Friday’s lab work a success! by Mayeesha

So far, things look great: from using the TapeStation on our DNA extracts, we know that there are small fragments of DNA (likely ancient DNA, or aDNA) preserved in the samples that we brought from our C.ohioensis specimens at JMM– Heather treated us to ice cream to celebrate! We do not yet know for sure whether the ancient DNA is endogenous giant beaver DNA (which is what we want) or whether it’s environmental DNA (we don’t want that). Ancient DNA is difficult to find in specimens that are so old, for various reasons including DNA damage due to poor preservation. At least, now we know that our samples probably contain aDNA and for now, that’s reason enough to celebrate!

For our next step on Thursday, we started a long-range PCR with the modern beaver DNA. (Just a heads up, from here onwards this post is going to have a lot of scientific details, so you can skip to the last paragraph if you’re not into all the details). The whole mitochondrial DNA of a modern beaver is about 16,000 base pairs (bp) long. So, we ordered primers that would amplify the entire mitochondrion in two large overlapping pieces that are 6,000 bp and 11,000bp. We ran a long range PCR to amplify those fragments. We were a little nervous whether the long range PCR would work at the first attempt, because Heather told us that they fail more often than not. We left the products in the thermocycler overnight because it takes ~6 hours to run a PCR that can amplify such long fragments!

Every day, we plan our work in such a way that we finish all our ancient DNA lab work first, before moving on to the modern lab, in order to reduce contamination from modern DNA and PCR products. So, the next morning (Friday), we couldn’t go in to look at our long range PCR results until after we finished any work we wanted to do in the ancient DNA lab. So, first we suited up in our smurf suits and started working on our single-stranded DNA library prep. This method has been optimized to retain very small DNA fragments, which is important for working with the small fragments of aDNA. Since the library prep is a long process, we decided to break up our work over 3 days and on Friday, we did the first three steps:

  1. Ancient DNA is often damaged by the Cytosine base being deaminated and converted into Uracil, artificially changing the DNA sequence from a Cytosine to a Thymine. Since we want accurate DNA sequences, we cut the damaged DNA at any Uracil site.
  2. We then dephosphorylated and denatured the DNA using heat to separate the two DNA strands from each other so we could work with single stranded DNA.
  3. We then added a short DNA sequence, called an adaptor, to each DNA strand. This adaptor has a biotin molecule on it, which will make it easier to use in later steps, which we will explain in future posts!

This is a good stopping point because ligation products can be stored for several days at 20°C.

After finishing up our work in the aDNA lab for the day, we went back to our long range PCR. Heather was very nervous about how the PCR would turn out because we had limited time on hand before she has to return to the U.S. and so we really needed the first trial to work out. I, on the other hand, was (way too) excited about the pretty purple gloves they had in the modern lab!

Luckily, our PCR did work on the first try (see the photo below)! The 6kb piece was amplified very nicely (right side of the photo on top and bottom left) but the 11kb needs to be cleaned by running a second gel because some other smaller pieces of DNA were also amplified (top left). After cleaning, we will chop the DNA to ~500bp fragments which will be used to make baits for hybridization capture (more details on this later).

long range PCR success

This photo shows the PCR products and a DNA ladder run out on a gel to visualize the results of our long range PCR. It worked!!!

Since our long range PCR worked, it was time for another celebration! We know that we are still many steps away from getting our final results which means there are many steps where things could go wrong and we could end up with no results. So far, nothing had gone wrong, and that’s what we were going to celebrate!

Categories: Uncategorized | Leave a comment

Holiday Weekend in Germany by Jacob

Mayeesha and I have decided to write about different things this time, so make sure to read both posts! She is going to discuss our lab work and provide updates on our research. I’m going to talk about our weekend.

After work on Friday, we went out with some of the other scientists. We went to a nearby restaurant and got flammkuchen (direct translation: flame cakes), these weird, flat German cream-cheese pizzas. While we ate, I got a chance to talk to some of the scientists I hadn’t met before. I really like the staff here. They’re all so funny. Afterwards, we went to a cafe in downtown Potsdam to get dessert.

