Science in the City

The follow-up to the successful first sequencing of the RpoB and RpoC loci on Monday has been a hurried attempt to get as many of the loose ends from the other genetic ’sites of interest’ processed as possible in the time I have remaining at JCU.  With the experience I got going through the first runs, I attempted to do a gel cleanup of the YCF5 region for all my samples yesterday.  To do this, I pour all of the amplified DNA product I’ve collected into an agarose gel, then run current through it to separate the chemicals in the solution by size.  Once separate, one can physically cut the gel under UV light, and transfer the trapped DNA product into a tube.  

Chemicals and heat are used to dissolve the gel, which is washed away before the residual DNA is adhered to a thin membrane, called a column.  A separate set of processes then releases the DNA from the membrane into a collecting tube where it is stored.  The normal next step is to see how much DNA is in each sample (quantification) before I can send it for sequencing.  But that’s not what I’ll be doing next!

Besides from working a bit more independently on the gel cleanup, the other challenge of working with the new marker is that it is about half the size of the other two.  This means that it’s harder to isolate, since it moves more quickly through gels and is more diffuse when trying to cut it out a band.  Fortunately, a shorter section of DNA means that less is required to successfully complete the sequencing reaction, but I was nervous as ever about how this run would ultimately turn out.  The gels during clean up yesterday didn’t have the clear banding I saw in the prior two times I’d done this step.   I was tempted to quit while I was ahead, but that wouldn’t be very sporting of me!

Anyhow, after running the final ‘check’ of the product this morning, I learned that whatever DNA was in the gel had been lost in the heavy cleaning process.  Very faint bands yet remained, but it didn’t look like enough for us to process for sequencing.  This means I’m 80% back to the starting point, should I continue seeking to get this one done before I leave.  I’m going to give it the old college try, running the PCR this afternoon, and maybe getting through the gel cleaning, though it’s not likely possible in a half day’s effort.  If I do the clean-up tomorrow, by lunch I might be back to where I thought I was this morning, which means that I’d have to get the results by e-mail.  Certainly not the end of the world, but a bit of a downbeat on this one front.  As Dr. Blair suggested, beginner’s luck never lasts! 

At the same time, I’m also attempting to work out with Kor the correct temperature to run the amplification of the Accd loci, which would give us a fourth piece of barcode information for distinguishing seagrasses.  Since we’re seeking to optimize the process for future work, the relative interest on this piece of the process shifts a bit.  Rather than getting the ‘answer’ per se, we’re trying to find the best process to getting that answer for each species, as a reference for the future.  We’d certainly like the genetic info, but first we need to find the best way of achieving it, going forward.  So alas, it looks like the main body of my accomplishment here is done.  I barcoded the 11 species we looked at for RpoB and RpoC, and I became familiar with the processes (and pitfalls!) of this line of work.  I’ll be returning home much more experienced in my lab skills, particularly with DNA manipulation, and hope to put that to productive use in my employment and teaching!