Wednesday, January 14, 2009

PCR is Magic

Over the summer I attempted to sequence COI from a few samples of the genus I'm working with.  I had little to know luck doing this on my own in an abandoned lab at Iowa State.  Although I had various friends helping troubleshoot, I was taking away their time and supplies and spending limited funds on a seemingly endless struggle to sequence what is often considered a relatively easy gene to sequence (well know, "universal" primers are published, etc.).  So, I headed east to a lab at the Cornell Lab of Ornithology, the Evolutionary Biology lab to work with Irby Lovette and his army of students and lab managers.  They offered open arms.  I continued to have minimal success, then had help developing primers from the success I did have.  I went gang busters yesterday finishing the PCR on the remaining samples using these new primers only to continue to have minimal luck (23 out of 36 samples showed promising results after running a gel).  I guess this is par for the course and something that people perhaps don't always talk or write about in the literature.  It's frustrating for sure.  PCR is really magical to me.  Not that I don't understand the basic process, but that there seem to be complexities beyond what I can understand.  Why some samples work and some don't under the "same" conditions (or at least as humanly possible) is amazing.  That it even works at all is amazing.  So I've progressed and I'm moving on to other genes, narrowing down the
remaining COI samples, aligning and analyzing what few sequences I do
have and learning a lot.