We slept in late Saturday morning. I went on a run through some of the parks in the city. Potsdam is thirty percent parks and forests and thirty percent lakes and rivers, so the running trails are beautiful and plentiful. In the afternoon, we went to Sanssouci Park, the summer palace of Frederick the Great. It was very touristy, but very fun. We explored the gardens, which are incredibly elaborate and pretty. I love that in Germany most people keep their dogs off leash, so there are always dogs running around with which you can play. We also went grocery shopping. Most places are closed Sundays and holidays, so we had to make sure we bought enough food for the rest of the weekend.

Fitting in with the statues at Sanssouci Park

Fitting in with the statues at Sanssouci Park

Neues Palais in Sanssouci, built from 1763-1769 was a place to receive royal guests for Frederick the Great.

Neues Palais in Sanssouci, built from 1763-1769 was a place to receive royal guests for Frederick the Great.

Sunday, we went into Berlin to see a parade for the karneval der kulturen. The floats were pretty cool, with music and dancing and some crazy costumes. Interestingly, each float had a long rope around it held up by a group of people that walked with the float.

Note the people holding up a rope around the upcoming float!

Note the people holding up a rope around the upcoming float!

It was a long and slow parade though, so I explored the neighborhood after a while. I bought some bratwurst, which was delicious and the most German food I’ve had so far. After we all tired of the parade and the 1.3 million people attending it (!), we walked through a large park (Volkspark Hasenheide) south of the parade for a while. It was very busy and had a full carnival with rides and lots of sugary food. Then, Heather and Mayeesha went back to Potsdam, while I stayed and wandered around the city a little more. I wasn’t able to find any of the sites I wanted though, so I’m going to go back next weekend with a better plan. I especially want to see the Reichstag and the Holocaust Memorial.

My trip back to Potsdam was uneventful until I had to use the bathroom. The only bathroom in the train station cost one euro, which is ridiculous. I wasn’t about to pay to use a bathroom, so I jumped the turnstile. That set off an alarm, so I ran and hid in a stall. A security guard came in and tromped up and down in his heavy boots, looking for me, while I crouched on top of the toilet seat, so he couldn’t see my feet under the door. It was pretty scary. Luckily, my hiding place was too good and I was able to escape, without spending my hard-earned euro.

We had Monday off for some unknown German holiday. Heather and Mayeesha had work to do, so they mostly hung around the hotel. I went on a long run in the morning, leaving Potsdam and running through the farms in the surrounding countryside. I ran probably around 15 miles, so I was pretty tired and spent the rest of the day eating and napping. Heather was working on a paper, which she finished, so we celebrated by eating nachos. We feel rested and recharged, and are excited to continue our project and our adventure!

Categories: Research | Leave a comment

Hallo! from Jacob

Hallo from Deutschland! Yesterday we began our research at the University of Potsdam. Our first day at the lab we met the scientists and made a schedule. This is a large lab group of ~25 people, all here working on different projects. Everyone is incredibly nice and friendly. Our work is facilitated by George Xenikoudakis, a PhD student. In the next two weeks, we’re aiming to work through half our samples with Heather taking point, and then the following two weeks Mayeesha and I will work with George to process the other half.

There are multiple sections of the facility, with different rooms for different procedures. It’s really important not to contaminate the labs, so if you go into the PCR room or modern lab, you can’t go back into the ancient lab until you’ve changed and showered.

Today, we donned our protective suits (and gloves and mask and hairnet and crocs and more gloves) and began the DNA extraction process, working in the ancient lab. We ground our samples into powder and added buffers to begin digestion, and left them heating in an incubator overnight. Tomorrow we’re going to finish the aDNA extraction, and test to see whether we have any DNA in our samples.

Jacob Harris in his ancient DNA smurf suit

My cool suit

We worked a little with our modern specimens as well, running them through the TapeStation machine, which through a gel electrophoresis-type process created a distribution showing the frequencies of different size DNA fragments. Longer fragments work better later in the process, and luckily our samples look like they have plenty of those. We’ve ordered the primers we need to start the PCR, and hopefully they’ll arrive soon.

