Goodness! I've been busy! Here's a post I started quite awhile ago, so the time line is all off (for example, my first trip up to UVM was more like five weeks ago, not three). I promise I'll write a bit more sometime soon...
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Well, first off, my trip to UVM nearly three weeks ago. I spent the Tuesday of that week extracting DNA from a fungus I thought was G. destrctans. It took most of a morning, and when I went to read the final DNA concentration, it was fairly low. You really don't need much DNA to run the PCR reaction on, so T and I agreed that I probably had enough, and that I should try running the PCR up at the Vermont Genetics Network, since it had failed to work so many times in the lab in Marlboro. Luckily, I needed to drive up to Burlington that coming Thursday (for car shuffling), so I got in touch with the guys at VGN and they graciously offered to help me out when I got up there.
When I arrived at UVM (a wee bit late, I always forget how long the drive takes), the first thing we decided to do was to test my sample using the nanodrop. The nanodrop is a cool little machine that takes a very tiny drop of your sample and tests it's absorbency at a bunch of different wavelengths, very similar to the spectrophotometer here in Marlboro, but on a tiny scale. The results from this showed that I really didn't have any DNA, and we decided it wouldn't be worth the resources to try and run PCR on the sample. Instead, they suggested I come up the next week with my agar plates of fungus and we do the DNA extraction and the PCR in their lab.
This would have all worked out nicely, except we had a major snow storm the next week, and instead of driving to Burlington, I hung around Marlboro and dug myself out, then went and helped my parents dig themselves out. And, of course, rescheduled my visit to Burlington for the next week!
This past Wednesday, I finally made it up to Burlington, with fungus, DNA extraction reagents, PCR primers, and PCR reagents in tow!
(to be continued some other day when I have more time)
Saturday, March 6, 2010
Saturday, February 27, 2010
Filler.
Well, it's been a rather long time since I managed to write anything here. And sadly, I'm not actually going to write anything tonight! But, coming soon, there will be a detailed description of my adventure up to the Vermont Genetics Network at the University of Vermont, hopefully in time for me to make another post about my second adventure up to the Vermont Genetics Network, which is coming up this Wednesday! For now, I'm enduring and enjoying (depending on the circumstances) the snow that we received this week!
Now, on to more important things. Like eating ginger chews.
Now, on to more important things. Like eating ginger chews.
Monday, February 15, 2010
Ups and Downs
I just realized. I have agar in my hair.
First the ups.
This morning I thought I'd take a look at some of the fungi that's been growing in agar plates on my stairs for the past few weeks. I looked at it all recently, but I thought it was time to take a sample of it, stain it, and look at it under the microscope. I had one agar plate that was growing something that looked a little like G. destructans, but I really was trying not to get my hopes up, since I've grown lots of things that I thought *might* be G. destructans, and turned out not to be.
So, I cut a sample off the plate, put it on a slide, stained it, threw a cover slip on, and looked at it under the microscope. At first, it just looked like all the other things I've looked at- kind of stringy, with little blobs on the ends of some of the strings. However, after taking a closer look, I thought I might see some of the curved conidia, which are found on G. destructans. The only problem was that with the cover slip, I couldn't magnify the sample as much as I wanted to. So, I cut a new sample off the plate and stained it. Then, I waited around while the stain dried, which took a long time, despite my holding the slide over the red-hot incinerator and singeing my hand (not really). Finally, the slide was dry, and I could look at it under the microscope at a higher magnification. And there they were! Curved conidia that looked quite a bit like the ones in the pictures of G. destructans that have been published! Yay!
What all this means is, I'm back to where I was last April! Brilliant! Now, if I can just finish up everything else and write it all up, I might graduate in May. Excellent.
Ah well, on to downs.
After taking lots of pictures of the fungus under the microscope, I decided to finish up some prep work for biology lab. A. was away today, so I was suppose to run bio lab, which shouldn't have really been a difficult task, except that I hadn't really looked over the procedure quite as throughly as I might have liked. Anyway, we needed more agar plates, and since the agar was already made and autoclaved, all I had to do was heat it in the microwave, and pour it into plates. Usually an easy task. One I've done lots of times. Of course, today, it didn't go quite like that.
I had one flask in the microwave and was waiting around for it to boil. It finally did, and I put on an oven mitt to remove the very hot flask full of boiling agar from the microwave. I had the flask just barely out of the microwave when it slipped through my oven mitted hand, and crashed down on the counter. Had the flask been full of cold water, it might have just sloshed a little and made a minor mess on the counter in the immediate vicinity. But no, it was full of boiling agar, so it boiled and sloshed violently all over the counter, the spectrophotometer, some boxes of gloves, the floor, my arm, a bit on my face, and into my hair. *sigh* Agar is such a pain to clean up. Once it cools a little, it congeals, and then one is stuck cleaning up a weird smelling jelly that has slightly adhered to the surface it's landed on.
And that, my friends, was my time in the lab today.
(Actually, my time in the lab continued on through bio lab where I made a bit of a fool out of myself by not really knowing what I was doing... but that seemed like a less fun story to tell.)
Time for a shower!
First the ups.
This morning I thought I'd take a look at some of the fungi that's been growing in agar plates on my stairs for the past few weeks. I looked at it all recently, but I thought it was time to take a sample of it, stain it, and look at it under the microscope. I had one agar plate that was growing something that looked a little like G. destructans, but I really was trying not to get my hopes up, since I've grown lots of things that I thought *might* be G. destructans, and turned out not to be.
