Student Research at Duke

It looks like I’ve hit my metaphorical stumbling block.

The miscreant samples that I previously mentioned– the ones that didn’t work out perfectly in the first try– have been giving me more headaches than I care to blog about. But here’s a (not so brief) summary; please excuse me if I start ranting…

After the first round of sequences were analyzed, I tried re-PCRing the ones that didn’t amplify correctly. Some worked the second time, but most of the samples still showed nothing. So I took a step back, and I re-extracted those samples and then ran PCR on the new extractions. But nope, no luck there either. So I ran a whole-genome amplification on these samples, hoping to increase my DNA concentration and bypass the issue of bad tissue samples. But STILL no luck; after running PCR on the whole-genome samples, it appears that while I did have success in increasing my DNA concentration, the troublemaking samples still won’t amplify.

I’ve run out of bullet points on my list of things to do if the samples don’t work…so I’m racking my brain to think of what has been going wrong. Right now I’m trying PCR again, but with a lower annealing temperature…or maybe my reagents are weird and I need a new stock. Hopefully I’ll think of something else to test or find out what’s actually going wrong with my rogue samples.

Either way, I’m feeling the pressure of deadlines (abstracts are due next week, and posters next weekend) and the stress of many failed attempts. And now I’m wondering…what happened to my summer vacation?

I haven’t posted much about the progress of the Tomato frog project, but there is lots of good news to report.

I’ve been busy the past two weeks or so with the samples: first isolating DNA from tissue samples (muscle, liver, toes, other unidentifiable body parts), then running PCR on the extracted DNA for a gene in cytochrome b of the mitochondria, and finally sequencing the subsequent PCR product. I received my sequences back from the Sequencing Lab (located conveniently just down the hall in BioSci) at the beginning of the week. I edited the sequences using this pretty awesome program called Sequencher; then I used another fancy program to compare the sequences to each other and also to Tomato frog samples from a previous study.

For various reasons along the way, some samples didn’t work as well as I had hoped. Maybe they were bad samples, maybe the primers didn’t work so well, or maybe I contaminated the samples along the way. (I’m hoping the last reason is not the case, but when a handful sequenced samples register as “Homo sapiens” with the online genome database, I get worried).

So anyways, I started with 36 frog samples and only 12 of them made it all the way through to sequencing. For the 24 other miscreants, the process starts over again: re-sequencing, re-PCRing, and even re-extracting.

I don’t mind all the re-do’s, though, because we got pretty good data from the 12 successful samples. I’ll try to briefly explain what we think we’ve found…

A previous paper looked at two species of Tomato frogs from eastern Madagascar and suggested that the genomes were so similar, maybe these two distinct species shouldn’t be separate at all. Our project looked at ONE species of Tomato frog from various populations in western Madagascar. When we compared the frogs to each other, we found an enormous amount of genetic variation– and after comparing the eastern frogs to the western frogs, we found that there was more variation WITHIN our single western species than there was between the previous paper’s TWO separate eastern species.

So what does this mean? Hopefully, that what we thought was one species located along the western coast is actually two — or even more — distinct species. But of course, we’ll need to sequence the rest of the samples to confirm this.

Anyways, I was pretty excited when I saw what had come out of four weeks of non-stop pipetting. Looks like hard work might actually pay off…