've also been exercising hobbies: in particular, training a hawk and courting the odd (mostly very odd) bloke, both of which are exactly like PCR optimisation: you do something, then wait for a bit, a change happens. You have no idea what exactly caused that change to happen. If it's a good change, you gingerly carry on doing what you were before, until you get a bad change. If it's a bad change, you try something else until you get a good change. At an agonisingly slow pace, you lurch towards success.
I am getting there, though. I am managing (I think) to amplify the right region of DNA, but one of the problems is the presence of inhibitors in my spider extracts that reduce the capacity of the PCR to amplify the DNA. I'm circumventing this somewhat by doing two PCR cycles, but in my latest PCR run I got three bright, crisp bands; the lowest, where the primers are helpfully sticking to themselves to create hairpins, and two higher ones, one of which is the one I want and the other of which is an unwanted region of the gene. So I am amplifying the right region (I think), but I'm also amplifying other regions. I am going to try and raise the annealing temperature, which may help make the primers stick to what they're supposed to stick to, and not stick to other things. I have tried this before, a lot, but this time it might work because I (dare to) think the other problems have been solved and the only problem now is specificity.
Every PCR that I do seems to yield better results, even if the improvement is only slight. I am going in the right direction, and I just need to be patient and keep going, and not anger the god of PCR by feeling sure it will work. Then I will get DNA sequences, and be able to start constructing my tree.