Preparations of a Lab Rat

Welcome to the first text post of the summer! I’m Natalie Hofmeister, one of two Natalies in this group, and this summer I’ll be writing about my work in the Rubenstein lab here at Columbia. Given that most of us are in transit or getting settled at field sites, I’m starting us off with a run-down of what I’ve been doing to prepare for the summer. In a two-year Master’s program like Columbia’s, we complete the bulk of our thesis research during the summer in between academic years. However, there is a lot of preparation to be done before the summer begins: writing grants, planning methods, ordering equipment and supplies, and – of course – lots of reading. For me, preparation meant a semester of primer design and testing before I could get to data collection.

My thesis focuses on signatures of evolution in the glucocorticoid receptor, a gene that is critically important in the production of the stress response. To look at variation in this gene (NR3C1, which stands for nuclear receptor subfamily 3, group C, member 1*), the first step was designing primers for each of the eight exons. Beginning in January 2014, I spent many hours on the computer and in the laboratory, designing and optimizing primers. Luckily, I had a great teacher (Joe Solomon, thanks for your help!), and after a few months I now have primers for all eight exons and I’m ready to start collecting data.

*Finding that all of these strange combinations of letters and numbers in molecular biology actually mean something definitely made life a lot easier to understand.

Superb starlings, one of the species I study (Pedro Fernandes, Cornell Lab of Ornithology).

Superb starlings, one of the species I study (Pedro Fernandes, Cornell Lab of Ornithology).

So, what goes on behind-the-scenes? Many molecular projects go through the same steps I did this spring, so I’ll try to demystify what we do in the lab.

1. Half of my time this spring was spent designing primers and aligning NR3C1 sequences on Geneious. Obviously, I needed to begin my project by learning to use the software that could analyze all of my sequences. So, I started at the computer, trouble-shooting in Geneious. First, I aligned the superb starling transcriptome (constructed by Joe Solomon) to putative sequences of NR3C1 in the zebra finch and the chicken. Once I developed a hypothesis of the starling NR3C1, I designed primers for each exon using the built-in commands in Geneious. The image below shows an example of a primer with the selection criteria we use to judge which primer is best; generally speaking, you want a primer with about 45% GC content and low scores for dimer and hairpin formation.

An example of primer design in Geneious from the manual (v5.3.6).

An example of primer design in Geneious (Geneious Manual v5.3.6).

 

2. I spent the other half of my spring testing those primers. Usually, in testing primers you start with a temperature optimization to test the primers at many different annealing temperatures, and then proceed to magnesium optimizations to clean up the bands on a gel. Unfortunately, these optimizations don’t always get you pretty bands. Below, I show two unpretty gels and one lovely gel (with the bands boxed in red).

These are a few of the gels I ran to test primers this spring. The left-most gel was one of the worst (see the streaking), while the gel on the right shows clear and bright bands.

These are a few of the gels I ran to test primers this spring. The left-most gel was one of the worst (see the streaking), while the gel on the right shows clear and bright bands.

I used touchdown PCR to get the beautiful bands in the gel on the far right. I’d been working on a particularly troublesome exon for a few weeks when my PI suggested I try touchdown PCR (TD-PCR). This protocol gradually decreases the annealing temperature of a reaction, literally “touching down” by one degree in each subsequent cycle, which selectively amplifies the product that amplified at the highest annealing temperature. For more information on TD-PCR, see Korbie and Mattick 2008.

 

Next step: moving on to this guy! I'll be sequencing on a machine like this one (the ABI 3730) at AMNH this month.

Next step: moving on to this guy! I’ll be sequencing on a machine like this one (the ABI 3730) at AMNH this month.

 

Now that I’ve finished up this preliminary work, I’ll be shuttling back and forth between the Rubenstein lab and the Sackler lab at AMNH, where I’ll complete the actual sequencing of NR3C1 (using a machine like the one shown above). Stay tuned for some (hopefully forthcoming) results!

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