Everything in Place?
The project seems to be where it needs to be. Since the last post, I ordered new “degenerate” primers, which consist of a mix of almost all possible sequences that would match the rbcL gene of the algae of interest. The folks at Midland Certified Reagent Co. were very helpful, and the primers arrived quickly.
As can be seen below, we get a much more robust signal from the presumed Avrainvillea with the new primers. There was, however, no signal for Bryopsis species 1. Because that batch of DNA consistently amplified in the past, I am going to attribute the failure to technical issues for the moment.
As was true for the previous run, the lane for Elysia clarki still had no signal. The repeated failure to get a PCR product from this extract suggests that it is not a simple technical glitch. The obvious culprit is mucus. Even though we used a small amount of tissue, it produced copious mucus, some of which may have remained in the sample after purification. The polysaccharides in mucus are expected to interfere with the PCR reaction, so that could be our problem.
Because his lab group has successfully extracted and sequenced DNA from Elysia species, I contacted Gregor Christa from Heinrich-Heine Universität Düsseldorf. He suggested a very simple fix: dilute the DNA template. It seemed too easy, but I performed another run yesterday, and, lo and behold, it worked! The undiluted DNA (& mucus) gave no signal, but the diluted DNA amplified nicely. I guess the dilution reduced the concentration of mucus enough to allow the reaction to proceed, while the sensitivity of the PCR reaction allowed amplification of the diluted DNA.
Everything seems to be on track. The next step is to send some of our PCR products out for sequencing, hopefully within the next few days.