Weeks Eight/Nine: So much to do, so little time

These past two weeks have been so busy, as I'm not only trying to finish up my lab work but also work on a presentation and paper for my internship.  As usual, the majority of my lab work has been running PCR tests and cloning samples when necessary.  

I have some new primers that I've been working with, nicknamed "Dub 3" that seems to amplify a second unique strain of E. in the variable D2 region.  The primers don't seem to amplify much in the Columbia River Estuary, but two of my ocean samples amplified really well.  This gave me the idea to extract DNA from several ocean samples taken during a few cruises at various times of the year (April, July, August, and November) to see if these would amplify as well.  In fact, after extraction and PCR, all six of the extracted samples amplified!  This is pretty exciting news as it seems this possible second strain (I still haven't gotten sequence results back yet, hopefully I will Week 10) is only present in the ocean, an idea no one had thought of before.  I extracted 24 new ocean samples on Friday and next week will be running the same PCR to see if I get amplification in all samples.

I also started something called qPCR or rtPCR; quantitative (or real time) PCR that lets you get an idea of the amount of gene copies being amplified.  Essentially, you can tell if the amplification in a sample is a lot or a little, helping you to understand if there is more abundance of, in my case, E. in one sample versus another.  My initial run had positive and negative results.  Unfortunately, contamination is very easy when doing qPCR because the machine is so sensitive.  I had some amplification in my negative controls, meaning I need to redo my qPCR next week.  However, my standards, which are based on the cell solutions we send over for sequencing (minipreps) had very good results.  While setting up this reaction is very tedious, I'm looking forward to re-doing it next week and hopefully getting some good results for my presentation and paper.

It's crazy to think I have only one week left!  There's still so much I want to do, but I know I made the most of my time here.  Until next week, Deirdre