Hello? Do you have any DNA?

Finally! A day where I felt my time spent in the lab was actually productive! My goal for the day was to extract DNA from one of my random fungi samples (I ended up using a fairly recent one out of the fridge, since I forgot my larger stash on the stairs at home), run PCR, then run the sample on a gel to see if I had intact DNA that could be sequenced.

I got a pretty good start to the day, doing most of the DNA extraction before lunch. After lunch (yes, I actually ate lunch in the dinning hall for the first time in quite awhile), I tested the DNA in the spectrophotometer and- for once- I actually had quite a bit! I quickly mixed it up with the needed reagents for PCR and put it in the thermocycler to run for about an hour and a half.

When the PCR was done, I made up an agarose gel and ran my samples. To do this, you mix your sample with a dye, then put it on a gel (with a standard), which is then attached to an electrical current. The current causes the DNA to separate in the gel based on size, and then you can determine the size of your DNA strand based on how far into the gel it has run and the size of the standard you've put in.

Anyway, after running a gel, we usually take pictures of them. But today, the camera didn't feel like turning on, so we just peered into the light box at it. I probably should have run the gel longer, but it did look like I had some intact DNA, which is pretty encouraging, considering how much trouble I was having with it last semester.

Finally, while the PCR was running, I got to have a bit of fun. First, Thursday afternoon is chemistry lab, so while my samples thermocycled, I watched the chemistry students use Thin Layer Chromatography (TLC) to separate some of the components of leafy greens. Thin Layer Chromatography is a technique used to separate components of a mixture by placing a small sample of the mixture at the bottom of a TLC plate (the stationary phase, usually made of silica, alumina, etc. with a plastic or metal back), then placing the plate in a mobile phase (often some kind of solvent), which moves up the plate via capillary action, taking the mixture with it and separating it into components along the way. This was particularly cool looking with the leafy greens because the solvent left bands components in fun shades of green.

ANYWAY, the second fun thing I got to do, was set up and test (a.k.a. play with) the new mini gas chromatograph! Gas chromatography is similar to TLC in that it separates a mixture into it's components. Except, gas chromatography uses a gas as the mobile phase and has a specially coated column that the gas (and sample) move through that acts as the stationary phase. As the sample moves out of the stationary phase, it is recorded, resulting in a graph with a bunch of peaks, each representing one of the components of the mixture. As always, I'm sure googling "gas chromatograph" will lead you to an article with more detail. Or, there's always Wikipedia.

Our new gas chromatograph is pretty awesome since it's very easy to use (you just inject a tiny, tiny, drop of your sample using a syringe), and it's hooked up to a computer that records the data! I realize that for many people, this would not be all that exciting, but for our lab, it's a pretty big deal. Our old gas chromatograph printed directly to a printer, making it difficult to transfer the graphs produced to a digital format. Mostly, I'm just excited to use a machine that works pretty much as soon as you take it out of the box, after spending last spring trying to get the HPLC to work (HPLC- high pressure (or performance) liquid chromatograph- yup, quite a bit like the gas chromatograph, but uses liquid as the mobile phase).

Whew! That was a lot of chatter that was not related to Plan. Sorry.

One final though for the day- I was encouraged this evening to get an email from my Plan Sponsor (like... a thesis advisor or some such, the professor I'm working with most at Marlboro) asking a few more questions about the work I'd done in the lab today since he'd been thinking about ways to check and see if the gel was running correctly. One of the things I love about Marlboro is having professors who put so much time and energy into their students projects! It's nice to know that I'm not the only one who think about my Plan after leaving the college in the evening. Thank you T! (I figured I'd just go with initials if I'm not talking about myself- for the sake of privacy and such)

That was far too much typing. Sorry about that, I'll have to learn to make shorter posts...

The Fungi Under the Stairs

Or, on the stairs, as the case may be.

So, my attempt at isolating the correct fungus continues. My bat wings and swabs on agar plates are still sitting on the stairs, and so far, they have not produced what looks like the correct fungus. So, in hopes of better results, I took some agar plates, sterile swabs, and test tubes with sterile water with me into Bat's Den cave down in Massachusetts. Bat's Den is a known hibernaculum, and the Mass and NY DEC have been doing bat counts there for the past few winters.

