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Adventures in Concentrating Virus sample and Isolating/Extracting viral DNA from BSL Sediment... Plus Other Adventerous News!
Submitted by Jennifer Newsted on Mon, 08/12/2013 - 2:06pm
This week I’m going to talk about what I’ve been doing with the sediment samples we took from Boiling Springs Lake at Lassen Volcanic National Park. Then, I’ll divulge the latest news about my forthcoming adventures!
Remember this? It's the sediment collected from Boiling Springs Lake in July.
Since the sediment had a chance to settle the first thing I did was decant the supernatant into glass bottles. Then I took the remainder of the slurry, balanced it in 50mL conicals (aka Falcon tubes) and spun them down to separate the supernatant (liquid) from the sediment (solid) using the Jouan centrifuge to extract the rest of the supernatant.
I don't really want to talk about this next picture. Alright, I'll talk about it. One of the things that Portland CC's Bioscience Technology Program taught us was how to change the filter inside of a pipet aid. They also taught us that the goal is to NOT have to change one. I think we still owe the Singer Lab on B2 a filter.
After that I used the TFF (Tangential Flow Filtration) to filter the supernatant. The TFF concentrates particulates larger than 10kDa. So, basically this was like squeezing all of the actual water out of the supernatant. The TFF is attached to a pump that keeps the flow moving horizontally across the filter membrane rather than filtration that works by passing all the material through the membrane. This way, the microbes we want don’t get stuck to the filter. Instead they get pushed over the top of the filter and back into the filtrate being concentrated. (I’ve attached a pdf of the Millipore technical brief which explains it much better than I do and has a perfect graphic on the bottom of page 1 illustrating this.)
The TFF filtration took me from about 3 liters of supernatant to approximately 50mls each of supernatant “A” and supernatant “B” (B had been sediment resuspended in MgCl). The concentrates were again spun in the centrifuge to eliminate any remaining sediment contained in the supernatant. Then things got a little painful. Of course by painful, I mean still fun. Just really slow fun.
I had to filter the concentrate using a 35mL syringe and a 0.2 micron filter. After about 10 seconds of filtration, the filters clogged. I immediately activated my problem solver mode. I found a lead brick and a younger undergrad to push down on the lead brick which was balanced over the syringe to get the rest of the supernatant through. While it was a bit painful in execution, it was absolutely hysterical to watch.
The keeper of the VivaSpin columns, grad student Eric Iverson (photobombed above by Gulliver - BioMedCentral), originally handed out two columns to me. It was so dramatic. I imagined that must be similar to what it felt like for families receiving their weekly ration during the great depression. I shouldn't joke though, these things aren't cheap. VivaSpin columns are filters that look like conical tubes. They can hold 15mls at a time and have a traditional type filter that uses gravity (through centrifugation) to filter the supernatant. This time our molecular weight cutoff was 2k.
Lab math time kids! I have approximately 150mls of concentrated sample. The Vivaspin columns hold 15 mls of sample at a time. Now here’s the fun part: they filter approximately 2.5mls every half hour AND the Jouan only spins for thirty minute increments. My final volume needed to be concentrated to 0.5ml of each supernatant. Get the answer yet?
It took me the better part of the whole week. If someone tells you to filter that much supernatant, you want to have a blog to write and about 600 photos to edit through, or a whole lot of simultaneous projects on which to be working because it is going to take a long, long time.
They look like a cohesive little group of rebels in their version of a hot tub (ice bucket) sitting around laughing at me. Don't they?
Next I added extraction buffer which contained components to normalize ionic strength, a chelator that regulates how ions bond with metals, and some detergent to lyse the cells and SDS to denature the proteins and stop DNAase from killing my goal precipitant.
Here’s where things got sort of wild. After adding sodium acetate and cold isopropyl alcohol to precipitate the DNA I had to centrifuge my tubes. No problem. I’m no stranger to this process. When I took the tubes out and couldn’t see any pellets I freaked out a bit! After my anxiety attack subsided, I was able to confirm with Dr. Stedman that it’s normal not to see the amount of DNA precipitating in amounts this small even though it’s concentrated. So apparently this is normal. (Say what? Invisible pellets indeed!)
I’ve finally pooled all of the samples and am going through the incubation on the last precipitation. Once that incubation is done, I’ll be able to spin again. Maybe there will be a pellet. I don’t know since as of right now I’m not quite there yet. Personally, I’m rooting for a pellet which will at least tell me all the work I’ve been doing for the last 3 weeks has been correct. That there is in fact, virus genomic material present. Dr. Stedman says I probably won’t know for sure though until after my next adventure here: PHI-29 Amplification. This is going to be pretty cool since this amplification works while NOT knowing the sequence of the sample therefore we're blind on the exact primers that would normally be designed to do PCR. Am I rusty on my PCR? We shall see! (Update: I did see a pellet. Or I talked myself into believing I saw a pellet. PHI amp is tomorrow.)
So, that about sums up the learning student-y section of this week’s blog. Here comes the big news!
My ongoing adventures are taking me out of beautiful Portland, Oregon. I’ll be transferring to University of Nevada, Las Vegas for the fall term. Which means I’m moving in nine days. I will miss my adventures here at the Stedman Lab but I am confident that my future adventures are promising and filled with all kinds of learning!
The lab of Dr. Brian Hedlund at UNLV has offered me an undergraduate for-credit position. This is awesome news since it means I won’t be getting to Las Vegas, going into lab withdrawal and stalking the labs at UNLV with my face pressed up against the glass begging them to let me pipet something. I know it sounds like I’m exaggerating, but I think if I hadn’t found a lab that would take me in it may have really come to that. Especially since it's too late to get into an actual Bio class!
Dr. Hedlund’s lab also studies extremophiles. Hot springs ecology, single cell genomics, biofuels and microbial dark matter are some of the projects currently going on in the lab. I’m sure I’ll be filling in wherever I’m needed at first, but ultimately I would like to work on the microbial dark matter project and learn more about biochemical processes of these crazy little microbes. Which is in itself is an area I’ll admit, I am weak in, but I’m definitely eager to learn more about. I’m excited to start though and the lab appears to be full of plenty of accomplished researchers, and students from whom to learn! Yay!!!
But wait, there’s more!!! In addition to my moving adventures, settling into a new school, new lab, new home, I’ve also been invited by Bio-Link.org to attend the Advanced Technological Education Conference in Washington DC this October. In addition to speaking about the blog, I will also be working on a poster based on the work I’ve done in the Stedman lab to demonstrate the type of preparation community college technical program bioscience students receive and how it fits into an academic lab. This means that the good Dr. Ken Stedman doesn’t get to get rid of me so easily. Muahahahahaha!
Speaking of Dr. Stedman, he will be discussing his favorite topic, Viruses from Hell, as part of an OMSI Science Pub. The Extreme Virologist will be speaking at the Venetian Theater and Bistro, in Hillsboro on Monday, August 26th from 7-9 p.m. If you’re in the area I highly recommend going along for a listen. There’s a $5 suggested donation, but if you have any interest in virology or extremophiles I can assure you it will be easily worth it. I am so bummed to be missing it!
Uh, does anybody in Las Vegas want to help me unload my moving truck? :)
Gulliver has been up to more photobombing! Jeremy's is my favorite. Dr. Stedman is standing right behind Jeremy laughing because he knows what I was up to before Jeremy did. Too bad he got cut off. :(
Gulliver also helped oversee my SDS PAGE gels I made for Eric.
Gulliver must be a lucky charm. They didn't leak. My first try was solid.