So, I cut a sample off the plate, put it on a slide, stained it, threw a cover slip on, and looked at it under the microscope. At first, it just looked like all the other things I've looked at- kind of stringy, with little blobs on the ends of some of the strings. However, after taking a closer look, I thought I might see some of the curved conidia, which are found on G. destructans. The only problem was that with the cover slip, I couldn't magnify the sample as much as I wanted to. So, I cut a new sample off the plate and stained it. Then, I waited around while the stain dried, which took a long time, despite my holding the slide over the red-hot incinerator and singeing my hand (not really). Finally, the slide was dry, and I could look at it under the microscope at a higher magnification. And there they were! Curved conidia that looked quite a bit like the ones in the pictures of G. destructans that have been published! Yay!
What all this means is, I'm back to where I was last April! Brilliant! Now, if I can just finish up everything else and write it all up, I might graduate in May. Excellent.
Ah well, on to downs.
After taking lots of pictures of the fungus under the microscope, I decided to finish up some prep work for biology lab. A. was away today, so I was suppose to run bio lab, which shouldn't have really been a difficult task, except that I hadn't really looked over the procedure quite as throughly as I might have liked. Anyway, we needed more agar plates, and since the agar was already made and autoclaved, all I had to do was heat it in the microwave, and pour it into plates. Usually an easy task. One I've done lots of times. Of course, today, it didn't go quite like that.
I had one flask in the microwave and was waiting around for it to boil. It finally did, and I put on an oven mitt to remove the very hot flask full of boiling agar from the microwave. I had the flask just barely out of the microwave when it slipped through my oven mitted hand, and crashed down on the counter. Had the flask been full of cold water, it might have just sloshed a little and made a minor mess on the counter in the immediate vicinity. But no, it was full of boiling agar, so it boiled and sloshed violently all over the counter, the spectrophotometer, some boxes of gloves, the floor, my arm, a bit on my face, and into my hair. *sigh* Agar is such a pain to clean up. Once it cools a little, it congeals, and then one is stuck cleaning up a weird smelling jelly that has slightly adhered to the surface it's landed on.
And that, my friends, was my time in the lab today.
(Actually, my time in the lab continued on through bio lab where I made a bit of a fool out of myself by not really knowing what I was doing... but that seemed like a less fun story to tell.)
Time for a shower!
Tuesday, February 9, 2010
PCR continues...
Sadly, after another week of tweaking things in the PCR, I have nothing more to show than I did two weeks ago. As usual, I clocked many hours in the lab, changing something small, running the PCR, running a gel... and getting no results on the gel. Over and over and over and over again.
And so what am I doing next? Well, first I want to check and make sure the primers that I'm using for the PCR are actually working. To do this, I need some sort of control template (or sample) that I can extract the DNA from, then run PCR on with the same primers I've been using. One of guys at UVM suggested using a olive colored fungus that grows on a potato dextrose agar plate if you leave it out for an hour. So, last week I spent a small part of my lab time making more potato dextrose agar (PDA) plates.
Making PDA is actually kind of fun. You take 200 gram of potato (roughly half of a large potato), cut it up, put it in one liter of boiling water, and boil it for 30 minutes. Then you strain the liquid off using cheese cloth (or, in my case, a plastic-y table mat that had small holes- we use them in the sinks in the lab so that if you drop a piece of glassware, it has something to protect it from shattering on the hard sink surface). Then, you add some dextrose and some agar, and you throw it in the autoclave. Mostly, I just find boiling potatoes in the lab kind of amusing.
Anyway, I made the agar, I poured the plates, I left one out for an hour than left it at room temperature to grow olive green fungus. Only, none has grown. So... I'm currently twiddling my thumbs, waiting for fungus to grow.
In the mean time, I swabbed one more sick bat and smeared the swabs onto PDA plates, in hopes that G. destructans would enjoy PDA. The plates are now on my stairs, and I am waiting. Waiting. Waiting. Waiting.
I suppose that's all for now. Just sitting around waiting for some fungus to grow...
And so what am I doing next? Well, first I want to check and make sure the primers that I'm using for the PCR are actually working. To do this, I need some sort of control template (or sample) that I can extract the DNA from, then run PCR on with the same primers I've been using. One of guys at UVM suggested using a olive colored fungus that grows on a potato dextrose agar plate if you leave it out for an hour. So, last week I spent a small part of my lab time making more potato dextrose agar (PDA) plates.
Making PDA is actually kind of fun. You take 200 gram of potato (roughly half of a large potato), cut it up, put it in one liter of boiling water, and boil it for 30 minutes. Then you strain the liquid off using cheese cloth (or, in my case, a plastic-y table mat that had small holes- we use them in the sinks in the lab so that if you drop a piece of glassware, it has something to protect it from shattering on the hard sink surface). Then, you add some dextrose and some agar, and you throw it in the autoclave. Mostly, I just find boiling potatoes in the lab kind of amusing.
Anyway, I made the agar, I poured the plates, I left one out for an hour than left it at room temperature to grow olive green fungus. Only, none has grown. So... I'm currently twiddling my thumbs, waiting for fungus to grow.
In the mean time, I swabbed one more sick bat and smeared the swabs onto PDA plates, in hopes that G. destructans would enjoy PDA. The plates are now on my stairs, and I am waiting. Waiting. Waiting. Waiting.
I suppose that's all for now. Just sitting around waiting for some fungus to grow...
Thursday, January 28, 2010
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...
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...
Tuesday, January 26, 2010
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.
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.
Wednesday, January 13, 2010
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.
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.
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