In the cave, we noticed that while there are far fewer bats then in previous years, those that remain seem to be much healthier then in past years. While is great, since it might mean that White Nose Syndrome has run it's course and the bat populations can start building back up (possibly), it made finding infected bats to swab rather difficult. I thought I could get away with swabbing just two of them in the main room (which is right at the entrance to the cave), but after swabbing one once, it flew away. I ended up leaving my swabs, etc. in the main room while hands and knees crawling and belly crawling back into a few smaller rooms. Typically, the room with the longest crawl had an infected bat, so I had to crawl back out, and back in again with my sampling kit.

In total, I ended up taking samples (via sterile swabs) off of 4 bats that looked to be infected. I then took the sterile swabs, placed them in the test tubes, and sealed them back up, hoping to keep them a sterile as possible. I can't say that caves are the most sterile environments, and taking samples did turn out to be a bit difficult. Anyway, I was glad to get a chance to go to Bat's Den for samples, and because it's a pretty interesting cave that is currently only open to bat counts.

Anyway, after returning to the lab, I smeared the samples on more agar plates and today, I added them to the collection on the stairs. Actually, that collection has grown to the point where, in combination with the caving gear and shoes that also live on the stairs, it's getting to be a fire hazard. I'm hoping that this fire hazard might pay off by growing the fungus I'm looking for. Otherwise, it's back to another cave for another sample.

I've also managed to make a bit more progress in terms of figuring out why my PCR hasn't been working. One of the professors from the Vermont Genetics Center (I'll talk about them later. Maybe when I get around to talking about RNA and gene chips and the like) was down at Marlboro College today helping out with the Molecular Biology Lab (which I'm auditing, despite the fact that I took it two years ago. I like lab work, what can I say?) and took a look at my PCR protocol. He changed a few lines in the protocol on the thermocycler (the machine that you put a template and a primer in and it produces more of the template by cycling through different temperatures... yup, not explaining that very well... ), but he said that everything else I was doing was good, so I should get results the next time I try and run PCR (Thursday perhaps?). He also suggested going back to the potato dextrose agar plates, so the lab will soon be filled with the lovely smell of boiling potato!

Sorry that post was not particularly interesting... other parts of my life have been a little crazy recently, and I'm really looking forward to doing some reading and falling asleep. Not reading for Plan, of course, but reading the seventh book in Patrick O'Brian's Aubrey/Maturin series- which I highly recommend if you enjoy sailing and adventure and nautical history and the like. Not to mention, a 21 book series keeps you going for a least a little while! Plus, it's a nice change from my usual reading genre, "popular science." What a surprise.

Wait, what are you doing?

So, what am I actually doing for my research?

Well, in the northeastern US bats have been dying off at alarming rates due to White Nose Syndrome. Right now, researchers are speculating that the bat deaths are caused by G. destructans, a newly discovered, cold-loving, white fungus that grows on the noses and wings of the bats while they're hibernating in caves. Somehow, this is linked to the bats waking up more often, searching for food, and eventually dying of starvation (more or less...). I was curious if there was anything that could be gained by looking at what RNA 'pathways' were turned on when the bats are exposed to the fungus.

To look at what RNA pathways are expressed in a cell when it's exposed to something, like a fungus, you need to be able to compare the normal pathways to those after the cell has been exposed. (This is all a rather complicated process, but basically, RNA is the precursor to protein, so by looking at RNA pathways you can begin to see how a cell is responding to something- yes, I know I'm leaving out lots of details and really simplifying it... sorry.) For a few different reasons, I decided to use mouse cells instead of bat cells (mostly because gene chips for bats don't exist, their genome hasn't been sequenced yet). I bought a cultured cell line from ATCC, and started growing up the cells last summer (for those of you who don't know, regular cells, say, skin cells from your hand, would die after dividing only a few times. Cultured cells are essentially given cancer, so they continue to divide for more like 20 times- again with the simplifying).

Now, I have my cells that I'm going to expose, but what about the fungus? Well, that I tried to get a sample of by hiking up to the Mt. Aeolis bat cave and collecting some dead bats from outside the gate. I took these back to the lab, swabbed them, rubbed the swabbed on agar plates, put the bats in the deep freeze and tried to grow some fungus. And this is where my life in the lab goes downhill.