I’m still a little jetlagged, so I didn’t say much and kept bumping into things. I think I’ve made an excellent first impression.

Potsdam is a beautiful city. I’ve been running every morning, and the trails and paths are awesome. I’ll be running on a forested dirt trail, and suddenly find myself in the ruins of a Prussian castle. There are walking and bike paths everywhere, so it’s really easy to run and explore. The food is great too. The bread is especially delicious, and I get these excellent sandwiches for lunch from the school cafe.

I’ve been learning so much. Being immersed in a lab environment is a great experience, and our work is clarifying a lot of what I’ve been studying, both last week and in CGI. I’m really looking forward to continuing our research, and hopefully not knocking anything else over.

Categories: Uncategorized | Leave a comment

Arriving in Germany (by Mayeesha)

On Tuesday morning, we set off to go to the University of Potsdam where we are going to work with a team of researchers working under Prof Dr Michael Hofreiter. We are really excited to be given this opportunity to work in his ancient DNA lab because the DNA extraction method we will be using here is fairly new and is the best-suited method for our research. Matthias Meyer was one of Dr Hofreiter’s graduate students at the Max Planck Institute for Evolutionary Anthropology in Leipzig, and his group developed a new DNA extraction method in 2013 that is specific for short fragment DNA. This method has been used to sequence DNA that is ~300,000 years old (before this method was developed, the oldest DNA that could be extracted was ~120,000 years old!). That’s so cool! We spent the rest of the day ordering primers for generating mitochondrial genomes and chalking out a timeline for our experiments. Since we handled modern samples that day, we could not go into the ancient lab.

Today (Wednesday), we showered and put on clean clothes right before coming to the university. This is important because we are going to be working in the ancient lab so we have to be as clean as possible, so that we can reduce contamination in the ancient DNA lab. Before entering the ancient lab, we put on hairnets, masks and blue suits (hooded onesies!), clean clogs and double gloves. The sign on the lab door had the perfect description of what we looked like:

IMG_0135

(For a more realistic photo, check out Jacob’s post!)

In the lab, we cleaned the work surfaces and prepared our samples by crushing with a mortar and pestle. We added 1mL of extraction buffer to our samples, vortexed them and left them in and incubator at 37°C to be digested overnight. We then prepared the buffers that we will use tomorrow to finish our DNA extraction. We also got to use the Tape Station to measure the lengths of the DNA fragments we extracted from modern beaver tissue and now we know that our fragments are long enough to be used as baits in our hybridization capture method.

IMG_0142

George showing us how to use the TapeStation (above)

IMG_0144

Jacob and I are happy to see that our modern beaver samples have long enough DNA fragments (above)

I am looking forward to extracting DNA from our samples. Ideally, we should have sampled from the petrous bone as it is known to contain best-preserved DNA but this bone was missing in all of our giant beaver specimens. So, as our next best alternative, we brought mostly samples from dentin in teeth of the giant beaver fossils. This may or may not work; we won’t know until we do our extraction. For now, I am going to keep my fingers crossed and hope that we are able to sequence at least some giant beaver DNA from our ancient samples.

Categories: Uncategorized | Leave a comment

Off to Germany!

My name is Jacob Harris, and I just finished my freshman year here at Earlham. I’m so excited to be part of this research. Mayeesha, the museum director Heather, and I are studying DNA from C. ohioensis, the ancient giant beaver. Because the DNA is so old, it’s incredibly unstable and difficult to sequence and study. We’re sampling some of the specimens in the Joseph Moore Museum, and then we’re going to Germany to work in a specialized aDNA (ancient DNA) lab!

