Oh, the bitter taste of failure.
In order to create the well resolved phylogeny I’m looking for, I have to acquire protein-coding gene data. Over the past few days I have attempted to amplify the genes minichromosome maintenance complex 7 (MCM7) and RNA polymerase II (the largest subunit, called RPB1), without success.
You can see from these images that there a couple of faint bands in the MCM7 attempt, but some of them are obviously too small to be the right product. On the other hand, there were no bands at all from the RPB1 run (except for the positive control). On the plus side, at least I didn’t have any negative control issues.
Now comes the process of trying to figure out what is going wrong. Could the primers need adjustment? Perhaps the PCR thermocycling program is off? Would a nested procedure work better? Should I try adding Betaine or DMSO to the reaction mix? The positive control band for both reactions indicates that I probably made my master mix correctly. What I suspect is more likely the problem is simply a lack of product. Because these protein-coding genes generally exist in low copy numbers within the genome to begin with, it is more difficult to successfully amplify them even under ideal conditions. Additionally, we have to use degenerate primers, which have reduced sequence specificity. Not to mention the intrinsic problems associated with genetic work on these organisms (e.g. small amount of extracted DNA, divergent sequences, etc.).
Back to the drawing board…
Nicole Reynolds — 8/13/13