I grew lots of fungus over the summer. The first one seemed like maybe it was G. destructans, but then something else grew that looked more like it, so I switched over to culturing that one. I had already decided to run PCR on the samples to make sure I had the right fungus, but the PCR wasn't really working and I wasn't getting any results (ugg. Sick of explaining molecular biology terms. I'm sure Wikipedia has an article on what PCR is. Basically, it's used to amplify a strand of DNA so that it can be sequenced and compared to an existing sequence of DNA, thus verifying that you do or do not have the species you think you have). After many months of trying to fix the PCR and many months of growing the fungus, I read an article that said something along the lines of "G. destructans DOES NOT grow at room temperature." And included a picture of what it should look like. Oops. Not growing the right fungus.

Now, briefly, back to the mouse cells. They were growing and because I wanted to get things moving along, I decided to expose them to the fungus without being totally positive that the fungus was G. destructans. I did this, and afterwards, went on to try and extract their RNA. The only problem was that I extracted the RNA lots and lots and lots of times, and every time the concentration came out to about a tenth of the concentration that I needed. After many months of trying, I finally got the concentration up to about half of what I needed, but that was about the same time I realized I had the wrong fungus.

So... where does that leave me now? Well, back in December I took the bats out of the deep freeze, cut some of their wings off and put them on agar plates, which I have been trying to keep at around 14 degrees Celsius in hopes that they will grow fungus. Turns out, 14 degrees C is both the temperature at which G. destructans grows best and the temperature about half way up the stairs to my apartment (varying a bit based on the outside temperature) and so that is where they have been sitting for the past month (except when I went home to my parent's house for a few days and they lived in their bedroom, also around 14 degrees C). But, today, they moved. I took them into the lab, looked them over, and realized they're still not growing what I want them to. So, more bat wings were cut off, different types of agar were used, and now the wings are back on my steps.

Wow. That was a longer post then I had intended. Sorry everyone.
I realize I've still left a lot out, like, what happens after I get this RNA? Will I ever get this RNA? Is this a wild goose chase? When exactly will I give up and realize that many Plans at Marlboro involving complicated lab work have failed and that mine is one of them? Yeah, I'll write about some of that later. Now, bed.

What is this?

Over the past few months, I've had a few people show some interest in the research I'm doing for my Plan at Marlboro College. So, to avoid answering questions like "What are you doing for Plan?" "How is Plan going?" "What's up with those bats these days?" etc. over and over again, I thought I'd try and keep everyone up-to-date with a blog.

First off, for those of you who don't know, 'Plan' is Marlboro College's senior thesis equivalent, which many have likened to writing a master's thesis. I'm starting this blog rather late, since I've already finished one semester 'on Plan' and starting next week, I'll be plunging into the second semester of Plan, and my final semester at Marlboro (unless some rather unfortunate or fortunate things happen between now and May).

Second, here's a rather brief description of what my Plan is about. My Plan consists of three different sections: a review-type paper comparing White Nose Syndrome in Bats to Colony Collapse Disorder in Bees and Chytridiomycosis in Amphibians (worth 30% of my grade), a research article/lab write up type paper that will come from the research I've been doing since last May, which includes exposing cultured mammalian cells to G. destructans (the fungus associated with White Nose Syndrome (worth 50%), and finally, a biochemistry exam (worth 20%). At some later date I'll give a bit more description on what exactly I'm doing for my research and what of that I've done so far.

After finishing all my research and writing, I get to send it all off to an outside examiner (someone who's knowledgeable in the field and is not associated with the college) who will (presumably) read it, and then come for my orals, where I'll babble about White Nose Syndrome and molecular biology for awhile, and then stand anxiously outside the room while the outside examiner and my Plan sponsor decided on an appropriate grade.

Finally, I should note two things. First, I'm not a writer by any means, that's part of the reason why I'm a science student. Thus, I apologize in advance for spelling errors, grammatical mistakes, and generally poor writing. Second, I am on Plan while writing this, so I doubt I'll bother to update it very often. I'd like to make a goal of once a week, but something makes me think it won't even happen that often.

Okay- for next time, a review of what I've actually been doing in terms of research for the past few months.