The first two days of the project, we focused on learning background information about ancient beavers. We reviewed beaver phylogenies, and read current studies. Scientists are debating whether beaver populations in the Midwest and Southeast were the same species. These articles focus on the morphological differences, mainly altered bone structures in the skull and different dental patterns. Ancient beavers in Florida tend to have more smushed-shape skulls and broken lines on their molars, whereas beavers in the Midwest, the traditional C. ohioensis, have an elongated skull and jaw. John Iverson came by and gave us an anatomy lesson, which really helpful.

We hope that by sequencing the DNA we can add a genetic element to the discussion. Unfortunately, it’s very difficult to get DNA from ancient specimens. What little DNA is left is likely heavily contaminated with bacterial and human DNA, even with extensive precautions. Temperature changes and UV radiation fragment and destroy DNA as well, and so ancient DNA can be seriously damaged. Specimens in places with more stable temperatures tend to preserve DNA more effectively, and so we’re looking for specimens found underwater. One thing we hope to prove with this project is that we can get DNA from common specimens, which would set us up for further research. In this project, our methods are just as important as our findings.

Today we started the DNA extraction process. We took samples from areas of our specimens more likely to contain DNA, primarily dentine from the interior of molars and incisors, where odontoblasts grew new teeth cells. Between each sampling we cleaned our equipment with bleach and changed gloves, so hopefully we avoided contamination. We asked to borrow specimens from other museums, and they’re being shipped. We’ll take samples from them as well. I’m excited to find out if we managed to sample any DNA, and I can’t wait to go to Germany!

Categories: Uncategorized | Leave a comment

Summer Research on Ancient DNA

Jacob Harris and I, Mayeesha Ahmed are doing summer research at the Joseph Moore Museum, supervised by museum director Dr. Heather Lerner. We will be working with ancient DNA extracted from Giant Beaver (Castoroides ohioensis) specimens to find out as much as we can about this large rodent from the Pleistocene.

The Joseph Moore Museum is known for having the most complete (7/8) giant beaver fossil in the world! So naturally we were interested to find out more about this extinct species. The first step on our exciting mission was to dig up what others have found out about giant beavers, and we learned some pretty interesting stuff! Despite being called the giant beaver, the Castoroides’ habits were similar to that of a muskrat. They lived in ponds and lakes surrounded by swamps and fed on swamp vegetation. They were found in the Northeastern and Midwestern US as well as the Southeast (Florida and Georgia) and even as far up north as Alaska and Canada! Some experts say that unlike modern beavers, the giant beaver was not a woodcutter. However there are others who disagree. There are also questions about whether there was more than one species of giant beaver as significant physical differences could be found in the parts of Castoroides skulls such as the lamdoidal crest, basisphenoid, mesopterygoid fossa.

Lamboidal basisphenoid mesoptery-what-fossa? These were some of the difficult anatomy terms we were coming across, which was making it difficult to understand the papers pointing out these interesting differences that are so relevant to our research. Looks like we need some help…

But not to worry! We invited an expert, John Iverson, to tell us more about these structures and he very generously helped us out! We learned how to identify the distinctive parts of the Castoroides skull and take note of the differences that have been observed in the papers we read. By looking at the pictures of Castoroides dilophidus from the Pleistocene of Florida and comparing them to our Castoroides ohioensis specimens, we were able to see significant differences in their bone structures that suggest that they could have been two different species. But John pointed out that these differences could have been due to geographical range or time period in which they lived. How can we find out what it actually was? Ancient DNA analysis will tell us!

But where can we find this ancient DNA? So, the next day we did some research to find more information about where in the tooth we may be able to find endogenous DNA (DNA that belongs to the giant beaver we are sampling from, and not environmental DNA). We found out that if we remove the enamel of the tooth we could expose the odontoblasts in the pulp cavity that are likely to contain DNA. So then, we gathered all the materials we need for ancient specimen sampling: bleach and ethanol for sterilizing the workspace, gloves, wipes, dremel, tubes to collect samples in. Next, we finally started doing some hands-on work on taking samples from the specimens. But we had to be very careful in order to minimize risks of contamination. We are going to pack these samples up and take them with us to Michael Hofreiter’s ancient DNA lab in Germany for extraction! We also extracted modern beaver DNA, which was done in our own lab.

