After the exhilaration of obtaining positive results on the cruise, I'm back to Wilmington and back to reality. 6 months in, and I'm still struggling to optimise a reaction to amplify and sequence a gene of interest from the Mycale genus of sponges. It doesn't help that learning all these protocols and their steps require constant repetition and I'm always away this summer. Was happy to have amplified the ITS gene before Key Largo, but the cloning got stymied by a bum cloning kit. The next two attempts didn't succeed, and today, I couldn't even get the PCR reaction working, although I tried twice. What frustrates me is that unlike in ecological research, I don't really know how to proceed to troubleshoot all these problems. The protocol is like a giant black box, and when there's a negative result, I don't know if it's due to my error, insufficient DNA, annealing temperatures and what have you.

Just got reminded not too gently that I should start purchasing my own reagents, but I'm assailed by self-doubts. Have half a mind to can this project and concentrate on the chemical component of my project instead. On the other hand, it worked before, it probably can work again, and I've collected so many samples from so many people it seems like such a waste. Where to find the motivation to keep striving though?


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