Weeks Six/Seven: Sequences, Sequences, Sequences

The last two weeks have been very busy, it's crazy to think I only have 3 weeks left now.  I spent most of my time doing more PCRs, growing cells, and analyzing sequences.  As I mentioned a few weeks ago, I have some new primers that I've been working with, rather successfully.  Nicknamed "Dub 2" primers, these amplify the variable d2 region plus a non-variable region on each end.  This allows us to determine if there are different strains of Euduboscquella in our samples that would otherwise not have been amplified using just the unique d2 primers.  After I got some sequences back from these new primers, I found what he had hoped:  a possible new variable region (new strain?) that only matched up about 70% with the original unique sequence.  In order to make sure this wasn't a fluke or an error on my part, Pete and I designed new primers that were specific for this new unique element.

Unfortunately, once these new primers came in, my PCR results were anything but ideal.  One (or both) of these primers wasn't specific enough and amplified lots of unwanted DNA, some up to 1000 base pairs, whereas my target is close to 300 bp.  In order to test if my primers were causing the issue, I re-did the PCR in two different ways; I used the new "Dub 3" forward primer and the old "Dub 2" reverse primer and vice versa.  This seemed to work better, and it now appears that this unique element was found in one of my ocean samples.

Next week, I'll be doing more PCR using the combination of "Dub 2" forward and "Dub 3" reverse to see if I can amplify this sequence in any other samples.  Until next time, Deirdre