Because of it’s age and many environmental factors, ancient DNA is very difficult to extract. I am really hoping that our samples contain DNA we can use for analysis and I am looking forward to learning more about ancient DNA in Germany

Categories: Uncategorized | Leave a comment

Young Scientist Symposium at the University of Michigan

The last weekend in March, JMM students (and students from the EC Evolutionary Biology class and alum, Nick Pondelis) donned mardi gras beads and set off to attend the Early Carrier Scientist Symposium at the University of Michigan. We extended our trip by a day so we could see the University of Michigan Museum Zoology collections and learn about the graduate program. First, we visited the exhibit museum and gift shop on our own. Then, we went to the Paleo collection where we learned about how paleontologists have adapted dentist’s tools, jeweler’s equipment, or made the equipment themselves to help them prep fossils.

2015-03-27 14.51.43

In the bird collection we saw unique, rare and type specimens. We also inhaled the scent of the Hawaiian honeycreepers and confirmed that they really do smell like a canvas tent! One particularly interesting specimen was a bird prepared in fluid with its bones stained red and its cartilage stained blue. It was then stored in glycerin to help keep the birds shape. The colors were vibrant and at least one of us would like to learn how to perform that kind of a prep!

We had dinner with two current graduate students, Delaney Cargo and Lisa Walsh. We learned about research that they were working on and their paths to graduate school. We especially enjoyed discussing animal behavior and Lisa Walsh’s research about Opossums. We talked about how they were able to get enough specimens for research and all of us had fun Opossum stories to share. We were also able to discuss different research that we had done and found communal interests in ecology and conservation, even though everyone had divergent interests.

2015-03-27 17.27.162015-03-27 16.12.52

Cindy Carl, the Graduate Program Coordinator of the Ecology and Evolutionary Biology Department (EEB) and Gina Baucom, Assistant Professor, talked to us about applying to graduate school, and specifically EEB. They gave us some tips for getting into graduate school, including contacting specific people in the department before applying and that it is best to have talked to a couple of potential advisors. They also advised us to be question-driven, specific, but not too specific, and enthusiastic when talking with a potential advisor.

Two of the presentations that we listened to on Saturday were Justine Garcia and Georgiana May.

Justine Garcia looked at insects’ symbiosis, specifically the broad-headed bug species, to determine partner specificity. She asked, “do gut microbes live in certain organisms and can they be transferred to other organisms?” Garcia found that microbes can be found in multiple species and can switch between organisms. She also looked to see from where the bugs were getting their microbes and found they were coming from plant leaves, plant nodules, and soil.

As the last presenter on Saturday, Georgiana May was a Keynote presenter. Her presentation was entitled Microbial interactions drive the evolution of virulence in pathogens and was based around an experiment she did on maize and two microbes that lived in it, one being an endophyte, and one being a fungal pathogen. She found that within a maize plant, the endophyte gains a growth benefit from the fungus’ presence, and the fungus gains a reproduction benefit from the endophyte’s presence.

by

Elisabeth Sorrows

Categories: Uncategorized | Leave a comment

Big Ol’ Salamanders (and a Big New one, too!)

by Chris Angell

This week, the discovery of a new Triassic amphibian species has made headlines across the ‘net. Touted as “a Triassic terror” and “super salamander,” Metoposaurus algarvensis was a six-foot-long aquatic beast that lived in Portugal alongside some of the early dinosaurs. It probably ate mostly fish and may have lived like a modern crocodile. Despite the hype surrounding Metoposaurus, this “super salamander” is not the biggest (or the most interesting) extinct amphibian we know of!

_81840122_algarvensis

Metoposaurus algarvensis by Marc Boulay

Frogspawn_closeup

Frog eggs by Tarquin at the English language Wikipedia

Before we go any further, let’s be clear about what animals we’re talking about. Amphibians (from the Greek for “double life,” due to their life cycle that spans both land and water) are vertebrates with four legs and moist skin, which return to water to breed. Unlike reptiles and birds, whose eggs are covered by a protective shell, amphibian eggs are jellylike and usually have to stay wet to survive. Frogs and salamanders make up most of today’s amphibians, although there is a third, lesser-known group called the caecilians.

Metoposaurus belong to a group of early amphibians called Temnospondyls, whose relationship to modern frogs and salamanders is unclear. They may have been the ancestors of modern amphibians (or it might have been another group called Lepospondyls). But what is clear is that 250 million years ago, Temnospondyls were fearsome creatures!

A six-foot long salamander might sound huge, but Metoposaurus was far from the biggest amphibian during their heyday. The longest amphibian to ever walk the Earth (or rather, swim the rivers and ponds) was the Brazilian Prionosuchus, a 30-footer, which looks strikingly similar to today’s gharials. Coming in second is the 16-foot-long, Australian Koolasuchus, with its flat, rounded head. “Suchus,” in both of their names, comes from the Greek word for crocodile, referencing their size and lifestyle.

Prionosuchus_DB

Prionosuchus by ДиБгд

428px-Diplocaulus_vale21DB

Diplocaulus by Dmitry Bogdanov

Probably the most striking extinct amphibian is Diplocaulus, from Morocco: a three-foot-long Lepospondyl with a boomerang shaped head. Nobody knows for sure why its skull was shaped this way. Maybe it worked like an airplane wing, generating some lift as Diplocaulus swam through the water. Or perhaps it was defensive: you would have to have a pretty big mouth to swallow something with a head like this!

There were some pretty big and crazy amphibians in the past, but don’t worry! We’ve still got a few gigantic salamanders of our own. The appropriately named “Giant Salamanders” are a family that can be found in Asia and the United States. The American species, the hellbender, can reach up to two feet in length and can be found from New York to Mississippi and as far west as southern Indiana. But the Asian giant salamanders put ours to shame! The Chinese giant salamander, the largest living salamander species, can be up to six feet long! Unfortunately these salamanders are highly endangered due to habitat destruction and overhunting for use in Chinese medicine.

giant_chinese

Giant Chinese Salamander by James Joel

Categories: Uncategorized | Leave a comment

Bats emerging

We have a new visitor at the Joseph Moore Museum, a little brown bat that emerged on campus a little too early. Have you seen bats around recently?

IMG_9391IMG_9397 The fluctuating temperatures may cause some bats to come out of hibernation early and some bats arouse during the winter months to hydrate at infrequent intervals. In particular, bats who are suffering from white nose syndrome may especially need hydration. Many bat populations are experiencing drastic declines from white nose syndrome and a recent study found evidence that dehydration may play a role in increasing mortality from white nose syndrome.

If you find a bat, to keep the bat and yourself safe do not touch the bat with your bare hands. Also, do not force your bat out into the cold.  In this cold weather the bats will freeze almost immediately if let outside. They will fare a lot better once overnight temperatures are sustained at ~40 degrees. Call the Animal Care Alliance (765)488-1342. They are located at 4101 National Rd. West Richmond, IN. We’ll be sending our bat visitor their way too! Overnight, our public safety was able to coral the bat safely into a carrier, where there is water and a comfortable branch for resting.

 

Categories: Uncategorized | Leave a comment

Joseph Moore Museum Open House

The Joseph Moore Museum will be open from 10am-5pm February Saturday 25. Only 5% of the Joseph Moore Museum Collections are in the museum come see behind the scenes in the collection rooms.

There will be skinning demonstrations, cool new games like Senat, face painting, the opportunity to try eating bugs and much more. These opportunities will be occurring throughout the day Saturday February 25.

New Measures will be performing at 2 pm.

There will be planetarium shows at 10:30 am, 12:30 pm, 2:30 pm, and 4:30 pm.

Categories: Uncategorized | Leave a comment

Blog at WordPress.com. The Adventure Journal Theme.

Follow

Get every new post delivered to your Inbox.

Join 771 other followers

%d bloggers like this: